WO2008012724A2

WO2008012724A2 - Optical tracking and position determination for detection methods and systems

Info

Publication number: WO2008012724A2
Application number: PCT/IB2007/052800
Authority: WO
Inventors: Marius I. Boamfa; Erik M. H. P. Van Dijk; Reinhold Wimberger-Friedl; Christianne R. M. De Witz
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2006-07-20
Filing date: 2007-07-13
Publication date: 2008-01-31
Also published as: WO2008012724A3

Abstract

A tracking system (106) is described for tracking areas on a substrate (104) using an irradiation beam. The irradiation beam typically has an irradiation beam projection (202) on the substrate (104) whereby at least one dimension of the irradiation beam projection (202) is substantially larger than a wavelength of the irradiation beam. The tracking system (106) typically comprises a detecting means (108) adapted for detecting a variation of an optical characteristic of at least part of the irradiation beam induced by interaction with different regions (204, 206) of the substrate having different irradiation modulating properties, for locating said irradiation beam with respect to said substrate (104). The tracking system typically is adapted to cooperate with a substrate. The invention also relates to a position determination system for obtaining position related information from a modulation of the irradiation beam induced by the substrate, a corresponding method and substrates for use therewith.

Description

Optical tracking and position determination for detection methods and systems

FIELD OF THE INVENTION

The present invention relates to methods and systems for optical tracking of a beam, e.g. used in a detection system, with respect to its position on a substrate. More particularly, the present invention relates to methods and systems for optical tracking of a substantially large beam with respect to its position on a substrate e.g. in optical detection systems such as detection systems for biological, bio-chemical or chemical particles on a substrate. It also relates to corresponding substrates and methods of making the same. The present invention also relates to a position determination system and method for obtaining position related information from a modulation of the irradiation beam induced by a substrate, and substrates for use therewith.

BACKGROUND OF THE INVENTION

In general, molecular diagnostics of a bio-sample, usually a liquid analyte mixture, typically comprises screening of the bio-sample for detection of certain biological components, referred to as target particles, such as genes or proteins. This is done by first allowing selective bindings to be formed between target particles and capture probes that typically are attached to a solid surface. Such selective binding is also known as hybridization. The hybridization step is then typically followed by a washing step, where all unbounded target particles are flushed away. Finally a detection step is performed for detecting the presence of target particles captured by the capture probes. Taking into account the probability for capturing the target particle to the capture probe, a quantitative analysis of the concentration of target particles present in the bio-sample can be determined. The detection step is typically based on detection of fluorescent labels attached to the target molecules. In order to cope with the small concentration of target molecules typically present in the bio-samples, it is important that the fluorescent detection is very sensitive, ideally close to the ultimate detection limit of single fluorescent label sensitivity. To reach this high sensitivity, the detection step is typically time consuming.

Methods of maintaining an illumination beam on tracks of e.g. an optical disc are known. A typical optical tracking method used may be based on the generation of a tracking error signal referred to as push-pull signal for the illumination beam. Such a tracking error signal typically is generated based on the interaction of the spot with grooves or some other tracking structure placed on the disc surface. US Patent Application 2005/0265153 Al discloses such a tracking system and method for guiding an optical beam on tracks on an optical disc e.g. in optical disc players or writers. The tracking system includes a photo detector comprising two areas for detecting reflected irradiation signals of an optical spot, thereby generating two output signals. Using these output signals, the position of the illumination beam is adjusted such that the irradiation beam is kept on track. The tracking system described furthermore comprises components to take into account whether tracks are written or unwritten. Such a system typically is based on grooves present in an optical disc, adapted for tracking beams having a diameter of the order of the wavelength of the illumination beam used. Such tracking thus typically is based on the diffraction of a beam on structures with dimensions comparable with the wavelength of the used light.

SUMMARY OF THE INVENTION

At present in the field of molecular diagnostic the fluorescence detection is addressed by different methods, such as scanning the sample surface with a tightly focused beam and temporary detecting a fluorescence response, statically irradiating a large sample area and spatially detecting a fluorescence response, or scanning the sample surface with an elongated beam and detecting spatially and temporary a fluorescence response. Using an elongated beam for scanning the sample surface allows to obtain a high throughput while providing sufficient illumination power on the substrate and obtaining good sensitivity.

It is an object of the present invention to provide good or alternative methods and systems for optical tracking of a beam on a substrate, e.g. used in a detection system, with respect to its position on a substrate.

It is another object of the present invention to provide a position determination system for obtaining position related information from a modulation of the irradiation beam induced by the substrate, a corresponding method and substrates for use therewith.

It is an advantage of embodiments of the present invention that systems and methods are provided that can be used for a large number of irradiation beam configurations, e.g. irradiation beam configurations having an irradiation beam on the sample that has at least one dimension substantially larger than the wavelength of the light used. It is an advantage of embodiments of the present invention that structures to be scanned and applied on a substrate for tracking an irradiation beam thereon may be substantially larger than the wavelength of the light used.

It is an advantage of at least some embodiments of the present invention that position information with respect to the scanned surface can be obtained accurately. It is an advantage of at least some embodiments of the present invention that position information can be obtained independent of a scanning stage accuracy, a stability and a scan velocity.

The above objective is accomplished by a method and device according to the present invention. The invention is defined by the independent claims. The dependent claims define advantageous embodiments.

The present invention relates to a substrate for use with a tracking system, the substrate adapted for being scanned with an irradiation beam in a scan direction, e.g. either by movement of the substrate relative to the beam or the beam relative to substrate or a combination of these two, the irradiation beam having an irradiation beam projection on the substrate having at least one dimension larger than a wavelength λ of the irradiation beam, the substrate comprising a plurality of alternating first regions and second regions wherein the first regions and second regions are substantially extending along a first direction suitable for being substantially the scan direction, the first regions and second regions having a length in a second direction substantially perpendicular to the scan direction adapted such that at least one of the first regions and second regions has a length in a second direction substantially perpendicular to the scan direction which is substantially larger than the wavelength λ of the irradiation beam, the first regions and second regions having a substantially different optical property for modulating the irradiation beam. It is an advantage of embodiments of the present invention that methods for optical tracking of a beam and optical tracking systems can be provided whereby the irradiation beam substantially is larger than the wavelength of the light used.

The substrate may be especially useful in the field of biosensors. The substrate may optionally comprise binding sites, e.g. spotted binding sites. The binding sites may e.g. be adapted for binding chemical, biological and/or bio-chemical particles to be detected.

The substantially different optical property may be provided using sub- wavelength patterns. It is an advantage of embodiments of the present invention that standard techniques may be applied for creating different optical properties.

The present invention further relates to a substrate (104) comprising: a first region having a first optical property and a second region having a second optical property, the first optical property being different from the second optical property, the first region and the second region extending along a first direction, the first region having a first length along a second direction and the second region having a second length along the second direction, the second direction being perpendicular to the first direction; binding sites capable of binding chemical, biochemical and/or biological particles;

The first and second region thus provide an optical contrast. Such an optical contrast may be advantageously used by a tracking system according to the invention of a sensor or measurement device. The change of the optical contrast providing information on the position of the projection of an irradiation used in the tracking system on the substrate.

In an embodiment the binding sites are present at least in the first region. This first region may then be specially reserved for comprising the binding sites whereas the second region may serve to provide the optical contrast.

In an embodiment along a third direction perpendicular to the first and second directions, a finite distance exists between the first region and the second region. The first and second region form a step on the surface of the substrate. The first and second region may have a surfaces that are not parallel. In an embodiment the second region extends on a first side of the first region and a further second region extends along the first direction on a second side of the first region. At least tow second regions can now be used to guide the irradiation beam over the first region.

In an embodiment the second region is at least partially discontinuous in the first direction. Partially discontinuous is to be construed such that when following along the first region it is interrupted at least over part of its first length, where the first length is measured in the second direction. As such, a clock signal may be provided as will e elucidated hereafter.

In an embodiment at least on of the first and second regions comprises a sub wavelength pattern.

In an embodiment the second region and the further second region are both discontinuous such that the discontinuity of the first region is not in phase with the discontinuity of the further second region at least along part of the distance over which the second region and further second region extend in the first direction. This is advantageous for providing information with respect to stabilisation of the irradiation beam.

Any of the first regions and/or the second regions may comprise additional information. Such additional information may be a position address and/or a clock signal. The additional information may be provided in optical modulating structures. The optical modulating structures may comprise one or more reflective structures, absorption structures or phase modulating structures. The additional information may be indicative of position related information of the optical modulating structures with respect to the substrate.

The substantially different optical property may be a substantially different reflectivity, transmission, scattering behavior or absorption behavior or any combination of these.

The present invention also relates to a tracking system for tracking areas on a substrate using an irradiation beam, the irradiation beam having an irradiation beam projection on the substrate whereby at least one dimension of the irradiation beam projection is substantially larger than a wavelength λ of the irradiation beam, the tracking system comprising a detecting means adapted for detecting a variation of an optical characteristic of at least part of the irradiation beam induced by interaction with different regions of the substrate having a different irradiation modulating property, for locating the irradiation beam with respect to the substrate. The different regions may be at least a first region and a second region.

It is an advantage of the present invention that the tracking system is robust to noise or mechanical interference. Alternatively worded, the tracking system as described above and hereinafter may be a tracking error detecting system. Tracking thereby may be finding the relative position, e.g. determining a tracking error signal, but it also may include responding to such an error signal to correct the relative position. The tracking system may be a tracking system adapted for tracking an irradiation beam in a detection system for detecting chemical, biological and/or bio-chemical particles, such as e.g. a biosensor. The tracking system thus may be a tracking system for tracking areas on a substrate comprising binding sites for binding chemical, biological and/or bio-chemical particles to be detected. The detecting means may be adapted to simultaneously detect part of the irradiation beam modulated by a first region of the substrate and part of the irradiation beam modulated by a second, adjacent region of the substrate, the first and second region having a different irradiation modulating property. The variation in the optical characteristic of the irradiation beam may be induced by a variation of an area of the first region of the substrate and a variation of an area of the second region of the substrate It is an advantage of embodiments of the present invention that the irradiation beam can be tracked with high accuracy, e.g. based on the intensity distribution.

The irradiation beam may be an at least piecewise elongated irradiation beam having an irradiation beam projection with a length in a direction perpendicular to a scan direction by which the substrate is scanned with the irradiation beam, the length being substantially larger than the wavelength λ. It is an advantage of embodiments of the present invention that an irradiation beam having a length substantially longer than the wavelength λ can be used, resulting in a high throughput for detecting samples and shorter overall detection time or improved signal to noise ratio, thus resulting in efficient systems. An optical system such as e.g. a detection system may also comprise a means for scanning the irradiation beam over the substrate, e.g. either by moving the beam, moving the substrate or a combination of the two.

The tracking system furthermore may comprise a position correcting means adapted for correcting the position of the at least piecewise elongated irradiation beam with respect to the substrate in view of a detected optical characteristic variation of the at least part of the irradiation beam. It is an advantage of embodiments of the present invention that a correct position of an irradiation beam can be obtained and maintained.

The tracking system may comprise an automated feed-back loop between the detecting means and the position correcting means. It is an advantage of embodiments of the present invention that the system can operate in an automated and automatic way.

The optical characteristic of the at least part of the irradiation beam may be any of: the amount of reflected light, the amount of transmitted light, the amount of scattered light or the density of scattered light of at least part of the irradiation beam induced by interaction with the substrate. It is an advantage of embodiments of the present invention that the tracking system can be applied in a large number of optical systems, e.g. detection systems, such as both in systems working in reflection or operating in transmission or in both of these. It is also an advantage that different optical properties of the substrate may be used in embodiments according to the present invention.

The tracking system may furthermore be adapted to detect additional variations in the optical characteristic of the irradiation beam and may comprise a processing means for determining additional information from the additional variations in the optical parameter. It is an advantage of tracking systems according to embodiments of the present invention that additional information also can be obtained using such tracking system.

The detecting means may be any suitable optical detector, e.g. a split detector, a single spot detector or a pixelated detector. It is an advantage of tracking systems according to embodiments of the present invention that standard detectors can be applied in the tracking system.

The tracking system may furthermore comprise a rotating means for rotating the irradiation beam with respect to a rotating axis substantially inclined to the substrate.

It is also an advantage of embodiments of the present invention that an automated and/or automatic tracking of areas to be scanned by the irradiation beam is obtained. It is furthermore an advantage of embodiments of the present invention that they provide a tracking system that may be part of an optical system such as e.g. a detection system, thus avoiding the need for manually tracking the area to be scanned by an irradiation beam of an optical system such as e.g. a detection system using mechanical means. It also is an advantage that a substantially high irradiation power can be used. It furthermore is an advantage of the present embodiment that large areas can be scanned, without positioning problems of the irradiation beam. The latter results in a good image quality e.g. substantially without blurring of the image, e.g. by scanning neighboring areas at least partly more than once. It is a further advantage of the present invention that the tracking system is robust to noise or mechanical interference.

The detecting means may furthermore be adapted for stabilizing the orientation of the irradiation beam with respect to the scanning area of the substrate.

The present invention also relates to a method for tracking a position of an irradiation beam on a substrate, the method comprising scanning the substrate by guiding the relative position of the substrate and an irradiation beam having an irradiation beam projection on the substrate with at least one dimension substantially larger than a wavelength λ of the irradiation beam, and detecting a variation in an optical characteristic of at least part of the irradiation beam induced by interaction with different regions of the substrate having a different irradiation modulating property, for determining a position of the at least piecewise elongated irradiation beam with respect to the substrate. The method further may comprise correcting the position of the at least piecewise elongated irradiation beam on the substrate in view of a determined irradiation beam location.

The method further may comprise stabilizing the orientation of the irradiation beam with respect to the scanning area of the substrate. The present invention also relates to a detection system for detecting light emission sites on a substrate, the detection system comprising an irradiation source for generating an irradiation beam having an irradiation beam projection on the substrate whereby at least one dimension of the irradiation beam projection is substantially larger than a wavelength λ of the irradiation beam, and a tracking system comprising a detecting means adapted for detecting a variation of an optical characteristic of at least part of the irradiation beam induced by interaction with different regions of the substrate having a different irradiation modulating property, for locating the irradiation beam with respect to the substrate. It is an advantage of embodiments of the present invention that detection systems with high accuracy may be obtained. It is also an advantage of embodiments of the present invention that detection systems are obtained that can be operated in an automated and/or automatic way. It is a further advantage of the present invention that the tracking system is robust to noise or mechanical interference. The detection system furthermore may comprise a detection unit adapted for spatially detecting a luminescence response obtained by scanning the sample on the substrate and an evaluation unit that is adapted for memorizing locations with a specific luminescence response as candidates for an occupied binding site and to classify such a candidate as a detected occupied binding site if it shows a predetermined response behavior in two or more scans.

The present invention also relates to a method for manufacturing a substrate for use in a detection system for detecting of light emission sites on a substrate, the substrate being adapted for being scanned with an irradiation beam having an irradiation beam projection on the substrate having at least one dimension larger than a wavelength λ of the irradiation beam, the method comprising providing alternating first regions and second regions in the substrate, wherein the first regions and second regions are substantially extending along a first direction suitable for being the scan direction, the first regions and second regions having a length in a second direction substantially perpendicular to the scan direction adapted such that at least one of the first regions and second regions has a length in a second direction substantially perpendicular to the scan direction which is substantially larger than the wavelength λ of the irradiation beam, the first regions and second regions having a substantially different optical property for modulating the irradiation beam.

In one aspect the present invention provides efficient methods and systems for detecting optical emission sites using a scanning irradiation beam having an irradiation beam projection on a substrate comprising the optical emission sites, with a length of the irradiation beam projection being substantially larger than the wavelength of the irradiation beam and methods and system for tracking areas to be scanned with such an irradiation beam.

It is an advantage of embodiments of the present invention that an efficient and highly sensitive detection system is obtained, allowing to accurately position the irradiation beam.

In another aspect, the present invention also relates to a position determination system for determining position related information of an irradiation beam on a substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the position determination system using an irradiation beam for irradiating the substrate for detecting presence of chemical, bio-chemical and/or biological particles, and the position determination system comprising a detection sub-system for detecting a modulation of the irradiation beam after interaction with the substrate, and for deriving from said modulation position related information of said irradiation beam with respect to the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam reflected at the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam transmitted at the substrate.

The position determination system may comprise a filter for filtering the irradiation beam after interaction with the substrate from a luminescence response from said chemical, bio-chemical and/or biological particles. It is an advantage of embodiments according to the present invention that the amount of stray radiation can be limited.

The present invention in one aspect also relates to a method for determining an absolute position of an irradiation beam with respect to a substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the method comprising irradiating the substrate with an irradiation beam for detecting the presence of chemical, bio- chemical and/or biological particles, detecting a modulation of the irradiation beam after interaction with the substrate, and deriving from said modulation position related information of said irradiation beam with respect to the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam reflected at the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam transmitted at the substrate.

The method furthermore may comprise, prior to detection of the modulation of the irradiation beam after interaction with the substrate, filtering the irradiation beam after interaction with the substrate, from a luminescence response from said chemical, biochemical and/or biological particles. Deriving position related information may comprise deriving absolute position related information with respect to the substrate.

Deriving position related information may comprise recognizing part of the modulation as start and/or end indication of the information. Deriving position related information may comprise deriving at least one digit value from the modulation.

The present invention in another aspect furthermore relates to a substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the substrate comprises optical modulating structures for modulating an impinging irradiation beam, the modulation being indicative of position related information of the structures with respect to the substrate. It is an advantage of embodiments of the present invention that position related information can be embedded in the substrate and does not need to be derived completely from the position of the components of the detection system. The latter improves the accuracy of the position determination. The substrate may be for use with a biosensing system.

The optical modulating structures may comprise at least one of reflection structures, absorption structures or phase modulating structures.

The optical modulating structures may be encoded with a first part encoding the position related information and a second part indicating a start and/or end of said first part. It is an advantage of embodiments of the present invention that accurate recognition of information can be provided.

The optical modulating structures may comprise different portions with different optical modulating properties for indication of different values of a plurality of digits. It is an advantage of embodiments of the present invention that numerical information can be encoded in the optical modulating structures. Such numerical information may be binary or may use digits having more than 2 possible values.

The optical modulating structures may comprise first optical modulating structures representative of a position in a first direction and second optical modulating structures representative of a position in a second direction, the first and second optical modulating structures being provided in an alternatingly manner. It is an advantage of embodiments of the present invention that information regarding the track as well as information regarding the position in the track can be provided that can be read out using the same system.The present invention, in another aspect, also relates to a detection system for detecting light emission sites on a substrate, the detection system comprising an irradiation source for generating an irradiation beam for irradiating the substrate for detecting presence of chemical, bio-chemical and/or biological particles, and a position determination system comprising a detection sub-system for detecting a modulation of the irradiation beam after interaction with the substrate, and for deriving from said modulation position related information of said irradiation beam with respect to the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam reflected at the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam transmitted at the substrate.

The present invention, in another aspect, furthermore relates to a method for manufacturing a substrate for detecting of light emission sites on a substrate, the substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the method comprising providing optical modulating structures for modulating an impinging irradiation beam, the modulation being indicative of position related information of the structures with respect to the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam reflected at the substrate. The irradiation beam after interaction with the substrate may for example be part of the irradiation beam transmitted at the substrate. The substrate may be for use with a biosensing system. It also is an advantage of device embodiments of the present invention that these can be based or can be built similar to known optical storage pick-up systems such as compact disc device (CD-ROM) or digital versatile device (DVD) writers/recorders, e.g. using standard optical components used in such optical storage pick-up systems. Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

The teachings of the present invention permit the design of improved methods and apparatus for detecting biological, bio-chemical or chemical particles.

The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic illustration of a substrate adapted for wide band tracking according to a first embodiment of the first aspect of the present invention.

Fig. 2 is a schematic illustration of a substrate adapted for wide band tracking according to a second embodiment of the first aspect of the present invention. Fig. 3 is a schematic illustration of a substrate adapted for wide band tracking according to a third embodiment of the first aspect of the present invention.

Fig. 4 and is a schematic illustration of a substrate with embedded position information providing features according to a fourth embodiment of the first aspect of the present invention. Fig. 5 to Fig. 7 are schematic illustrations of a substrate with embedded position information providing features in a substrate adapted for wide band tracking, according to a fifth embodiment of the first aspect of the present invention.

Fig. 8 shows a schematic representation of a patterned substrate provided with position information providing features according to a further embodiment of the first aspect of the present invention.

Fig. 9 shows a schematic illustration of a patterned substrate provided with both relative and absolute position information providing features according to an embodiment of the first aspect of the present invention.

Fig. 10 shows an enlarged view of part of the relative position information providing features of Fig. 9, together with a corresponding position of the tracking detector projected in the sample plane.

Fig. 11 shows an enlarged view of part of the absolute position information providing means indicated in Fig. 9.

Fig. 12 shows a schematic representation of a patterned substrate with in the different wide band tracks alternatingly horizontal absolute position information and vertical absolute position information, according to an embodiment of the first aspect of the present invention.

Fig. 13 is a schematic flow chart of a method for tracking a wide band according to a first embodiment of a second aspect of the present invention. Fig. 14 is a schematic overview of a tracking system according to embodiments of the third aspect of the present invention.

Fig. 15 is a schematic top view of a split detection set-up as can be used in embodiments according to the third and fourth aspect of the present invention. Fig. 16 is a schematic overview of a detection system with a tracking system according to embodiments of the fourth aspect of the present invention.

Fig. 17 is another schematic overview of a detection system with a tracking system according to embodiments of the fourth aspect of the present invention. In the different figures, the same reference signs refer to the same or analogous elements.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

Furthermore, the terms first, second and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art.

With an at least piecewise elongated irradiation beam, there is meant an irradiation beam having a projection on the substrate that is spatially continuous or discontinuous and that is substantially elongated in one direction of the projection on the substrate. At least piecewise elongated may correspond with a single continuously elongated beam component or with a number of spots located with respect to each other such that they are spread substantially throughout the whole elongated beam component on said substrate, meaning that said spots are present in at least 50%, preferably at least 70%, more preferably at least 90% of the whole elongated region. There thus is meant that the total irradiation beam component irradiates a region or substantial parts thereof that is substantially elongated in one direction.

Whereas the following description, embodiments and examples are mainly oriented to a tracking system, detection system, positioning system and substrate based on reflective measurements or reflective properties, the present invention also includes embodiments relating mutates mutandis to systems/devices operating in transmissive mode and to corresponding methods. Modulation in or by substrates usable in transmissive mode may, alternatively or in addition to reflective structures and phase structures, also comprise absorption structures. In other words, structures on the substrate are envisaged that influence the transmission of an irradiation beam through the substrate, thus allowing modulation of the transmitted irradiation beam.

In a first aspect, the present invention relates to substrates 104 for use with a tracking system for tracking an irradiation beam on a substrate. The irradiation beam typically has a projection, being a cross section of the beam with the substrate, on the substrate having at least one dimension being substantially larger than the wavelength of the irradiation beam. The dimension of the irradiation beam projection 202 on the substrate 104 substantially larger than the wavelength of the irradiation beam may be the dimension in the direction substantially perpendicular to the scan direction S (as shown in Fig. 1, Fig. 2 and Fig. 3) of the irradiation beam. Such an irradiation beam may e.g. be an at least piecewise elongated irradiation beam, although the invention is not limited thereto. In other words, substrates 104 according to embodiments of the first aspect of the present invention are adapted for being scanned with a large irradiation beam, i.e. an irradiation beam with an irradiation beam projection 202 on the substrate 104 having at least one dimension substantially larger than the wavelength λ of the irradiation beam. Exemplary embodiments are shown in Fig. 1, Fig. 2 and Fig. 3. The substrates 104 are preferably adapted such that tracking areas to be scanned with a substantially large irradiation beam becomes possible. Typically, the latter is performed by patterning the substrate 104. In order to assist in tracking an area to be scanned with an irradiation beam, i.e. to provide an irradiation beam at the correct position with respect to areas of the substrate 104 to be scanned, the substrate 104 typically comprises a first region 204 and a second region 206 extending along a first direction suitable for being a scan direction S. Those skilled in the art will know that alternatively and without loosing the effect of the invention a plurality of alternating first and second regions can be used. The first regions 204 and second regions 206 thus may have a band-like shape. The first regions 204 and second regions 206 typically may be positioned adjacent to each other. The first regions 204 typically have an optical property that is different from the second regions 206, such that the irradiation beam used for scanning a substrate 104, e.g. during a detection action, is modulated differently in the different regions 204, 206. In order to be able to track a location of an irradiation beam with respect to the substrate 104, typically at least one of the first regions 204 and second regions 206 have a length in a second direction perpendicular to the first direction, i.e. perpendicular to the typical scan direction, that is substantially larger than the wavelength of the irradiation beam. Typically the length of the regions to be scanned, e.g. the first region having a first length Dfirst region, or a combination of the first region and the second region having a second length, may be slightly smaller than the at least one length of the irradiation beam projection being substantially larger than the wavelength λ of the irradiation beam. The first length is preferably between 0.5 micrometer and 1000 micrometer. More preferably the first length is inbetween 10 micrometer and 250 micrometer and most preferably it is between 50 and 150 micrometer. The optical properties of the first regions 204 and second regions 206 may be any of: the reflectivity, the transmission, the absorbance, the scattering capacity or any other suitable feature allowing to modulate the at least piecewise elongated irradiation beam or at least part thereof. Different optical properties for the first regions 204 and for the second regions 206 may be obtained in many different ways. For example, the different optical characteristics may be obtained by selecting different materials used, by covering the regions 204, 206 with different sub-wavelength patterns or by covering the area with different thin layers. The different regions 204, 206 also may show a different surface roughness or different thickness such that phase modulation is different for radiation reflected or transmitted by the first regions and the second regions. Thus, in case systems are used with a transmission set-up, the thickness of the substrate in the different regions 204, 206 may be different, resulting in different absorbance or transmission of the irradiation beam. Typical examples of using material selection may be using metallic regions having a high reflectivity for the first regions 204 and using non-metallic regions or metallic regions with low reflectivity for the second regions 206 or vice versa. Applying different sub-wavelength patterns may offer the advantage that sub-wavelength patterns can be easily manufactured in a replication process that is compatible with optical storage replication methods. The substrate 104 may be a flat plate and may comprise a base of glass or polymer.

The substrates may be used for a variety of applications. The substrates may e.g. be used for biological, bio-chemical or chemical detection in samples, they may be used as optical storage devices, they may be used in other optical applications, etc. The substrates may be also further adapted for use in these different applications. E.g. in the case of substrates suitable for biological detection, bio-chemical detection or chemical detection, the substrate 104 typically may comprise capture elements, for capturing different particles from the sample. The substrate may have e.g. capture elements with a surface density between 0.01 and 10⁶ elements per μm², preferably between 1 and 10⁴ elements per μm². The substrate with capture elements in contact with the sample or the substrate after it has been in contact with the sample, typically may be screened for certain components, e.g. biological components such as oligonucleotides, DNA, RNA, genes, proteins, carbohydrates, lipids, cells, cell components such as external cell membranes or internal cell membranes, bacteria, viruses, fungi, protozoa, etc. also called the target particles. Luminescent labels are typically attached to the target particles and thus assist in the detection of target particles. Such labels can be, for instance, fluorescent, electroluminescent, chemo luminescent particles, etc. The optical variable particles may be any entity that is capable to bind to a binding site mechanically, electrically, chemically or otherwise. It may be single molecules or a plurality of molecules, preferably a collection of between 1 to 10⁸ molecules and/or quantum dot-like labels. If a plurality of molecules is used, typically a stronger response to the irradiation is obtained, resulting in a better signal-to-noise ratio. The latter illustrate one of the vast amount of applications in which these substrates may be used, such as e.g. in clinical diagnostics, point-of-care diagnostics, advanced bio-molecular diagnostic research, biosensors, gene and protein expression arrays, environmental sensors, food quality sensors, optical storage applications, etc. Capture elements may be applied only to those regions of the substrate that will be scanned with the wide irradiation beam, i.e. the irradiation beam having an irradiation beam projection 202 on the substrate 104 having at least one length being substantially larger than the wavelength of the irradiation beam.

Substrates 104 according to embodiments of the present invention thus may be patterned with alternating bands exhibiting different optical responses, at least one type of the alternating bands having a length in a direction perpendicular to the scan direction that is larger than the wavelength of the irradiation beam used, so as to allow wide band tracking or in other words localizing irradiation beams with a size substantially larger than the wavelength of the irradiation beam used. By alternating bands exhibiting different optical responses is meant that bands with certain optical properties are separated by bands with distinctively different optical characteristics. Thus adjacent bands on the substrate are such that the optical response of one band is distinctively different from the optical response of the adjacent bands. The first aspect of the present invention will be further illustrated by way of a number of exemplary embodiments, the present invention not being limited thereto.

In a first embodiment according to the first aspect, the substrate 104 comprises alternating first regions 204 and second regions 206 extending in a first direction suitable for being a scan direction S. The first regions 204 and second regions 206 furthermore have a size in a second direction perpendicular to the first direction, i.e. perpendicular to the suitable scan direction, that is substantially larger than the wavelength of the irradiation beam. The size in the second direction preferably is slightly smaller than the width in the direction perpendicular to the scan direction of the projection of the irradiation beam on the substrate. The first regions 204 and second regions 206 may have a band- like shape. The latter is illustrated by way of example in Fig. 1. Typically the irradiation beam will be scanned over the substrate 104 such that the irradiation beam extends over the full width of one region on the substrate 104, while edges of the irradiation beam projection irradiate one or both adjacent regions. Both regions having a first type of optical characteristics and second type of optical characteristics can be scanned in this way. By detecting intensity variations in the irradiation beam or part thereof after interaction with the substrate, the position of the irradiation beam with respect to the substrate, e.g. with respect to the first or second regions can be determined and possibly controlled. In the present embodiment, the first regions 204 and second regions 206 thus are alternating bands having the same width, the width of the bands typically being slightly smaller than the length of the irradiation beam projection in the direction perpendicular to the scan direction S. In this case, typically both first regions 204 and second regions 206 are to be scanned.

In a second embodiment according to the first aspect, the substrate 104 comprises alternating first regions 204 and second regions 206 as described in the first embodiment according to the first aspect, but one of the first regions 204 or second regions 206 have a size in a second direction perpendicular to the first direction that is substantially smaller than the corresponding size of the other type of regions. The first regions 204 and second regions 206 may be band- like shaped. The latter is illustrated by way of example in Fig. 2. Typically the irradiation beam will be scanned over the substrate 104 such that the irradiation beam extends over the full width of the broadest region, while edges of the irradiation beam projection irradiate both adjacent regions. In this embodiment, typically only one type of regions will be scanned over their full width during a single scan movement, while regions of the other type of regions are basically used for tracking the irradiation beam. In other words, the substrate may be patterned with a first type of regions having the same optical properties, separated by narrower bands with different optical properties.

In a third embodiment, the substrate 104 comprises alternating first regions 204 and second regions 206 as described in the second embodiment, whereby the irradiation beam will be scanned over the substrate 104 such that the irradiation beam is scanned over the full width of the smallest region, while side portions of the irradiation beam projection irradiate substantial parts of the adjacent regions. The latter is illustrated by way of example in Fig. 3. In other words, the substrate 104 may be patterned with a first type of regions 204, e.g. band-like shaped regions, having the same optical properties, each with an embedded, central second type of region 206 having different optical properties.

In a further embodiment according to the first aspect, the present invention relates to a substrate 104 comprising first regions and second regions as described in the above embodiments, whereby in at least one of the regions of the first type 204 and/or at least one of the regions of the second type 206 additional information is embedded. Such additional information may be or correspond with a position address, such that the irradiation beam can not only be localized with respect to first regions and or second regions but also with respect to its relative position with respect to the full substrate. Such additional information also may be a clock signal or other information allowing the system to uniquely identify different areas of the substrate surface while scanning it. Such additional information thus may be provided by way of features providing a specific modulation of the irradiation beam different from the modulation in the first regions or second regions. The information may be provided by introducing a specific intensity change in the irradiation beam or part thereof detected after interaction with the substrate or by introducing a specific intensity distribution change in the irradiation beam or part thereof detected after interaction with the substrate. Such additional information could be applied in any suitable way, such as example in barcode shape. The latter is illustrated in more detail hereafter.

In a fourth particular embodiment of the first aspect of the present invention, the first regions 204 and/or the second regions 206 may comprise optical modulating structures. Such structures may e.g. be patterned structures. The structures comprise or are encoded with position related information of the structures with respect to the substrate. Such additional information may be e.g. an absolute position of the features with respect to the substrate and/or a relative position of the features with respect to the substrate. These structures may be reflective and/or absortion and/or phase modulating structures and may form a reflective and or absorption and/or phase modulating structure layer. The structures may be such that separate reflective parts are formed as is illustrated in Fig. 4 and 5. However, according to other embodiments, the structures alternatively or in combination therewith may comprise features altering the phase of the incident light, e.g. using holes. The structures may comprise a periodic structure of features. Fig. 4 illustrates the principle of this fourth embodiment. In the example given, only the second regions 206 comprises patterned structures. As can be seen from the figure, different second regions 206 may comprise different patterns. It has to be understood that according to other embodiments also only the first regions 204 may comprise patterned structures or both the first regions 204 and second regions 206 may comprise patterned structures. It can be seen that the layer of binding sites 208 and the optical modulating structures can be provided in a stacked way, resulting in a small distance between both and thus in the possibility for simultaneous detection of particles and detection of position related information. Fig. 5 illustrates a specific embodiment of phase or reflective or absorption structures wherein both first regions 204 and second regions 206 are introduced for tracking a wide irradiation spot. The phase or absorption or reflective structures in the first regions 204, which in the present example precede and follow the area comprising binding sites 210, may comprise absolute information with respect to their position on the substrate, and optionally about the area comprising binding sites 210 they border. The phase structures in the second regions 206, which in the present example run above and below the area comprising binding sites 210 comprise relative position information, providing position information with respect to e.g. neighbouring structures or structures providing absolute position information. In addition to tracking, the different regions used for tracking and/or the modulation structures embedded therein can be used to stabilize the orientation of the optical spot (excitation line) with respect to the scanning direction/scanned track. For example, using detectors at both ends of the wide irradiation spot to detect reflection of the irradiation spot, the phase shift of the detected signals corresponding to each of the two structures adjacent the area 210 can be used as a direct indication of the orientation of the projection of the irradiation beam, e.g. the excitation line, with respect of the modulation structures. If for example the two structures adjacent the area comprising binding sites 210 and extending along the scanning direction are identical, the phase shift of the two detected signals is zero when the excitation stripe is perpendicular to the scanning direction. It is to be noticed that whereas the modulation structures are of the order of the wavelength of the irradiation beam, the bio-spot can have much larger dimensions, i.e. a number of orders larger. Fig. 6 presents a similar example as described in Fig. 5, but wherein a combination of reflective and phase structures or absorption and phase structures is used. In one example, the reflective layer provides a high irradiation beam reflection and the phase structure underneath ensures the desired modulation of the reflected signal. Although alternative arrangements may be used, it is advantageous to have a coarse patterning of the reflecting structure and the a fine patterning for the phase structures.

Fig. 7 presents an example that makes use only use of phase structures. The same structure of absolute and relative information as described for Fig. 5 can be present. The phase modulating structures may be designed so as to reflect only small amounts of radiation. They may be made of a fine structure, with typical dimensions of sub-wavelength resolution. The reflection contrast between these structure and the adjacent surface then may be used for focusing purposes.

Fig. 8 and Fig. 9 illustrate a further example of a substrate 104 according to an embodiment of the present aspect, wherein the first regions 204 and second regions 206 may be formed by patterning the surface of the substrate with a reflective structure. The reflective structure may comprise stripes, which may preferably be metallic. As can be seen from Fig. 8, the stripes, which form the second region 206, may most preferably be substantially parallel to each other and organized in meta-tracks, to allow the wide band tracking as described above. The distance between the stripes, or in other words, the width Df_irstregion of the first region 204, may be slightly smaller than the at least one length of the irradiation beam projection 202 being substantially larger than the wavelength λ of the irradiation beam. According to the present example, the stripes which form the second region 206 may be sub- patterned with, for example, a periodic structure of holes 220 as is illustrated in Fig. 8 and Fig. 9. This periodic structure of holes 220 may serve for periodically modulating the irradiation beam after interaction with the substrate. The irradiation beam after interaction with the substrate may for example be a reflected irradiation beam reflected by the substrate or part thereof or a transmitted irradiation beam transmitted at the substrate or part thereof. This irradiation beam may be used as a relative position mark. In the first region 204, absolute position marks 222 may be located. The absolute position marks 222 may be placed periodically and may be located in the middle of the first region, i.e. at equal distances di and d₂ from the two neighboring second regions 206, as can be seen in Fig. 8 and 9. Making use of the length base generated by the relative position marks (reflection from the side of the line) the content of the absolute position marks may be correctly read. According to the present embodiment, the patterned structures embedded in the substrate may be of metallic nature. However, structures of any other nature can serve equally well for the purpose of the present invention, such as for example phase structures, phase change materials, dielectric layers, absorption structures or a combination thereof. Fig. 10 shows a detail of an extremity of an excitation radiation beam 202 scanning over a patterned reflecting structure as provided in the second region 206, as described above (see Fig. 8 and 9). The the excitation radiation beam 202 after interaction with the substrate or part thereof is imaged on a split detector 224. For easy understanding of the tracking principle, in Fig. 10, the detector 224 is projected into the substrate plane. A signal (B-A)/(A+B) may be used as servo control for the tracking actuator, with A and B electrical signals as detected by the two parts of the split detector 224. The control value, i.e. average value to be received when the projection of the irradiation beam is on track may advantageously be set to 0.33. This corresponds with a 2:1 ratio of radiation intensity on the two parts of the detector 224, i.e. (B-A)/(A+B) = (2-l)/(l+2) = 0.33. This would make the irradiation beam 202 to cover about 75% of the total width of the reflecting structure, as can be seen when distance q is compared to the full width w of the reflecting structure, e.g. for the beam position pi. In a region with less reflective area, as indicated by the reflective area present at beam position p2, the ratio of the reflective regions generating an impinging beam on the different parts of the tracking or localization detector advantageously is selected having the same ratio as for the reflecting region corresponding with beam position 1. In this way the normalized tracking signal is about constant while the excitation lines scan across more reflective and less reflective regions, i.e. corresponding with beam positions pi and p2. According to embodiments of the invention, the structure may be designed such that the total intensity A+B never goes to zero. As the radiation beam 202 scans across a region of less reflective area, i.e. corresponding with beam position p2, the total intensity A+B measured by the detector is modulated with a depth of about 50% compared to a region of more reflective area, i.e. corresponding with beam position pi . When normalization is performed, this does not affect the servo-tracking signal. This modulation may nevertheless be used as relative position sensor by using the absolute and not the normalized values. When a repetitive pattern is used, a clock can be generated from it. The number of pulses then indicates a relative motion equal to the number of structures scanned. Encoding of an absolute position mark 222 as used in the examples of Fig. 8 and Fig. 9 is illustrated by means of Fig. 11. In this example position encoding may be done on nine bits 226, which are embedded between five control bits 228, i.e. from one side 3 bits (110) and from one side 2 bits (01). The control bits 228 ensure that the signal is correctly read irrespective of the scanning direction. The optical modulation structures may comprise or be encoded with the position related information by a code embedded in the pattern of the optical modulation structure, which may be binary or which may be based on digits having more than two possible values, e.g. based on differences in optical modulation. E.g. the coding may be normal binary code, namely a one bit (1) value represented by a reflective region and a non-reflective region representing a zero bit (0) . The bit values may also be represented by the inverse reflectivity properties, or, in case of more than two values, by more than two different reflection coefficients of a region. The modulation structures may more generally also comprise an information part where a plurality of regions corresponding with a plurality of bits or digits are provided and a reference part indicating the begin and/or the end of the information part. The reference part may be the same for each set of modulation structures. The simple binary or digit code can advantageously be used when relative position marks are present allowing to generate a clock. Alternatively an external clock, linked to the scanning or more particularly scanning speed, may be used. The clock may assist in accurate decoding irrespective to the scanning speed, and independent of speed variations. The information enclosed in the modulation structure may be absolute position information. Different modulation structures may be encoded to represent different types of information, whereby at least one bit or digit may be indicative of the type of information that is provided. E.g. in a binary encoded modulation structure providing absolute position information, the first bit of the information part may encode wherether horizontal or vertical information is provided and the remaining bits may encode the position in case of horizontal info or the meta-track number in case of vertical info. As shown in Fig. 12, horizontal position information or vertical position information may be placed alternatively along the meta-track.

In a second aspect, the present invention relates to a method for tracking an area to be scanned, e.g. wide band, with an irradiation beam, e.g. used in a detection system for detecting light emission sites on a substrate 104, whereby the irradiation beam projection 202 on the substrate 104 has at least one size being substantially larger than the wavelength λ of the irradiation beam used. The method comprises scanning a substrate 104 by guiding an irradiation beam having a projection on the substrate 104 with at least one size substantially larger than the wavelength λ of the irradiation beam used. The method furthermore comprises detecting a variation in an intensity of an optical characteristic of at least part of the irradiation beam after interaction with the substrate 104 for determining a position of the irradiation beam with respect to the substrate. The latter allows to correctly position the irradiation beam with respect to the substrate, such that detection of light emission sites may be performed with a high accuracy. The method for tracking may be applied in a number of applications such as for example a biological, chemical or bio-chemical detection, optical storage or other optical applications, etc.

The method 700 for tracking an area, e.g. wide band, to be scanned with an irradiation beam will be discussed in more detail with reference to Fig. 12, indicating different steps, some of them being optional.

In a first step 702, scanning of a substrate 104 with an irradiation beam having an irradiation beam projection on the substrate with at least one size being substantially larger than the wavelength of the irradiation beam used. The irradiation beam may have a spherical shape, elliptical shape, etc. Preferably the irradiation beam has an at least piecewise elongated irradiation beam allowing to irradiate a substantially large part of the substrate 104 simultaneously in a similar way during a single scan movement.

During said scanning, the method for tracking 700 an area to be scanned with an irradiation beam according to the present embodiment controls the position of the irradiation beam with respect to the substrate. The latter may be performed in different steps.

In step 704 there is detection of a variation of an optical characteristic of at least part of the irradiation beam after it has interacted with the substrate 104. Such a variation of an optical characteristic may be a variation in intensity of radiation reflected by the substrate, a variation in intensity of light transmitted by the substrate, a variation in intensity or a variation in density of scattered light, etc. Depending on the specific property that is to be measured, detection may be done with a detecting means 108 being a single spot detector or a pixelated detector or a split detector, etc. E.g. if at least one portion of the irradiation beam is detected, whereby that portion of the irradiation beam comprises a first sub-portion modulated by a first region 204 on the substrate 104 and a second sub-portion modulated by a second region 206 on the substrate 104 having different light modulating properties, a shift of the irradiation beam typically will result in a change of the area of the different sub-portions and consequently in a change of the modulation of the irradiation beam. The latter thus typically results in a change in intensity of an optical characteristic of the at least part of the irradiation beam, resulting in a different detection. In optional step 706 the variation of an optical characteristic of at least part of the irradiation beam may be processed to derive position related information regarding the position of the irradiation beam with respect to the different type of regions on the substrate. This position related information then may be outputted or used in a further step. In optional step 708, the position related information may be used for correcting the position of the irradiation beam with respect to the different type of regions on the substrate. In this way, the irradiation beam can be kept in the specific area to be scanned, resulting in a higher accuracy as scanning of unwanted areas or not-scanning of wanted areas is avoided.

Steps 704 to 708 typically may be repeatedly performed in a type of feed-back loop, whereby positional information of the irradiation beam with respect to different regions on the substrate is obtained and whereby, based on the positional information, the position of the irradiation beam with respect to different regions on the substrate may be corrected. All of the steps may be performed in an automated or automatic way. The method for tracking thus may be an automated or automatic method for tracking.

In a further embodiment, the method for tracking also may comprise the step of detecting additional information embedded in the substrate 104. This step may be part of step 704, whereby a specific variation of the intensity of an optical characteristic of the irradiation beam after interaction with the substrate is detected. This specific variation may be substantially different from a variation of an optical characteristic that may be generated by a shift of the irradiation beam. Such a variation may differ in intensity, e.g. by providing strongly absorbing features as additional features while providing strongly reflecting regions with different reflectivity coefficient for tracking the areas to be scanned with the irradiation beam. Alternatively or in combination thereto, additional info may be provided by providing features causing variations of the optical characteristics of the irradiation beam having different temporal behavior, e.g. for a standard scan rate. For a given scan rate, additional variations may be present in a different time scale than variations caused by shift or drift of the irradiation beam. For example, additional variations may be detected only in a short time period, whereas shift or drift of the irradiation beam typically extends over a longer period. When such additional variations are detected, these may be processed and translated in additional information, e.g. to provide information such as e.g. a position with respect to the full substrate surface 104 or a clock signal or any type of signal allowing to uniquely identify different areas on the substrate 104. In another embodiment according to the third aspect, the present invention relates to a method for tracking a position of an irradiation beam on a substrate as described above, but wherein the method comprises, prior to scanning the irradiation beam on the substrate, adapting an orientation of the irradiation beam with respect to the scanning direction, in order to set the width of the irradiation beam in a direction perpendicular to the scan direction in agreement with the width of the first and second regions on the substrate. It is an advantage of such embodiments that these methods may solve for initial incompatibility between the detection systems used in the method and the patterning on the substrates used. In other words, by rotating the irradiation beam a more appropriate tracking method may be obtained as different parts of the irradiation beam may be detected during the tracking method.

In a third aspect, the present invention relates to a tracking system for tracking an irradiation beam on a substrate, especially an irradiation beam having at least one dimension substantially larger than the wavelength of the irradiation beam. By way of example, a schematic representation of the tracking system is shown in Fig. 13. More particularly, the tracking system thus typically is adapted for tracking a location of the irradiation beam having an irradiation beam projection 202 (as indicated in Fig. 1, Fig. 2 and Fig. 3) with a dimension substantially larger than the wavelength λ of the irradiation beam. The irradiation beam typically may be a UV, visible or infrared irradiation beam although the invention is not limited thereto and other types of electromagnetic irradiation also could be used. The wavelength referred to may be the average wavelength of the irradiation beam or the wavelength at which the maximum emission is obtained. Substantially larger than the wavelength λ of the irradiation beam may be larger than the wavelength λ of the irradiation beam, at least 2 times the wavelength λ of the irradiation beam, at least 10 times the wavelength of the irradiation beam, e.g. at least 100 times the wavelength of the irradiation beam . The irradiation beam may have any suitable cross-sectional shape or irradiation beam projection shape, such as a circular shape, an elliptical shape, etc. The irradiation beam having an irradiation beam projection 202 on the substrate 104 with at least one dimension substantially larger than the wavelength of the irradiation beam may for example also be an at least piecewise elongated irradiation beam, i.e. an irradiation beam having an at least piecewise elongated irradiation beam projection 202 on the substrate 104. The dimension of the irradiation beam projection 202 on the substrate 104 substantially larger than the wavelength of the irradiation beam may be the dimension in the direction substantially perpendicular to the scan direction S (as shown in Fig. 1, Fig. 2 and Fig. 3) of the irradiation beam.

For tracking, the tracking system 106 comprises a detecting means 108 adapted for detecting a variation in an optical characteristic of at least part of an irradiation beam after interaction with the substrate 104. The irradiation beam to be tracked may be generated by an irradiation source that is part of the tracking system or may be an irradiation beam or part thereof induced by an irradiation source that is not part of the tracking system 106 but that is part of an optical system in which the tracking system 106 is used. The variation of the optical characteristic of at least part of the irradiation beam thereby is induced by interaction with different regions 204, 206 (shown in Fig. 1, Fig. 2, Fig. 3) of the substrate 104, the different regions 204, 206 having different irradiation modulating properties. In other words, by detecting an optical characteristic of at least part of the irradiation beam after interaction with the substrate 104 and thus modulated by the interaction with the substrate 104, a localization of the irradiation beam with respect to the substrate 104 may be performed. A substrate 104 that typically may be used with the present tracking system has been described in more detail in the first aspect of the present invention.

The tracking system 106 may be operated in a transmission set-up or in a reflection set-up. In a transmission set-up, typically the optical characteristics of at least part of the irradiation beam may for example be the amount of transmitted light or the amount of absorbed light. In a reflection set-up, the optical characteristic of at least part of the irradiation beam may for example be the amount of reflected light, the amount of scattered light or the density of scattered light of at least part of the irradiation beam. The detecting means 108 may be adapted for simultaneously detecting part of the irradiation beam modulated by a first region 204 on a substrate 104 scanned and part of the irradiation beam modulated by a second adjacent region 206 of the substrate 104 scanned, whereby the first region 204 and second region 206 have different irradiation modulating properties. In other words, the detecting means 108 may be adapted such that at least part of the irradiation beam is detected that comprises two sub-components, each sub-component differently modulated during interaction with the substrate 104 to be scanned. Variation of an optical characteristic of at least part of the irradiation beam thereby may be induced by a variation of the area modulating the first sub-component and a variation of the area modulating the second subcomponent, e.g. when the beam shifts over the substrate 104. In this way a small variation in position of the irradiation beam with respect to the substrate 104, i.e. for example s small beam shift, may result in a detectable variation of the optical characteristic, thus resulting in a high accuracy with witch the irradiation beam can be tracked with respect to the relevant regions on the substrate 104. The differently modulated sub-components to be detected may stem from any place in the irradiation beam projection, such as e.g. the center of the irradiation beam, one side of the irradiation beam or two sides of the irradiation beam, thus allowing different patterns to be applied to the substrate to be scanned, all allowing to track the regions to be scanned. The detecting means 108 may be any suitable detecting means allowing to detect variations in optical properties of the irradiation beam induced by interaction of the irradiation beam with the substrate 104, such as e.g. a split detector, a single spot detector or a pixelated detector. Typically variations of optical properties of the irradiation beam are detected by a deviation of the detected optical characteristic from a predetermined value. By way of example, Fig. 13 illustrates part of a split detector with an incident irradiation beam after interaction with the substrate 104. Typically, with use of a split detector, differences between signals incident on a first active region 152 and a second active region 154 of the detecting means 108 are detected and used as output signal of the detecting means 108. Based on the signal detected by the detecting means 108, a location of the irradiation beam with respect to the substrate 104, or more particularly with respect to different regions 204, 206 on the substrate 104 may be derived. The tracking system 106 furthermore may comprise a position correcting means 110 adapted for correcting the position the irradiation beam. Such a position correcting means 110 may comprise means for altering the position of the irradiation means itself or it may provide correction signals, to provide a correction to the position of the irradiation means, to the positioning and scanning means of an optical system The correction signals then typically allow the positioning and scanning means to redirect the irradiation beam to the appropriate region of the substrate 104, i.e. the region to be scanned. In other words, it is an advantage of embodiments according to the first aspect of the present invention that imaging at least part of the irradiation beam that has interacted with the patterned substrate may allow obtaining useful signals for correcting the objective position of the irradiation beam with respect to the region of interest on the substrate.

In a particular embodiment, the tracking system 106 may also comprise an automated feed-back loop 114 between the detecting means 108 and the position correcting means 110 such that automated and/or automatic correction of the position of the irradiation beam with respect to the regions to be scanned on the substrate 104 is performed. This results in a detection system 100 that allows automated and/or automatic correction of shift and drift of the irradiation beam. In a further embodiment of the third aspect, the present invention relates to a tracking system 106 furthermore is adapted to detect additional variations in the optical characteristic of the irradiation beam, which provide additional information. The additional variations in the optical properties of the irradiation beam may be variations differing substantially from the variations caused by shift or drift of the irradiation beam. Such variations may e.g. be in a substantially different parameter range of the optical characteristic. E.g. if the optical characteristic of the irradiation beam used is an amount of irradiation reflected by the substrate, variations between different regions on the substrate may e.g. be caused by different reflectivity coefficients of the substrate, whereas the additional variations may be obtained by introducing features in these regions that are substantially not reflecting the irradiation beam. Alternatively or in combination thereto, the additional variations in the optical properties of the irradiation beam may vary from variations caused by shift or drift of the irradiation beam by their temporal behavior. For a given scan rate of the irradiation beam, additional variations may be present in a different time scale than variations caused by shift or drift of the irradiation beam. For example, additional variations may be detected only in a short time period, whereas shift or drift of the irradiation beam typically extends over a longer period. If the detecting means only detects additional variations over a short time period, they may be processed by information processing means 130 to derive additional information, whereas if the detecting means detects variations over a longer time period, these may be processed by the position correction means to correct a position of the irradiation beam. In this way a clear distinction can be made between additional variations relating to additional information provided on the substrate and position related variations relating to a shift of the irradiation beam. Any type of additional information may be provided in this way. E.g. additional information about the relative position of location with respect to the full substrate may be provided. The additional information may be for example a position address or a clock signal or any type of information that allows the system to uniquely identify different areas of the substrate while scanning it. The information processing means 130 may use processing power of a processor. The processor also may be used for other tasks in the system. In another embodiment according to the third aspect, the present invention relates to a tracking system 106 comprising the same characteristics and features as any of the tracking system as described above, but whereby furthermore a rotating means is provided for rotating the irradiation beam with respect to the substrate, such that the length of the irradiation beam in a direction perpendicular to the scan direction is such that appropriate different regions of the substrate are illuminated. By rotating the irradiation beam around an axis perpendicular to the substrate, the beam length may be adapted such that modulation of at least part of the irradiation beam area is such that different parts of the irradiation beam modulated differently by different regions on the substrate can be detected by the detecting means 108. Such a rotating means may be any suitable rotating means. For example for an elongated irradiation beam generated using a cylindrical lens, the rotating means may be a means for rotating the cylindrical lens.

The tracking system according to embodiments of the present aspect of the invention may be applied to any suitable optical system wherein tracking of an irradiation beam may be performed, such as e.g. in optical storage systems such as e.g. systems for reading and writing optical data to an optical carrier.

In a fourth aspect, the present invention relates to a detection system for detecting light emission sites on a substrate. In the present aspect a tracking system as described in the third aspect is implemented together with a detection system in order to perform tracking of an irradiation beam in a detection system. The detection system of embodiments according to the fourth aspect of the present invention are adapted for detecting light emission sites by scanning substrates with an irradiation beam having an irradiation beam projection on the substrate with at least one dimension substantially larger than the wavelength of the irradiation beam. The latter may result in a high throughput of samples to be scanned. Typically light emission sites may be detected by detecting a luminescence response from the substrate scanned. Such luminescence response may e.g. be a fluorescence response, although the invention is not limited thereto. An example schematic representation of a detection system according to embodiments of the fourth aspect is shown in Fig. 16 and Fig. 17, the invention not being limited thereto. The detection system 100 of embodiments according to the present invention typically comprises an irradiation source 102 generating an irradiation beam having an irradiation beam projection 202 (shown in Fig. 1, Fig. 2 and Fig. 3) on the substrate 104 with at least one dimension substantially larger than the wavelength λ of the irradiation beam. The wavelength referred to may be the average wavelength of the irradiation beam or the wavelength at which the maximum emission is obtained. Substantially larger than the wavelength λ of the irradiation beam may be larger than the wavelength λ of the irradiation beam, at least 2 times the wavelength λ of the irradiation beam, at least 10 times the wavelength of the irradiation beam, e.g. at least 100 times the wavelength of the irradiation beam . The irradiation beam may have any suitable cross- sectional shape or irradiation beam projection shape, such as a circular shape, an elliptical shape, etc. The irradiation beam having an irradiation beam projection 202 on the substrate 104 with at least one dimension substantially larger than the wavelength of the irradiation beam may for example also be an at least piecewise elongated irradiation beam, i.e. an irradiation beam having an at least piecewise elongated irradiation beam projection 202 on the substrate 104. The dimension of the irradiation beam projection 202 on the substrate 104 substantially larger than the wavelength of the irradiation beam may be the dimension in the direction substantially perpendicular to the scan direction S (as shown in Fig. 1, Fig. 2 and Fig. 3) of the irradiation beam. The irradiation source 102 typically may be adapted for emitting light at a predetermined wavelength or a predetermined wavelength range, suitable for exciting or irradiating the light emission sites, e.g. optically variable particles, present in the sample. For example, in the case where the generated luminescence response to be detected for detecting particular components in the sample is fluorescence radiation, the optical wavelength of the irradiation may typically span a region from UV to IR e.g. in the range from 200 nm to 2000 nm, or e.g. in the range from 400 nm to 1100 nm, the invention not being limited thereto. The irradiation source 102 used may e.g. be a laser device, LED or a, typically filtered, broad-spectrum irradiation source. The irradiation power present in the irradiation beam preferably may be such that each spatially distinctive irradiation area of the irradiation beam projection 202 on the substrate 104 may comprise an irradiation power such that about 10% to 90%, preferably about 30% to 80% of the saturation level of the luminescence is achieved. It is to be noted that although the irradiation source referred to in the above description is both used for exciting/irradiating emission sites on the sample and for tracking the areas of the substrate to be scanned by the irradiation beam, a separate irradiation source could also be used for tracking the areas of the substrate to be scanned by the irradiation beam. The latter would result in two irradiation beams used and would require a known positional correlation between the irradiation beam used for tracking and the irradiation beam for exciting/irradiating the emission sites on the sample to be detected.

In a particular embodiment wherein the irradiation beam is an at least piecewise elongated shape of the irradiation beam component as projected on the substrate 104, the at least piecewise elongated irradiation beam may be generated in any suitable way. It may be generated using an optional means for generating a single irradiation beam comprising a row of irradiation spots or an optional means for generating a line-shaped irradiation spot. Such optional means may e.g. be a means for generating an at least piecewise elongated irradiation beam comprising an irradiation beam projection on the substrate with a row of discrete irradiation spots or a row of distinctive, possibly overlapping, irradiation spots. Such an irradiation beam component may e.g. be obtained by the use of a phase plate or a diffraction grating in the path of the irradiation beam. The effect of such an element is that an initial irradiation beam is degenerated in a row of multiple irradiation spots. Such means also may e.g. be a means for generating an elongated irradiation beam, e.g. forming a continuous elongated irradiation area on the substrate. In other words, a means for generating a line-shaped irradiation beam as first elongated irradiation beam component may be provided. Such means for generating a line-shaped irradiation beam may be a cylindrical lens or a phase plate used in the light path of the laser and generating a line shaped irradiation area in the focal plane of the lens. An advantage of this way of generating an at least piecewise elongated irradiation beam is that these can be used with a single refractive element for focusing the irradiation beam on the substrate, as the row of multi- spots or the line-shaped spot may fit in the single refractive element. The latter allows the use of a conventional optical component for the refractive element, as e.g. used in optical datastorage systems. Alternatively, the at least piecewise elongated irradiation beam may be generated in any other suitable way. For example, a plurality of irradiation sources 104 may generate a plurality of irradiation beams e.g. through a plurality of optical elements on a substrate. Alternatively a line-shaped irradiation source may generate a line-shaped irradiation beam focused on a substrate. It will be obvious to the person skilled in the art that a plurality of ways are available to generate an at least piecewise elongated irradiation beam, using any or a combination of particular irradiation source(s) and particular optical components. It is an advantage of embodiments of the present invention that a high throughput for detecting in samples and a shorter overall detection time or improved signal to noise ratio can be obtained. The latter results in efficient systems.

The detection systems 100 according to embodiments of the fourth aspect of the present invention furthermore comprise a tracking system 106 for tracking a location of the irradiation beam having a an irradiation beam projection 202 with a dimension substantially larger than the wavelength λ of the irradiation beam. The tracking system 106 therefore comprises a detecting means 108 adapted for detecting a variation in an optical characteristic of at least part of the irradiation beam after interaction with the substrate 104. The variation of the optical characteristic of at least part of the irradiation beam thereby is induced by interaction with different regions 204, 206 (shown in Fig. 1, Fig. 2, Fig. 3) of the substrate 104, the different regions 204, 206 having different irradiation modulating properties. In other words, by detecting an optical characteristic of at least part of the irradiation beam after interaction with the substrate 104 and thus modulated by the interaction with the substrate 104, a localization of the irradiation beam with respect to the substrate 104 may be performed. The substrate 104 may be part of the detection system 100 whereby the substrate 104 may be cleaned such that sample can be removed after it has been scanned, or it cannot be part of the detection system 100 and be e.g. disposable. The substrate 104 comprising different regions 204, 206 having different optical properties for modulating an incident irradiation beam have been described in more detail in the first aspect of the present invention. The features as described in the first aspect of the present invention thus may apply to the first aspect of the present invention. In each case, the detection system 100 according to the fourth aspect of the present invention is adapted to be used with substrates 104 comprising different regions 204, 206 having different optical properties for modulating an incident irradiation beam, as described in the first aspect.

The detection system 100 may be operated in a transmission set-up or in a reflection set-up. In a transmission set-up, typically the optical characteristics of at least part of the irradiation beam may for example be the amount of transmitted light or the amount of absorbed light. In a reflection set-up, the optical characteristic of at least part of the irradiation beam may for example be the amount of reflected light, the amount of scattered light or the density of scattered light of at least part of the irradiation beam. The detecting means 108 may be adapted for simultaneously detecting part of the irradiation beam modulated by a first region 204 on a substrate 104 scanned and part of the irradiation beam modulated by a second adjacent region 206 of the substrate 104 scanned, whereby the first region 204 and second region 206 have different irradiation modulating properties. In other words, the detecting means 108 may be adapted such that at least part of the irradiation beam is detected that comprises two sub-components, each sub-component differently modulated during interaction with the substrate 104 to be scanned. Variation of an optical characteristic of at least part of the irradiation beam thereby may be induced by a variation of the area modulating the first sub-component and a variation of the area modulating the second subcomponent, e.g. when the beam shifts over the substrate 104. In this way a small variation in position of the irradiation beam with respect to the substrate 104, i.e. for example s small beam shift, may result in a detectable variation of the optical characteristic, thus resulting in a high accuracy with witch the irradiation beam can be tracked with respect to the relevant regions on the substrate 104. The differently modulated sub-components to be detected may stem from any place in the irradiation beam projection, such as e.g. the center of the irradiation beam, one side of the irradiation beam or two sides of the irradiation beam, thus allowing different patterns to be applied to the substrate to be scanned, all allowing to track the regions to be scanned.

The detecting means 108 may be any suitable detecting means allowing to detect variations in optical properties of the irradiation beam induced by interaction of the irradiation beam with the substrate 104, such as e.g. a split detector, a single spot detector or a pixelated detector. Typically variations of optical properties of the irradiation beam are detected by a deviation of the detected optical characteristic from a predetermined value. By way of example, Fig. 16 illustrates part of a split detector with an incident irradiation beam after interaction with the substrate 104. Typically, with use of a split detector, differences between signals incident on a first active region 152 and a second active region 154 of the detecting means 108 are detected and used as output signal of the detecting means 108.

Based on the signal detected by the detecting means 108, a location of the irradiation beam with respect to the substrate 104, or more particularly with respect to different regions 204, 206 on the substrate 104 may be derived. The tracking system 106 furthermore may comprise a position correcting means 110 adapted for correcting the position the irradiation beam. Such a position correcting means 110 may comprise means for altering the position of the irradiation means itself or it may provide correction signals, to provide a correction to the position of the irradiation means, to the positioning and scanning means 112 of the detection system 110 that typically is used for positioning and scanning the irradiation beam with respect to the substrate 104. The correction signals then typically allow the positioning and scanning means 112 of the detection system 110 to redirect the irradiation beam to the appropriate region of the substrate 104, i.e. the region to be scanned. In other words, it is an advantage of embodiments according to the first aspect of the present invention that imaging at least part of the irradiation beam that has interacted with the patterned substrate may allow obtaining useful signals for correcting the objective position of the irradiation beam with respect to the region of interest on the substrate.

As described above, the detection system 100 thus also may comprise a positioning and scanning means 112 for scanning the substrate 104 with the irradiation beam. The scanning direction S of the irradiation beam in principle may be according to any or a combination of any direction in the 2-dimensional plane of the sample surface. Scanning preferably occurs along Cartesian coordinates, e.g. along line segments, e.g. finite line segments, or a combination of line segments, e.g. finite line segments, in a Cartesian coordinate system. Such scanning typically includes for example x-y scanning, raster scanning, x-y scanning along line segments, e.g. finite line segments, with stochastically chosen directions. Although not excluded from the present invention, it thus is less preferably to perform scanning in a relative rotational movement, as the latter typically results in the need for scanning very large areas.

In a particular embodiment, the tracking system 106 may also comprise an automated feed-back loop 114 between the detecting means 108 and the position correcting means 110 such that automated and/or automatic correction of the position of the irradiation beam with respect to the regions to be scanned on the substrate 104 is performed. This results in a detection system 100 that allows automated and/or automatic correction of shift and drift of the irradiation beam.

Typically, detection systems 100 according to embodiments of the first aspect of the present invention also may comprise a detection unit 116 for detecting a luminescence response from a sample 104 to be studied, i.e. a luminescence response from the light emission sites to be detected. The detector unit 116, as shown in Fig. 2, may be adapted for spatially distinctively detecting one or more luminescence responses from different emission sites on different locations in an instantaneous stationary irradiation beam projection on the substrate 104. In other words, the emission sites may be detected spatially distinctive. The light emission sites may relate to all kinds of sites emitting light, such as e.g. luminescent labeled target particles, but also other light emitting sources such as e.g. electronic or microelectronic light sources or chemical or structural features of a device, sample or surface leading to generation of light emission, e.g. when illuminated. Preferably the spatially distinctive excitation areas are such that nearly always maximally one light emission site, e.g. occupied binding site, is present within each of the spatially distinctive excitation areas. The detector unit 116 typically may be a pixelated detector or a line of multiple single-pixel detectors. Such a detector unit 116 may e.g. be a charge coupled device (CCD) detector, a row of photon tube multipliers, a row of avalanche photodiodes, etc. Typically the detector may be a row-detector (n x 1 pixels) or a 2-dimensional detector (n x m pixels). The width of the detector typically is such that the number of pixels (n) is at least sufficiently large to spatially distinctively detect different areas of the at least piecewise elongated excitation field component. These different areas thereby are such that approximately always maximally one occupied binding site is present within the area detected by a single pixel during examination. A typical area detected by a single pixel may be sized between 0.0 lμm² and lOOμrn², preferably between O.lμm² and 25μm², such as e.g. lμm². The detection system may be equipped with a dichroic filter or a dichroic beam splitter 118 for blocking unwanted irradiation to be incident on the detector unit, as with the detector unit 116 only a luminescence response of the light emission sites is to be detected. The dichroic filter or beam splitter 118 suppresses the reflected excitation radiation that is directed to the detector unit 116 and does not substantially suppress the luminescence response coming from the sample and directed to the detector unit 116.

Detection systems 100 according to embodiments of the present invention typically also may comprise optical components 120 such as e.g. refractive elements, filters and/or beam splitters for guiding the irradiation to and from the substrate 104 and from and to the appropriate component. In a detection system 100 a common refractive element 122 may be used for guiding the irradiation beam on the substrate 104 and for receiving the luminescence response from the sample on the substrate 104. Such component also may be used for collecting a reflected or scattered irradiation beam from the sample and guiding it to the detecting means 106, i.e. in a tracking operation. Other typical components that may be present are a focus controlling means 124, e.g. a focusing servo system, for controlling the focusing of the irradiation beam used for exciting/irradiating the light emission sites. The focus controlling means 124 may be based on different focusing methods, such as for example, but not limited to Foucault wedge focusing. Furthermore a high frequency controlling means 125 and an auxiliary detector 126 such as e.g. a charge coupled device (CCD), may be used for optimizing the focusing functions.

The detection system 100 furthermore may comprise an evaluation unit 128 allowing determination of the presence of, a concentration of or the distribution of light emission sites, such as - but not limited to - e.g. the target particles present in the sample. The evaluation unit 128 also may be adapted for performing processing, e.g. statistical processing, of the obtained detection results. Typically such an evaluation unit 128 may comprise a processing means, such as e.g. a microprocessor, for processing the evaluation information and a memory component for storing the obtained and/or processed evaluation information. Furthermore typical input/output means may be present. The evaluation unit may be controlled using appropriate software or dedicated hardware processing means for executing the evaluation steps.

In a further embodiment of the fourth aspect, the present invention relates to a detection system 100 as described above, whereby the tracking system 106 furthermore is adapted to detect additional variations in the optical characteristic of the irradiation beam, which provide additional information. The additional variations in the optical properties of the irradiation beam may be variations differing substantially from the variations caused by shift or drift of the irradiation beam. Such variations may e.g. be in a substantially different parameter range of the optical characteristic. E.g. if the optical characteristic of the irradiation beam used is an amount of irradiation reflected by the substrate, variations between different regions on the substrate may e.g. be caused by different reflectivity coefficients of the substrate, whereas the additional variations may be obtained by introducing features in these regions that are substantially not reflecting the irradiation beam. Alternatively or in combination thereto, the additional variations in the optical properties of the irradiation beam may vary from variations caused by shift or drift of the irradiation beam by their temporal behavior. For a given scan rate of the irradiation beam, additional variations may be present in a different time scale than variations caused by shift or drift of the irradiation beam. For example, additional variations may be detected only in a short time period, whereas shift or drift of the irradiation beam typically extends over a longer period. If the detecting means only detects additional variations over a short time period, they may be processed by information processing means 130 to derive additional information, whereas if the detecting means detects variations over a longer time period, these may be processed by the position correction means to correct a position of the irradiation beam. In this way a clear distinction can be made between additional variations relating to additional information provided on the substrate and position related variations relating to a shift of the irradiation beam. Any type of additional information may be provided in this way. E.g. additional information about the relative or absolute position of location with respect to the full substrate may be provided. The additional information may be for example a position address or a clock signal or any type of information that allows the system to uniquely identify different areas of the substrate while scanning it. The information processing means 130 may use processing power of a processor used for other tasks in the system. In a particular embodiment, the system may comprise a position determination means as described in the sixth aspect of the present invention. In another embodiment according to the fourth aspect, the present invention relates to a detection system 100 comprising the same characteristics and features as any of the detection systems as described above, but whereby furthermore a rotating means is provided for rotating the irradiation beam with respect to the substrate, such that the length of the irradiation beam in a direction perpendicular to the scan direction is such that appropriate different regions of the substrate are illuminated. By rotating the irradiation beam around an axis perpendicular to the substrate, the beam length may be adapted such that modulation of at least part of the irradiation beam area is such that different parts of the irradiation beam modulated differently by different regions on the substrate can be detected by the detecting means 108. Such a rotating means may be any suitable rotating means. For example for an elongated irradiation beam generated using a cylindrical lens, the rotating means may be a means for rotating the cylindrical lens.

In a fifth aspect, the method of tracking an irradiation beam as described in the second aspect may be incorporated in a method for detecting light emission sites on a substrate. In the fifth aspect, the invention thus relates to a method for detecting light emission sites, whereby a substrate is scanned with an irradiation beam having an irradiation beam projection on the substrate with at least one dimension larger than the wavelength of the irradiation beam used. Besides the method steps for tracking the irradiation beam as described in the second aspect, the method for detecting light emission sites according to the firth aspect furthermore typically may comprise detecting a luminescence response from the sample, e.g. a fluorescence response and determining a qualitative or quantitative parameter of the sample scanned. The latter may e.g. be the amount of sample or the amount of particular components of the sample present.

In a sixth aspect, the present invention relates to a position determination system for determining position related information of an irradiation beam on a substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles. The substrate may be for use with a bio sensing system. The position determination system is adapted for using an irradiation beam used for irradiating the substrate for detecting presence of chemical, bio-chemical and/or biological particles. The position determination system furthermore comprises a detection sub-system for detecting a modulation of the reflected irradiation beam, reflected at the substrate, and for deriving from the modulation position related information embedded in the substrate indicative of a position of the irradiation beam on the substrate. The position related information may be absolute position related information with respect to the substrate or relative position related information with respect to the substrate. The position determination system may comprise a processor for deriving the position related information. The processor may be adapted, e.g. programmed, for decoding a modulation according to a predetermined algorithm, a neural network, look up tables, etc. The processor may be adapted for deriving absolute position information by e.g. detecting a spatial or temporal modulation of the reflected irradiation beam. E.g. when imaging is performed, a spatial modulation may be detected, whereas when scanning is performed a temporal modulation may be detected. A predetermined modulated part may be recognized as start and/or end of an encoded position information part. Deriving position related information may comprise deriving a plurality of bits or digits from a spatial and/or temporal modulated signal, which may be representative of a position. Deriving values for bits or digits may be performed by quantitative or qualitative assessment of the modulation of the irradiation beam, e.g. be determining optical values of the modulated irradiation beam within certain ranges and converting it to a corresponding value. When scanning is applied, the detection sub-system may be adapted for generating a clock from a periodic modulation of the irradiation beam after interaction with the substrate. The clock may be used for deriving a relative position of an irradiation beam or a speed of an irradiation beam with respect to a previously determined position or speed. The position determination system may be adapted for obtaining an irradiation beam after interaction with the substrate by filtering it from a luminescence response from chemical, bio-chemical and/or biological particles which may be detected simultaneously such that position information can be easily combined with chemical, bio-chemical and/or biological detection signals. The system therefor may comprise a filter. The position determination system may be combined with the tracking system as described in the third aspect, and detection of the irradiation beam after interaction with the substrate may e.g. be performed using the same detection sub-system. In a seventh embodiment, the present invention relates to a detection system for detecting light emission sites on a substrate. In the present aspect a position determination system as described in the sixth aspect is implemented together with a detection system in order to derive position related information of a reflected irradiation beam in the detection system. Such a detection system may be as described in the fourth aspect, or it may be an imaging system wherein substantially the full area of interest on the substrate is imaged simultaneously using an array detector or an array of detectors by flood irradiation of the substrate. The system may be a fluorescence detection system. It may comprise a filter for filtering the irradiation beam after interaction with the substrate, from the luminescence, e.g. fluorescence, response from chemical, bio-chemical or biological particles or labels connected thereto.

In an eighth embodiment, the present invention relates to a method for determining position related information of an irradiation beam with respect to a substrate. The substrate thereby comprises binding sites for binding chemical, bio-chemical and/or biological particles. The method may be suitable for use in biosensing applications. The method comprises irradiating the substrate with an irradiation beam for detecting the presence of chemical, bio-chemical and/or biological particles. The latter may be performed by scanning the substrate or may be performed by imaging the substrate. In the last case, the substrate is subject to flood light whereby substantially the complete or at least a large portion of the area of interest is irradiated at the same time. The method further comprises detecting a modulation of the irradiation beam after interaction with the substrate. The irradiation beam after interaction with the substrate may be for example a reflected irradiation beam, reflected at the substrate, or part thereof or a transmitted irradiation beam, transmitted through the substrate. The modulation may be a spatial modulation, e.g. when the irradiating is performed as imaging technique. The modulation may comprise a temporal modulation, e.g. when the irradiating is performed by scanning. Detecting the modulation may e.g. be performed using a detector suitable for detecting irradiation with the wavelength or in the wavelength range of the radiation of the irradiation beam. Detecting of a temporal modulation may be performed using any suitable detector, such as e.g. a single element detector but preferably a line detector or array detector. The detector used may be a detector as described for the tracking embodiment. Detecting of a spatial modulation, occurring when an imaging technique is used, may be performed using any suitable array detector. The resolution of the array detector may be adapted to resolve the spatial modulation provided by the substrate and representative of position related information. The method furthermore comprises deriving from the modulation position related information of the irradiation beam with respect to the substrate. The position related information may be encoded in the modulation. It may be absolute position related information with respect to the substrate or relative position related information with respect to the substrate. Deriving position related information may comprise decoding a modulation using a predetermined algorithm, a neural network, look up tables, etc. Deriving absolute position information may be performed e.g. by detecting a spatial or temporal modulation of the irradiation beam after interaction with the substrate. E.g. when imaging is performed, a spatial modulation may be detected, whereas when scanning is performed a temporal modulation may be detected. Deriving position related information furthermore may comprise recognising part of a modulation as start and/or end indication of an encoded position information part. Deriving position related information may comprise deriving a plurality of bits or digits from a spatial and/or temporal modulated signal, which may be representative of a position. Deriving values for bits or digits may be performed by quantitative or qualitative assessment of the modulation of the irradiation beam, e.g. be determining optical values of the modulated irradiation beam within certain ranges and converting it to a corresponding value. Assessment of part of the modulation may take into account a clock signal generated from a periodic modulation of the irradiation beam after interaction with the substrate. Detecting an irradiation beam after interaction with the substrate may be performed after filtering the irradiation beam from a luminescence response from chemical, bio-chemical and/or biological particles which may be detected simultaneously. The latter is an advantage as the thus obtained position information can be easily combined with chemical, bio-chemical and/or biological detection signals. The method for determining position related information may be advantageously combined with the method of tracking as described above, and one or more of the irradiating step, detecting step and optionally filtering step may be used in common by both methods. The same features may be used and similar advantages may be obtained. In a further aspect, the method also may be used in a method for sensing chemical, biological and/or biochemical particles, e.g. using a fluorescence technique. Such a method may be an imaging method or a scanning method. In one particular embodiment wide beam scanning as indicated above may be applied.

In a ninth embodiment, the present invention relates to a substratecomprising binding sites for binding chemical, bio-chemical and/or biological particles. The substrate may e.g. be for use with a biosensing system. The substrate furthermore comprises optical modulating structures for modulating an impinging irradiation beam, wherein the optical modulating structures encode a modulation indicative of position related information of the structures with respect to the substrate. The position related information may comprise information about a relative position of the structures with respect to the substrate and/or information about the absolute position of the structures with respect to the substrate. The optical modulating structures may be for example reflective structures and/or absorption structures and/or phase modulating structures. The structures may be patterned structures.

The reflective structures and/or absorption structures and/or phase modulating structures may be formed in a reflective and/or absorption and/or phase modulating patterned layer. The structures comprise or are encoded with position related information of the structures with respect to the substrate. The structures may for example be in a periodic pattern, a barcode pattern.

An advantage of phase structures is their ease of manufacturing as for example use can be made of injection moulding techniques or by photo-polymerisation replication. Reflective structures may require deposition of several layers during the fabrication process and furthermore, patterning of the reflective structure may also require additional manufacturing steps. Nevertheless, in some embodiments, reflective and phase structures can be combined in one functional layer. In that case, a reflective part, e.g. un-patterned, may take care of a high reflection of excitation radiation, e.g. excitation light, and a superimposed, patterned phase structure may offer the desired local modulation of the reflected radiation, e.g. reflected light for including the position related information. The latter may advantageous in order to have a sufficiently large part of irradiation beam reflected while also providing accurate modulation information. For example, the optical modulating structures may be manufactured using a thin metal layer on the substrate 104 with height modulation in the range of λ/(4n) or λ/(2n) (with n the imaginary part of the refractive index of the substrate 104). The optical modulation structures may be embedded in regions applied for tracking a wide irradiation beam, as described in the first aspect, and may comprise the same features and advantages as described in the fourth and further particular embodiments of the first aspect. For maximum modulation of the irradiation beam at least one dimension of a phase structure may preferably be in the order of λ/4 of the wavelength of the excitation radiation in reflection mode, and about λ/2 of the wavelength of the excitation radiation in transmission mode. Reflective structures with high reflection, i.e. with a reflection of higher than 80%, preferably higher than 90%, more preferably higher than 95% can be realized in a reflective, e.g. metal, layer with a thickness in the range of tens of nanometers. With these specification the phase and/or reflective structure layer can be made very thin. If the modulation structures are in a stack with the layer of binding sites, the limited thickness of the modulating structure allows fulfilling the required condition that the layer of binding sites is in the proximity of the embedded position information, such that they can be read out simultaneously by an impinging irradiation beam. The optical modulation structures advantageously are located near the binding sites for binding or capturing different particles from a sample and used in case of biological detection, bio-chemical detection or chemical detection. The latter may allow simultaneous detection of a luminescence response and an indication of the modulation structures, e.g. position related information, thus resulting in an accurate system. According to embodiments of the invention, the position information may be of absolute nature, relative nature or a combination of both. With absolute nature is meant that by reading the information embedded in the modulation structures the absolute position of the location can be retrieved. With relative nature is meant that by reading the information embedded in the modulation structures only the relative position, i.e. with respect to some previous positions, of the location can be retrieved. For example, a combination of a course grid of absolute position info and a finer grid of relative position marks may allow accurate mapping of the surface of the substrate. The principle of spreading the optical modulating structures carrying absolute position information more than the optical modulating structures carrying relative position information is illustrated, amongst others, in Fig. 6. It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. For example, whereas in the above embodiments substrates are described being suitable for use in a detection system with tracking system using an irradiation beam having an irradiation beam projection on the substrate with at least one dimension larger than the wavelength of the irradiation beam used, the present invention also relates to a method of manufacturing such a substrate. Such substrates may be manufactured by providing alternating first and second regions having different irradiation beam modulating properties in said substrate, the first and second regions extending in a first direction suitable for being a scanning direction and at least one of the first and second regions extending in a second direction perpendicular to the first direction over a width substantially larger than the wavelength of the irradiation beam used. The method furthermore may comprise providing capturing probes on the surface adapted for capturing sample components to be detected, e.g. using fluorescent labels attached to the sample components. The capturing probes may be provided in those first regions and/or second regions of the substrate that will be scanned, using the tracking system as described above. Which of the first regions and/or second regions of the substrate will be scanned depends on the specific configuration of first and/or second regions used, as is illustrated in the second aspect of the present invention.

Claims

CLAIMS:

1. A substrate (104) for use with a tracking system (106), the substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles and the substrate (104) adapted for being scanned with an irradiation beam in a scan direction (S), the irradiation beam having an irradiation beam projection (202) on the substrate having at least one dimension larger than a wavelength (λ) of the irradiation beam, said substrate (104) comprising a plurality of alternating first regions (204) and second regions (206) wherein said first regions (204) and second regions (206) are substantially extending along a first direction suitable for being substantially the scan direction (S), said first regions (204) and second regions (206) having a length in a second direction substantially perpendicular to the scan direction (S) adapted such that at least one of said first regions (204) and second regions (206) has a length in a second direction substantially perpendicular to the scan direction (S) which is substantially larger than the wavelength (λ) of said irradiation beam, and said first regions (204) and second regions (206) having a substantially different optical property for modulating said irradiation beam.

2. A substrate (104) according to claim 1 wherein said substantially different optical property is provided using sub-wavelength patterns.

3. substrate (104) according to claim 1 or 38 wherein any of said first regions (204) or second regions (206) comprise additional information.

4. A substrate (104) according to claim 3, wherein the additional information is provided in optical modulating structures.

5. A substrate (104) according to claim 4, wherein the optical modulating structures comprise one or more of reflective structures, absorption structures or phase modulating structures.

6. A substrate (104) according to claim 4, wherein the additional information is indicative of position related information of the optical modulating structures with respect to the substrate (104).

7. A substrate (104) according to claim 1 or 38, wherein said substantially different optical property is a substantially different reflectivity, transmission, scattering behavior or absorption behavior.

8. A tracking system (106) for tracking areas on a substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the tracking system using an irradiation beam, the irradiation beam having an irradiation beam projection (202) on the substrate (104) whereby at least one dimension of the irradiation beam projection (202) is substantially larger than a wavelength (λ) of the irradiation beam, the tracking system (106) comprising detecting means (108) adapted for detecting a variation of an optical characteristic of at least part of the irradiation beam induced by interaction with different regions (204, 206) of said substrate having a different irradiation modulating property, to thereby locate said irradiation beam with respect to said substrate (104).

9. A tracking system (106) according to claim 8, wherein said detecting means

(108) is adapted to simultaneously detect part of the irradiation beam modulated by a first region (204) of said substrate (104) and part of the irradiation beam modulated by a second, adjacent region (206) of said substrate (104), the first and second region (204; 206) having a different irradiation modulating property.

10. A tracking system (106) according to claim 9, wherein said variation in said optical characteristic of said irradiation beam is induced by a variation of an area of said first region (204) of said substrate (104) and a variation of an area of said second region (206) of said substrate (104).

11. A tracking system (106) according to claim 9, wherein said irradiation beam is an at least piecewise elongated irradiation beam having an irradiation beam projection with a length in a direction perpendicular to a scan direction (S) by which said substrate (104) is scanned with said irradiation beam, the length being substantially larger than said wavelength (λ).

12. A tracking system (106) according to claim 9, said tracking system (106) furthermore comprising a position correcting means (110) adapted for correcting the relative position of said at least piecewise elongated irradiation beam and said substrate (104) in view of a detected optical characteristic variation of said at least part of the irradiation beam.

13. A tracking system (106) according to claim 12, wherein said tracking system (106) comprises an automated feed-back loop (114) between said detecting means (108) and said position correcting means (110).

14. A tracking system (106) according to claim 9, wherein said optical characteristic of said at least part of the irradiation beam is any of: the amount of reflected light, the amount of transmitted light, the amount of scattered light or the density of scattered light of at least part of said irradiation beam induced by interaction with said substrate (104).

15. A tracking system (106) according to claim 9, wherein said tracking system (106) is furthermore adapted to detect additional variations in said optical characteristic of said irradiation beam and comprises a processing means (130) for determining additional information from said additional variations in said optical parameter.

16. A tracking system (106) according to claim 9, wherein said detecting means (108) is a split detector, a single spot detector or a pixelated detector.

17. A tracking system (106) according to claim 9, the tracking system (100) furthermore comprising a rotating means for rotating said irradiation beam with respect to a rotating axis substantially inclined to said substrate (104).

18. A tracking system (106) according to claim 8, wherein the detecting means

(108) furthermore is adapted for stabilizing the orientation of the irradiation beam with respect to the scanning area of the substrate.

19. A method (700) for tracking a position of an irradiation beam on a substrate, the method (700) comprising:

- scanning (702) said substrate (104) by guiding the relative position of the substrate (104) and an irradiation beam having an irradiation beam projection (202) on the substrate (104) with at least one dimension substantially larger than a wavelength of the irradiation beam,

- detecting (704) a variation in an optical characteristic of at least part of said irradiation beam induced by interaction with different regions of said substrate (104) having a different irradiation modulating property, for determining a position of said at least piecewise elongated irradiation beam with respect to said substrate (104) for use in tracking.

20. A method (400) according to claim 19, the method further comprising stabilizing the orientation of the irradiation beam with respect to the scanning area of the substrate.

21. A method (400) according to claim 19, further comprising correcting the position of said at least piecewise elongated irradiation beam on said substrate (104) in view of a determined irradiation beam location.

22. A detection system (100) for detecting light emission sites on a substrate (104), the detection system (100) comprising:

- an irradiation source (102) for generating an irradiation beam having an irradiation beam projection (202) on the substrate (104) whereby at least one dimension of the irradiation beam projection (202) is substantially larger than a wavelength (λ). of the irradiation beam, and - a tracking system (106) comprising a detecting means (108) adapted for detecting a variation of an optical characteristic of at least part of the irradiation beam induced by interaction with different regions (204, 206) of said substrate (104) having a different irradiation modulating property, for locating said irradiation beam with respect to said substrate (104).

23. A method for manufacturing a substrate for use in a detection system (100) for detecting of light emission sites on a substrate (104), the substrate (104) being adapted for being scanned with an irradiation beam having an irradiation beam projection (202) on the substrate having at least one dimension larger than a wavelength (λ) of the irradiation beam, the method comprising:

- providing alternating first regions (204) and second regions (206) in said substrate (104), wherein said first regions (204) and second regions (206) are substantially extending along a first direction suitable for being the scan direction, said first regions (204) and second regions (206) having a length in a second direction substantially perpendicular to the scan direction (S) adapted such that at least one of said first regions (204) and second regions (206) has a length in a second direction substantially perpendicular to the scan direction (S) which is substantially larger than the wavelength (λ) of said irradiation beam, said first regions (204) and second regions (206) having a substantially different optical properties for modulating said irradiation beam.

24. A position determination system for determining position related information of an irradiation beam on a substrate comprising binding sites for binding chemical, bio- chemical and/or biological particles, the position determination system using an irradiation beam for irradiating the substrate for detecting presence of chemical, bio-chemical and/or biological particles, and the position determination system comprising a detection sub-system for detecting a modulation of the irradiation beam after interaction with the substrate, and for deriving from said modulation position related information of said irradiation beam with respect to the substrate.

25. A position determination system according to claim 24, wherein the position determination system comprises a filter for filtering the irradiation beam after interaction with the substrate from a luminescence response from said chemical, bio-chemical and/or biological particles.

26. A method for determining an absolute position of an irradiation beam with respect to a substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the method comprising

- irradiating the substrate with an irradiation beam for detecting the presence of chemical, bio-chemical and/or biological particles

- detecting a modulation of the irradiation beam after interaction with the substrate, and - deriving from said modulation position related information of said irradiation beam with respect to the substrate.

27. A method according to claim 26, wherein the method furthermore comprises, prior to detection of the modulation of the irradiation beam after interaction with the substrate, filtering the irradiation beam after interaction with the substrate from a luminescence response from said chemical, bio-chemical and/or biological particles.

28. A method according to claim 26, said deriving position related information comprising deriving absolute position related information with respect to the substrate.

29. A method according to claim 26, said deriving position related information comprising recognizing part of the modulation as start and/or end indication of the information.

30. A method according to claim 26, wherein deriving position related information comprises deriving at least one digit value from the modulation.

31. A substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the substrate comprises optical modulating structures for modulating an impinging irradiation beam, the modulation being indicative of position related information of the structures with respect to the substrate.

32. A substrate as described in claim 31, wherein the optical modulating structures comprise at least one of reflection structures, absorption structures or phase modulating structures.

33. A substrate as described in claim 31, wherein the optical modulating structures are encoded with a first part encoding the position related information and a second part indicating a start and/or end of said first part.

34. A substrate as described in claim 31, wherein the optical modulating structures comprise different portions with different optical modulating properties for indication of different values of a plurality of digits.

35. A substrate as described in claim 31, wherein the optical modulating structures comprise first optical modulating structures representative of a position in a first direction and second optical modulating structures representative of a position in a second direction, the first and second optical modulating structures being provided in an alternatingly manner.

36. A detection system (100) for detecting light emission sites on a substrate (104), the detection system (100) comprising:

- an irradiation source (102) for generating an irradiation beam for irradiating the substrate for detecting presence of chemical, bio-chemical and/or biological particles, and

- a position determination system comprising a detection sub-system for detecting a modulation of the irradiation beam after interaction at the substrate, and for deriving from said modulation position related information of said irradiation beam with respect to the substrate.

37. A method for manufacturing a substrate for use in a detection system (100) for detecting of light emission sites on a substrate (104), the substrate comprising binding sites for binding chemical, bio-chemical and/or biological particles, the method comprising:

- providing optical modulating structures for modulating an impinging irradiation beam, the modulation being indicative of position related information of the structures with respect to the substrate (104).

38. A substrate (104) comprising: a first region having a first optical property and a second region having a second optical property, the first optical property being different from the second optical property, the first region and the second region extending along a first direction, the first region having a first length along a second direction and the second region having a second length along the second direction, the second direction being perpendicular to the first direction; - binding sites capable of binding chemical, biochemical and/or biological particles;

39. A substrate according to claim 38 wherein the binding sites are present at least in the first region.

40. A substrate according to claim 38, 39 , wherein, along a third direction perpendicular to the first and second directions, a finite distance exists between the first region and the second region.

41. A substrate according to claim 38, 39 or 40 wherein the second region extends on a first side of the first region and a further second region extends along the first direction on a second side of the first region.

42. A substrate according to claim 38, 39, 40 or 41 wherein the second region is at least partially discontinuous in the first direction.

43. A substrate according to claim 38, 39, 40, 41 or 42 wherein at least on of the first and second regions comprises a sub wavelength pattern.

44. A substrate according to claim 41 wherein the second region and the further second region are both discontinuous such that the discontinuity of the first region is not in phase with the discontinuity of the further second region at least along part of the distance over which the second region and further second region extend in the first direction.