EP0996854A1

EP0996854A1 - Wave field microscope, method for a wave field microscope, including for dna sequencing, and calibration method for wave field microscopy

Info

Publication number: EP0996854A1
Application number: EP98967010A
Authority: EP
Inventors: Michael Hausmann; Christoph Cremer; Joachim Bradl; Bernhard Schneider
Original assignee: Universitaet Heidelberg
Current assignee: Universitaet Heidelberg
Priority date: 1997-07-10
Filing date: 1998-07-09
Publication date: 2000-05-03
Also published as: AU9251898A; DE19830596A1; EP1008845A1; WO1999002974A9; WO1999002974A1; DE19830596B4

Abstract

The invention relates to two new wave field microscopes (type I and type II) characterized in that they each have a stimulation and illumination system comprising at least one real and one virtual light source and at least one lens (in the case of type II) or two lenses (in the case of type I). The light sources and lens(es) are positioned in relation to each other in such a way that they are suitable for generating one, two or three-dimensional wave fields in the object space. The calibration method provided for in the invention has been adapted to these wave field microscopes and allows for geometrical distance measurements between fluorochrome-marked object structures the distance of which can be less than the half-intensity width of the principal maximum of the effective point image function. The invention also relates to a method for DNA sequencing by wave field microscopy.

Description

Wave field microscope,

Wave field microscopy methods, also for DNA sequencing, and calibration methods for wave field microscopy

Description

The invention relates to a wave field microscope with an illumination or excitation system, which comprises at least one real and a virtual illumination source and at least one objective, illumination sources and objective (s) being arranged relative to one another in such a way that they are suitable for generating a one-dimensional standing wave field, with an object space, which comprises holding and maneuvering device (s) for an object, and with a detection system, which comprises an objective, an eyepiece and a detector, it also relates to a calibration method adapted therefor for geometric distance measurements between fiuorochrome-marked object structures whose distance is less than The half-width of the main maximum of the effective point image function can be, and it relates to a method based thereon for wave field microscopic DNA sequencing.

State of the art

Through the use of highly specific markers, such as DNA samples or protein probes, it is possible to use almost any small size in biological (micro) objects, especially in cells, cell nuclei, cell organelles or on chromosomes - hereinafter simply referred to as objects Mark (sub) structures. Structures in dimensions from a few μm (10 ^"6 m) to a few tens of nm (10 ^{" 9} m) can be represented specifically. The markers are usually coupled with fluorochromes or colloidal micro (gold) particles in order to facilitate their optical detection and imaging, or to enable them in the first place.

In order to be able to detect two markers separately from one another within the same object, the markers in question are often coupled with fluorochromes of different colors. The available color emission spectrum of the commonly used ones Fluorochromes range from deep blue to green and red to the infrared spectral range.However, fluorochromes can also be used which do not differ in their excitation spectrum or in their fluorescence spectrum, but in which the lifetime of their fluorescence emission is used as a parameter to distinguish them.The latter have the advantage that wavelength-dependent focal shifts do not occur Fluorochromochromes can also have a different emission spectrum and therefore have different spectral signatures, but they can be excited with the same photon energy, e.g. by multi-photon processes

The above-mentioned fluorochromes which can be bound or bound to specific (sub) structures in biological micro-objects are referred to below as fluorescent markers

If the excitation spectrum and / or emission spectrum and / or the fluorescence lifetimes of two fluorescence markers match, then these fluorescence markers have the same spectral signature with regard to the parameter in question. If the fluorescence markers differ in one or more parameters relevant to the measurement, they have a different spectral signature

In the following, fluorescence is understood to mean any photon interaction in which there are differences between the excitation spectrum and the emission spectrum of the same substance, which cannot be attributed to monochromatic absorption or scattering. This includes, in particular, multi-photon interactions, in which the excitation wavelengths can be longer than the Emis ion wavelengths

Furthermore, the term fluorescence is also used here for the closely related phenomena of luminescence, in particular phosphorescence.This includes in particular longer mean fluorescence lifetimes, for example fluorescence lifetimes in the range of up to several or many msec (milliseconds) closely related processes of luminescence, phosphorescence and fluorescence are regarded as equally relevant to the invention

The detection and imaging of fluorescent markers in extended biological objects and the quantitative localization with respect to defined object points / object structures (distance and angle measurements) is carried out using light microscopic measurement methods. The so-called “point spread function” (PSF) or “point response” plays here. of the microscope used or of the optical system in general, ie its ability to create an equally ideal point-like image from an "ideally point-shaped" object, the crucial role The point-image function is a characteristic feature of every imaging optic and a measure of its quality

Distance measurements between object structures depend essentially on the effective point image function - that is, the one given locally in the marked object point. This effective point image function in turn strongly depends on the respective local refractive index and the absorption in the object, in the embedding medium of the object, in the immersion liquid and possibly in the Cover glasses

The effective point image function generally differs significantly from the point image function calculated for the microscope used. The point image functions measured under technically optimized boundary conditions also generally differ from the effective point image functions which can be achieved in practical routine laboratory conditions in biological objects

Since these effective point image functions are usually not available, ideal calibration results are used to calibrate the distance measurements or calibration measurements carried out under standard conditions, such as reflection methods.Both methods, however, come at the expense of precision in three-dimensional distance measurement in biological systems Micro-objects, and the result is considerable uncertainty in the determination of the actual spatial distance between the object structures. In the case of biological objects, such quantitative large estimates contain uncertainties of up to several micrometers Until recently, there was a practically unanimous belief among experts that two object structures can only be separated if they have at least one

Half-width of the main maximum of the effective point image function are apart

It was only in 1996 that the authors of the present invention succeeded in providing a calibration method for far-field microscopy (and also for fluorofluorometry) with which it is possible to measure distances between object structures whose distance from one another is less than the resolution of the relevant far-field microscope, i.e. less than that Half width of the main maximum of the effective point image function are apart from each other, regardless of the position of the object structures in question in three-dimensional space, with high accuracy

This procedure includes the following steps

• Before, during or after the preparation of the object in question on or in an object holder, in particular object carrier plates, object carrier fibers / capillaries or object carrier fluids, the structures to be examined or located (measurement structures) are marked with fluorescent dyes of different and / or the same spectral signature, ie Structures to be located (measuring structures) that are in close proximity to one another, namely within the half-width of the main maximum of their effective point image function, are marked with fluorescent dyes of different spectral signatures, while those measuring structures whose distance from one another is greater than the half-width of the main maximum of the effective point image function, marked with fluorescent dyes of different or the same spectral signature. Two measuring structures to be located may always be marked with the same spectral signature if, for example, by their relat ive location or can be clearly identified by other criteria

• With the same fluorescent dyes, calibration targets of defined size and spatial arrangement are marked,

• The fluorescent calibration targets are prepared either together with the objects or separately on or in or in an object holder (slide plates, slide fiber / capillary, slide liquid or the like) • (Examination) object and calibration targets are examined under the same conditions, simultaneously or one after the other microscopically or by fluorofluorometry

• Two defined calibration targets with different spectral signatures are measured taking into account the wavelength-dependent imaging and localization behavior of the respective optical system (microscope or river fluorometer). The measured values determined in this way are equal to actual values and the known actual distance values equal to target values (ie compared to the target locations calculated on the basis of the geometry), and the difference between actual values and target values, namely the calibration value, is used to correct the offset caused by the optical system in the detection of different emission loci, in particular of the measurement structures

In other words, the distance measurement between the object (sub) structures (depending on the distance from each other) marked with different or the same spectral signatures - hereinafter also called measurement structures - is based on the highly precise localization of independent (calibration) targets with a corresponding spectral signature and with a known large and spatial arrangement, taking into account the wavelength-dependent imaging and localization behavior of the respective optical system, the calibration measurement between the (calibration) targets and the measurement in biological objects taking place under the same system and boundary conditions.These calibration targets have the same or one Higher multispectrality such as the (object) structures to be measured They can be arranged directly in the biological objects, or as separate preparations on a specimen holder (specimen slide plates or specimen carrier fiber / capillary or specimen carrier liquid or the like) exist or be part of an object holder

Two or more fluorescent measuring structures in intact, three-dimensional biological objects, the distance and extent of which is smaller than the full width at half maximum of the main maximum of the effective point image function, can be discriminated on the basis of their different spectral signature (fluorescence absorption wavelengths and or fluorescence emission wavelengths and / or fluorescence emission lifetimes), ie their distance from each other can be determined The distance measurement is reduced to the localization of the individual measuring structures and can now - with optical far-field microscopy or river fluorometry - be carried out with a much higher accuracy than the half-width of the main maximum of the point image function.The localization of the center of gravity of the measuring structures in question is adapted to the maximum intensity of their fluorescence signal D h from the measured (diffraction-limited) signal (^ intensity curve) of a fluorescence point (= fluorescent measurement structure) - taking into account the total information from the main and secondary maxima - the center of gravity (the barycentre) of the signal is determined and thus the location of the measurement structure With an error-free optical system and consequently ideal symmetry of the measured intensity distribution (= course of the intensity curve), the center of gravity (the barycenter) of the intensity curve colocalizes with the main within the localization accuracy maximum (= maximum 0 order of the diffraction pattern) of the measured intensity distribution This new calibration method allows using optical far field microscopy such as wave field microscopy (or also using scanning flow fluorometry) to measure geometric distances in biological micro-objects determining distances can be less than the full width at half maximum of the main maximum of the effective point image function in the object. Since the information content of the distance determinations carried out in this way corresponds to a distance measurement obtained with an increased resolution, one can (and will also in the following) abbreviate "resolution equivalent"

The multispectral calibration makes it possible to carry out in situ measurements of the imaging behavior of the system on the specific biological object. If the fluorescence lifetime is used as the sole parameter type and / or if the fluorochromes are excited with the same or the same photon energy (s), the calibration eliminates the in situ correction of the chromatic offset in the object plane For high-resolution far-field microscope types such as the wave field microscope and when using suitable fluorescent markers, this calibration method enables three-dimensional geometric distance measurements in biological objects down to molecular precision (ie resolution equivalent better than 10 nm) To determine actual and target values, to compare them and to determine the

Correction value / calibration value, the following method steps are preferably carried out

one or more calibration targets B with a distance greater than the full width at half maximum of the main maximum of the effective point image function from the center of gravity of the N measurement structures is / are marked with any spectral signature,

the distances d, k (i, k = 1 N, i ≠ k) of the centers of gravity of the spectrally separated diffraction patterns of the N measurement structures and the distances d, _{B of} the N measurement structures from the calibration target B are measured, using automated methods of image analysis,

for a measuring structure, the distances d _lk and d ^ are each measured in the plane of the narrowest point image function and all normal distances, for which purpose the object is rotated axially tomographically by a defined angle φ _m ,

optical aberrations from the calibration measurements are corrected, and a cosine function A, k cos (φ _m + θ _lk ) or A is added to the corrected measured distances d, k (φ _m ) and d, B (φ _m ), B cos (φ _m + θ, _B ) adapted with a suitable phase shift,

- The maxima A _lk and A, _{B of} the adaptation function of d or d _ß are divided by the magnification factor and determined as the Euclidean distance D or D _{lB of} the N measuring structures from one another or the distances of the measuring structures from reference point B.

For the determination of the maxima, the minima corresponding to these of the distance z, k, z & in the plane orthogonal to the plane of the d, _k , d, _B are preferably additionally used and evaluated analogously

The determination of all coordinates of the N measuring structures and their relative coordinates to the reference point B, ie the determination of the positions x "y" z, and X, y, Zk and the distances Xk - x,, yk - y,, z _k - z, and x _B - x,, yß - yi, z _B - z, takes place according to the invention on the basis of the microscopically measured 3D distances D, _k or D, _B , preferably using the following system of equations D ² _lk = (x _k - x,) ² + (y _k - y,) ² + (z _k - z,) ² D ² , B = (x _B - x,) ² + (v _B - y.) ² + (z _B - z , f = (XB - Xk) ² + (VB - yk) ² + (z _B - z _k ) ²

To safeguard the determined measurement results, the procedure described above should be carried out for several calibration targets B and the same N measurement structures

The coordinates and distances of the N measuring structures can be determined on the basis of the centers of gravity, which result from averaging the centers of gravity of the measurements to all reference points

In particular for graphic representations, the determined positions x ,, y ,, z, and X _B , V _B , Z _{B are} preferably folded using a point image function which has a half-width with the resolution equivalent achieved in each case

For the fluorochrome marking of measurement structures and calibration targets, those fluorochromes are preferably used which can be excited in the ultraviolet, visible and / or infrared light wavelength range and which emit in the ultraviolet, visible and / or infrared light wavelength range

Either biological calibration targets or non-biological or synthetic calibration targets can be used as calibration targets

The biological calibration targets are marked regions of the biological object with a known distance from one another. The marking (s) of the region (s) in question can (can) be carried out, for example, with suitable biochemical probes. The use of such biological calibration targets is compared to the use of synthetic calibration targets , for example calibration spheres, the practical advantage that, in addition to the optical boundary conditions of the object, additional effects caused by the preparation flow into the calibration, such as the ratio of the actual fluorescence signal to the unspecific background (which is determined by automatic image analysis algorithms)

Microspheres with the same or a higher multispectral signature than the measurement structures to be located are particularly suitable as non-biological or synthetic calibration targets.They are treated like biological objects.These calibration targets are preferably fixed on object holders in a defined spatial arrangement The slides in question are fabricated, which is particularly advantageous for routine use To eliminate the problem existing in all known far-field microscopy methods that the width of the main maximum of the point image function and thus the resolution limit depends on the relative position in space, ie z B perpendicular to the optical axis (= lateral) is narrower than in the direction of the optical axis (= axial ), the calibration method mentioned can be combined very well with the so-called microaxial tomography methods known in the prior art. With these microaxial tomography methods, the (biological) objects are arranged in capillaries or on glass fibers and defined in or under the microscope about an axis which is normally perpendicular to the optical axis of the microscope, are rotated, distance measurements are carried out in the direction which has the narrowest half-width of the effective point image function

A far-field light microscopy method which is particularly suitable for the detection and imaging of, in particular, very small substructures in biological objects identified by fluorescent markers, because it has the advantage over the known epifluorescence microscopy methods or confocal laser scanning microscopy that it Also in the axial direction - perpendicular to the wave fronts - depth discrimination and thus significantly improved resolution (its dimension can be much smaller than the wavelength of the light used for excitation with a high numerical aperture) is the wave field microscopy method in wave field microscopy as it is e.g. In US Pat. No. 4,621,911, fluorescent or luminescent specimens are illuminated in the optical microscope with a standing wave field (Standing Wave Field Fluorescence Microscopy, SWFM). A standing wave field arises (only) where light k The specimens are arranged in a zone of flat, flat wavefronts and excited to fluorescence or phosphorescence.The distance between the wavefronts and their phase can be varied (especially for imaging). The three-dimensional distribution of the fluorescent or luminescent material can be made from individual optical sections using computer image processing Object points can be reconstructed The plane wave fronts are arranged perpendicular to the optical axis of the detecting lens and are generated by coherent superposition of two laser beams at a defined angle θ to the optical axis of the microscope system, the angle θ determining the spacing of the wave fronts from one another - for a given wavelength and refractive index instead of two crossing laser beams, the wave field can also be generated by causing a laser beam to interfere with itself after suitable reflection at a certain angle (e.g. with a mirror). The plane wave fronts are characterized by the fact that the intensity curve is perpendicular to the wavefronts (co) is sinusoidal. The fluorescence or luminescence is either spectrally discriminated by appropriate optical filters and guided in different beam paths or detected confocally. The achievable resolution, ie the smallest yet measurable distance z between two point-shaped object structures, which are marked with fluorochromes of the same spectral signature, is either by the Abbe criterion (= the maximum 0 order of the diffraction pattern of a point object is located in the 1 minimum of the diffraction pattern of a second point object) or by the half-width of the main maximum of the effective one Point image function given It depends on the respective wavelength, the numerical aperture of the lens used, as well as on the local refractive indices of the objects, the embedding medium, the cover glasses that may be used and the immersion fluids that may be used

The known wave field microscopes are basically constructed as follows

(I) an illumination or excitation system consisting of at least one real and a virtual illumination source and at least one objective, which are arranged with respect to one another in such a way that they are suitable for generating a one-dimensional, sinusoidal, standing wave field,

(II) an object space with

Holding and maneuvering devices for the object, and (πi) a detection system consisting of at least one objective, at least one eyepiece and at least one detector, which is often a camera, in particular a CCD camera, which is positioned such that the CCD chip lies in the intermediate image plane

A disadvantage of this prior art wave field microscope, hereinafter referred to as "one-dimensional wave field microscope" ("SWFM"), or the wave field microscopy method that can be carried out with it, is that the periodically generated wave field (with epifluorescent detection in conjunction with optical sectioning methods) becomes one Ambiguity in the recording or imaging of an object structure leads, the extent of which in the direction perpendicular to the wavefronts is substantially greater than λ / 2n (λ = wavelength of the excitation, n = effective refractive index) This ambiguity initially makes effective use of the resolution improvement achieved by the interference pattern difficult

To carry out distance measurements and other investigations of the spatial relationships of three-dimensional objects, the known far-field microscopy methods, including one-dimensional wave field microscopy, can be combined with axial tomography.For this purpose, the biological objects to be examined, if necessary after being equipped with calibration targets, in or on a microcapillary or glass fiber as object holder or object holder prepared The capillary / fiber has a precisely defined diameter, with different diameters being possible. To fix this capillary / fiber on the microscope stage, a special holder is proposed, which consists of a rigid, preferably dorsiventrally flattened frame, mounted on or on which at least one bearing bush is in which a microcapillary or glass fiber can be rotated about its longitudinal axis (preferably with the axis of rotation perpendicular to the optical axis of the microscope) (Die Bearing bushing (s) should be arranged so that the axis of rotation of the capillary / fiber runs perpendicular to the optical axis of the microscope) The objects to be examined in or on the capillary / fiber are rotated by rotating the capillary / fiber directly, preferably by means of a rotary motor The present invention has for its object to develop a wave field microscope of the known type such that it is suitable for generating plane wave fields in more than one dimension with high variability in the distances of the interference maxima, and to develop the aforementioned calibration method in such a way that it can be used in combination with such a wave field microscope. Furthermore, a method for wave field microscopic DNA sequencing is to be created.

One solution to this problem is, on the one hand, to provide the so-called "multidimensional wave field microscopes" described below, and on the other hand to provide the calibrating method that is also described below and is adapted to the use of a multidimensional wave field microscope. Furthermore, a method for "fluorescence DNA sequencing" is provided

The "multidimensional wave field microscope" (type I) according to the invention is a wave field microscope of the type mentioned at the outset, which is characterized by the features listed below

(1) The lighting or excitation system comprises in two or all three spatial directions at least one real or virtual lighting source for coherent light beams and at least one reflector or beam splitter for coupling out partial beams or a further lighting source for coherent light beams, each of which is assigned at least one objective, and which are each suitable for generating light wave trains, the light wave trains of the one illumination source being aligned antiparallel or at variably adjustable angles to the light wave trains of the reflector or the other illumination source, in such a way that the light wave trains emitted by the one illumination source match those of the reflector or the other lighting source to interfere with a standing wave field with flat wave fronts

(2) The detection system comprises at least one detection lens suitable for epifluorescent detection and / or at least one detection lens suitable for scanning point detection, preferably a high numerical aperture, which with its optical Axis is arranged perpendicular to the wave fronts of one of the interfering wave fields and which can be identical to a lens of the excitation system. The detection lens suitable for epifluorescent detection is preceded by a flat (two-dimensional) detector, e.g. a camera, while the detection lens suitable for scanning point detection is preceded by at least one fixed confocal detection ring diaphragm and / or pinhole diaphragm and / or at least one fixed detection slit and a point detector, in particular, a photomultiplier, a photodiode or a diode array is arranged downstream. “High” numerical aperture is understood here to mean a numerical aperture> 1 and “low” numerical aperture means a numerical aperture <1.

In a particularly preferred embodiment of this wave field microscope (type I), a lens with a high numerical aperture is assigned a lens with a high numerical aperture or a reflector in at least one spatial direction, and either two lenses with a low numerical aperture or one are associated in one or both other spatial directions Objective low numerical aperture and a reflector assigned to each other.

In the other "multidimensional wave field microscope" according to the invention (type

II) it is a wave field microscope of the type mentioned, which is characterized by the following features.

(1) In at least one of the three spatial directions, the lighting or excitation system comprises at least one real or virtual lighting source for coherent light beams and at least one beam splitter for coupling out at least one partial beam, to which a common lens is assigned, into which the light beams or light wave trains of Illumination source (s) and the beam splitter (s) can be coupled in such a way that they generate two spaced focal points on the rear focal plane (facing away from the object space) and run in the space between the two focal planes at a variably adjustable angle to one another and to one one-dimensional standing wave field. (2) The detection system comprises at least one detection lens suitable for epifluorescent detection and / or at least one detection lens suitable for scanning point detection, preferably a high numerical aperture, which can also be identical to a lens of the excitation system. The detection lens suitable for epifluorescent detection is preceded by a flat (two-dimensional) detector, for example a camera, while the detection lens suitable for scanning point detection is preceded by at least one fixed confocal detection ring diaphragm and / or pinhole diaphragm and / or at least one fixed detection slot and a point detector, in particular a Photomultiplier, a photodiode or a diode array is arranged downstream.

In a preferred embodiment of this wave field microscope (type II), the illumination or excitation system in the same or one or both other spatial direction (s) each has at least one further real or virtual illumination source for coherent light beams and / or at least one beam splitter for coupling out at least one Partial beam, to which a further lens is assigned, through which the light rays (light wave trains) are directed into the object space and aligned in such a way that they can be combined with the light rays from the same or from the other two spatial direction (s) or interfere with the one- or two-dimensional wave field formed by them to form a two- or three-dimensional wave field.

A further, very advantageous further development of all the above-mentioned wave field microscopes according to the invention (type I and type II) is characterized in that the object space comprises an object holder in or on which the object with the measurement structures and / or possibly the calibration target (see ) is rotatably mounted about one or two axes running orthogonally to one another in the shaft field, with a rotatability through 360 degrees (2π) being preferred for at least one axis. With these multidimensional type I and type II wave field microscopes according to the invention, it is possible to carry out a temporally sequential and / or simultaneous detection of several object planes by one, two and / or three objectives (or their orthogonal axes). Precision distance measurements of point objects of the same or different spectral signature, whose distances are smaller than the half-widths of the main maxima of the effective point image functions can be carried out in (all) spatial directions

The fixed confocal detection ring diaphragm, pinhole aperture and / or the fixed detection slit (s) in combination with at least one suitable light intensity detector offers the advantageous possibility that the object in x- , y and z directions can be scanned by the wave field (object or stage scanning)

The type II wave field microscope according to the invention and the wave field microscopy that can be carried out therewith - in particular compared to the known one-dimensional wave field microscopy - also have the advantage that both the lateral resolution (ie perpendicular to the optical axis) and the axial resolution are significantly improved. For the first time there is discrimination of flat objects in the axial direction possible without the use of confocal systems. There is also the advantageous possibility that the object can be moved in the lateral direction during the examination or for recording / data registration. With the help of image processing and reconstruction methods, it can then be obtained from the thus obtained Multiple acquisitions gain a higher lateral resolution The type II structure according to the invention with an objective is also suitable for generating a one-dimensional wave field perpendicular to the optical axis of an epifluorescence microscope s and thus to improve the lateral resolution of the same

In a further embodiment variant of these “multidimensional wave field microscopes” (type I and / or type II), the illumination source (s) generating the multidimensional wave field and / or the reflector (s) and / or the beam splitter and / or the lens (s) and thus the multidimensional wave field around one or two axially orthogonal axes rotatably arranged or mounted

In order to image the lateral object areas of an object fixed in the two- or three-dimensional wave field onto the detector ring diaphragm, aperture diaphragm or the detector slit, each wave field microscope according to the invention (type I and / or type II) can be equipped with a scanner mirror which is arranged in such a way that it detects the maps the relevant lateral object areas with the desired, usually maximum, fluorescence intensity

In a particularly advantageous development of the multi-dimensional wave field microscope (type I and / or type II) according to the invention, which is suitable for two- or more-photon fluorescence excitations, the so-called "wave field microscopes with combined multi-photon fluorescence excitation", the respective illumination system comprises in at least one of the three spatial directions has a real illumination source for the two- or multi-photon excitation and in one or both other spatial directions a real and / or virtual illumination source for the two- or multi-photon excitation. The standing wave fields generated thereby (WFi, WF ₂ ,, WF,) have mutually different wavelengths (λi, λ ₂ , λ,), and the distances (d _l5 d ₂ ,, d,) between their respective wave maxima or wave minima 2n cosθi or d ₂ = λ ₂ / 2n cosθ ₂ or d, = λ, / 2n cosθ, (with n = refractive index in the object space, θi, θ ₂ , θ, = crossing angle of the light wave train of wavelength λi, λ ₂ , λ, with the optical axis) These wave fields WFι, WF ₂ W, are aligned with each other according to the invention in such a way that at least a maximum of two or all standing waves is at the same point, namely the location of a multi-photon excitation

Suitable illumination sources for two- or multi-photon excitation are known in the prior art and are described, for example, in the publication by W Denk, JH Strickler, WW Webb, "Two-Photon Laser Scanning Fluorescence Microscopy", Science, Vol 248, pp 73-76 ( 6 April 1990), the content of which is expressly referred to here. These lighting sources either produce different photons

Wavelengths or coherent photons of the same wavelength

A particular advantage of the combination of one and two or multiple photon excitation is the simultaneous excitation of fluorescent markers with different spectral signatures.This enables distance measurement errors due to the chromatic aberration in the object to be eliminated.With two-photon excitation, a fluorochrome molecule is excited when two photons simultaneously deliver the energy for the excitation of a molecule. The two photons involved in the excitation of the molecule can have the same or different wavelengths or energies. For a coincident excitation with different wavelengths (λi, λ ₂ ), the so-called "two-photons - Wave field microscopy "two wave fields with the wavelengths λi and λ must be installed in each respective spatial direction. The wave maxima or wave minima of the two standing wave fields (WF1, WF2) have the distances di = λi / 2n cosθi or d ₂ = λ ₂ / 2n cosθ ₂ (where n = Refractive index in the object space, θi, θ ₂ = angle of intersection of the laser beam of the wavelength λi, λ ₂ with the optical axis) Since the distances di and d ₂ are generally different, the two wave fields are aligned per spatial direction so that a main maximum of both standing waves in the same place is multiphoton effects can occur only where both wave fields overlap thus be only individual ^ι strip '' of the wave field one spatial direction used for multiphoton excitation ambiguities with objects of dimension great d occur only in dimensions with the condition kιdχ = k ₂ d ₂ (ki, k ₂ are integers) on A multi-photon excitation of the fluorescence-marked measurement structures and calibration targets thus makes the three-dimensional image unambiguous. The use of two- or multi-photon fluorescence excitation methods enables the object to be scanned or scanned more quickly, ie Object points, lines and planes and thus a better image quality, especially with moving objects Another very advantageous development of the multi-dimensional wave field microscope according to the invention is characterized in that an arrangement of light source, lens and an electrically conductive mirror, which is suitable for generating a one-dimensional, electrical wave field, is provided relative to the object holder holder, in such a way that the Measurement structures and / or calibration targets located in the object can be aligned if necessary (e.g. if they are located on or in molecular chains) by applying the electric field - before or during the microscopic measurement process (the molecular chains that are spatially aligned in this way can then be aligned are still fixed with immobilizing substances) This variant of the wave field microscope according to the invention is particularly suitable for wave microscopic DNA sequencing, the one-dimensional electric wave field being used for calibration in the DNA sequence serving serves

For the detection of the luminescent light, a CCD camera (s) is preferably used in a known manner. This can sit behind the detector ring diaphragm or aperture plate or the detector slot. Instead of the CCD camera or the CCD chip, the multidimensional wave field microscope according to the invention can also be used with an electronic image recording device be equipped in the state of the art, for example of confocal laser scan microscopes (CLSM). In principle, any light-sensitive detection device, in particular photodiode (s), photomultiplier, CCD cameras / chips, CCD, Arrays, avalanche diodes, (avalanche) diode arrays, two-dimensional (avalanche) diode matrices are arranged behind which behind the detector ring diaphragm or aperture plate or the detector slot in order to detect and document the fluorescence, and fluorescence lifetime measurements can also be carried out

The calibration method according to the invention is a calibration method of the aforementioned type, which is characterized by the following measures (1) The biological object with the fluorochrome-marked measurement structures and / or the fluorochrome-labeled calibration targets is sequentially or simultaneously with individual (separate), standing in two or three spatial directions orthogonal to each other and interfering to a two- or three-dimensional wave field interfering with each other, the fluorochromes are excited to fluorescence emission

(2) To detect the fluorescence intensity, a camera and / or one or more two-dimensional arrangement (s) made up of individual detectors, each with a circular, ring or slot-shaped diaphragm or an arrangement of several circular, ring or slot-shaped diaphragms, are used

(3) Either the object with the measurement structures and / or the calibration target (s) or the one- or two-dimensional wave field or both is gradually rotated around one or two axes orthogonal to one another during the measurement process, the fluorochrome-marked measurement structures and / or calibration targets being sequential or are simultaneously illuminated with one or two individual, orthogonal wave fields

In the case of simultaneous illumination, the micro-object with the measurement structures and the calibration target (s) is mounted rigidly or rotatable about an axis. Two or three standing, plane wave fields that run orthogonally to one another are brought to interference and illuminate the micro-object simultaneously. Two wave fields create planes with a two-dimensional one symmetrical grid of points of maximum and minimum intensity When using three wave fields, a three-dimensional spatial grid of symmetrically regularly arranged points of maximum and minimum intensity is created. Between the intensity maxima and minima there is a continuous intensity curve. With sequential illumination, the micro object is moved around in the wave field two axes rotated During or after the detection, the position of the wave field relative to the object can be changed. When using suitable fluorescent markers, the invention thus allows three-dimensional (3D) geometris distance measurements between fluorescence targets of the same or different spectral signature with molecular precision, ie with a 3D resolution equivalent up to better than 10 nm and with a 3D localization accuracy up to better than 1 nm In contrast to electron microscopy or optical and non- optical near-field microscopy, the three-dimensional structure of the object to be examined remains intact, since mechanical cuts are omitted.Thus, in three-dimensionally preserved micro-objects, 3D distance measurements can be carried out in a range smaller than the half-width of the main maxima of the effective point image functions can also be carried out under vital conditions of the biological object. In DNA sequencing, the production of gels and the electrophoretic separation of the DNA pieces can be omitted. Likewise, autoradiography can be dispensed with, since no radioactive labeling is carried out, and long DNA sequences ( eg> 1 kbp) can be easily analyzed. This method variant according to the invention also allows a significantly improved determination of morphological sizes (eg volume, surfaces), provided that the multis spectral fluorescence markers can be distributed in a suitable manner, for example on the surface of the object. For example, the volume of a spherical micro-object with a radius of a few 100 nm can be determined considerably better than that with conventional ones using morphological segmentation techniques, such as Cavalieri and Voronoi procedure, or methods of volume preserving gradual thresholding is possible

With the multidimensional wave field microscope (type I and / or type II) and the calibration method according to the invention, it is possible to carry out microscopic DNA sequencing. For this, the "wave field microscopy method for DNA sequencing" described below is proposed

(1) All complementary sub-sequences of the DNA sequence to be analyzed are produced in such a way that all sub-sequences begin at the same nucleotide of the sequence to be analyzed.

(2) The fragments to be analyzed are all labeled at the 3 'end with a reference fluorochrome marker α and at the 5' end and / or at defined intermediate points with a fluorochrome marker a, g, c, or t - depending on whether the nucleotide has the Base adenine (Marker a), guanine (marker g), cytosine (marker c) or thymine (marker t) carries - marked, wherein the fluorochrome markers a, g, c, t and α have different spectral signature, and each contain one or more fluorochrome molecules

(3) The labeled DNA sub-sequences are fixed on supports in such a way that they are present as a linear sequence and introduced into a one- or multi-dimensional wave field microscope

(4) The linear DNA sub-sequences are oriented to the standing wave fronts in such a way that an exact distance measurement (accuracy <1 * 10 ^'10 m) between α and a or g, c or t - after determining the intensity bary centers and calibration of the imaging properties - can be done by

(5) the signals of the fluorochrome markers are recorded step by step, spectrally separated from one another, and

(6) the DNA base sequence of the DNA piece to be analyzed is determined from the distances between the fluorescent markers and their spectral signature

With this method, which is completely new in the prior art, the length of DNA fragments can be measured with nucleotide precision, and their base sequence can also be determined exactly. Gel electrophoresis and subsequent band evaluation can be omitted

Exemplary and comparative examples for a more detailed explanation of the invention:

EXAMPLE 1: Construction of a multidimensional type I wave field microscope with a rotatably mounted object

The basis is a conventional "one-dimensional" wave field microscope, which is constructed, for example, with two opposing high numerical aperture lenses or with a high lens with a low numerical aperture lens or with a lens for two interfering laser beams. The lenses make two partial beams of a laser so that a one-dimensional sional standing wave field is generated. The fluorescence is detected via one or two objective (s) with a high numerical aperture. In each case in one or in both orthogonal directions to the optical axis of the detection objective, two further partial beams of the laser are obtained via objectives with a lower numerical aperture and / or focusing lens systems coupled in at a suitable distance and brought into interference with one another and with the one-dimensional standing wave field in such a way that a two- or three-dimensional symmetrical intensity pattern consisting of intensity maxima and minima arises

A microaxial tomograph is installed in this "multidimensional" wave field microscope for object storage

In axial tomography, a microcapillary or a glass fiber is used instead of the glass slide, which is rotatably mounted and accommodates the (biological) object (capillary) or to which the (biological) object is appropriately applied (capillary / fiber) manually or With a computer-controlled stepper motor, the capillary / fiber, which is usually arranged perpendicular to the optical axis of the detection objective, can be rotated around the fiber axis by a defined angle. Rotation through an angle of 360 degrees (2π) is possible. The carrier holder for the capillary / fiber is rotatably supported on a semicircle The axis of rotation is perpendicular to the optical axis of the detection objective

The spatial arrangement of the microtargets and their distance are detected by means of the calibration method according to the invention and digital image analysis. Ambiguities in intensity profiles, ie, for example intensity main and secondary maxima of fluorescent “point” targets, can be statistically evaluated with the aid of suitable computer algorithms and thus increase contribute to localization precision In the case of extended objects, ambiguities can be reduced by excitation with two or more photons with photons of different wavelengths EXAMPLE 2: Distance measurement between gene sections of chromosomes in a cell nucleus by means of multidimensional wave field microscopy, calibration method according to the invention and, if necessary, axial tomography

(I) In a cell nucleus, the chromatin of the individual chromosomes occupies defined regions. Within one or more such chromosomal regions, the structures to be located, ie the measurement structures, e.g. small chromosome sections such as genes or parts of genes, are determined using a method known in the art the fluorescence in situ hybridization specifically marked, namely with fluorochromes of various specific spectral signatures Mi, M ₂ , M ₃ , the distances between the marking locations (the marked measuring structures) are below the classic resolution, i.e. they are smaller than the half-width of the main maximum of effective point image function The (object) structures (measurement structures) are marked in such a way that the spectral signatures on the structures (measurement structures) to be located are represented with almost the same dynamics

The biological object is prepared on a glass fiber with a precisely defined diameter or in a round or rectangular capillary of defined dimensions

(II) In order to determine the distances, microscopic specimens with calibration targets are produced under the same physical and chemical test conditions as the object or the object structures to be located

(= Measuring structures)

A microinjectable spheres of a spectral signature (monochromatic) are used as calibration targets or as preparations with calibration targets. The spheres are labeled with a fluorochrome, ie monochromatically, according to known methods, and because of their size they are marked by the structures in the object (the measurement structures) to be measured (to be located). distinguishable Such calibration spheres are injected, which represent the spectral signatures of the measurement structures present in the object, but are otherwise preferably identical (in terms of size, geometry, material properties, etc.) In other words, the spectral signatures The measuring structures and the calibration targets are selected so that the fluorescence emissions they emit can be analyzed separately from one another under the given examination conditions.The monochromatic calibration spheres are injected and fixed in such a way that the individual spheres of different spectral signature are clustered directly on the glass fiber surface Arrange or capillary wall, preferably in a cross-sectional plane of the fiber or capillary When using precision fibers and / or capillaries, the spheres are consequently at defined distances from one another or from a reference plane, reference axis or reference line

b) micro-injectable test balls with multispectral signature (polychromatic) and the same spectral dynamics

The spheres are marked according to known methods with all spectral signatures occurring in the marked (object) structures (measuring structures) .As a result, they can be injected into any place in the biological object to be measured (here nucleus). A target geometry as in a) is not necessary , since the chromatic focal points should be located at the same point for each signature. In order to distinguish them from the marked (object) structures (measuring structures), the spheres can either belong to a different size class or have an additional spectral signature that is associated with the structures to be measured ie the measuring structures (according to the preparation protocol) do not occur

c) Simultaneously marked chromosome regions of known distance on a chromosome other than the one that carries the structures to be located (measuring structures). The calibration targets, i.e. here the chromosome regions with a known distance from one another, are made with the aid of a sample combination of DNA sequences that contain the different spectral signatures carries, marked differently A differentiation of the chromosomal calibration targets from the (chromosome) structures (measurement structures) to be located can be made, for example, by different fluorescence intensity or by a different intensity ratio between fluorochromes with different spectral signatures or by using an additional fluorochrome with a different spectral signature, which was not used in the fluorescent labeling of the measurement targets

It is also possible for the calibration targets to belong to a different size class than the measurement structures to be located

(III) The distance measurements are carried out with a multi-dimensional wave field microscope according to the invention, combined with a photomultiplier and / or camera and data processing system. A series of optical sections is taken from the biological micro-object, here a cell nucleus in the example. The measurement structures Mi, M ₂ , M ₃ , Mi have / = 1,2, JL spectral signatures The spectral signature of the calibration targets Ui, U ₂ , U ₃ ,, Uι differ from that of the measuring structures eg in volume, diameter, intensity or in the number of spectral signatures (/ = 1,2, JL +) The images of the optical sections are recorded separately for each spectral signature and, if necessary, the background is corrected. For evaluation, the calibration targets are identified and the chromatic offset is determined. For this purpose, the calibration targets are located under each spectral signature and the distances between the calibration targets with fluorochrome marking measured against different spectral signatures The measured locations (ie the measured target distances) are compared with the target locations calculated on the basis of the geometry (ie the actual target distances) and the spectral shift (shift) is determined therefrom

This shift is the calibration value for the measured distance values between the (object) structures to be located (measurement structures)

Since this offset depends on the optical properties of the preparation (e.g. refractive indices in the cores and the preparation medium), the calibration should be carried out in situ.This means in the present example that the calibration targets are next to the (chromosomal) structures to be examined and marked (measuring structures) ) should be at the core

On the other hand, the distances between the (object) structures (measurement structures) to be located are localized. In each spectral signature, the position of the centers of gravity of the measured intensity signals is first determined independently of one another. le, i.e. the distances between the different color signals or color points of the measurement structures in question, e.g. between the red fluorescent and the green fluorescent color point (from intensity maximum to intensity maximum or from center of gravity / bary center to center of gravity / bary center) are measured, and this measured value is measured with the the calibration targets determined (due to the different spectral signature) corrected in a highly precise manner. The corrected positions of the measurement structures are given in relation to a comparison point. This comparison point can, for example, be any fixed point in the object or the center of gravity of a calibration target (eg a marked chromosome region) or an otherwise marked chromosome territory. It can also be the center of gravity coordinate of all measurement structures within a chromosome territory

In the case of calibration targets in the form of microinjectable test spheres with a multispectral signature (polychromatic), the chromatic offset is determined from the difference in the location of the focal points for each signature. The necessary identification of the fluorescence emission belonging to a calibration target can be carried out, for example, by volume-preserving threshold values or by averaging the segmentation results with threshold value variation.

In the case of calibration targets in the form of fluorochrome-marked object regions with a multispectral signature (polychromatic), the chromatic offset is determined in the same way.

Centromere regions which are hybridized with a sample combination of such DNA sequences which all bind to the same chromosomal DNA sections but are labeled with fluorochrome with different spectral signature are also particularly suitable as fluorochrome-marked calibration regions. If the hybridization takes place under highly stringent conditions, there are two labeling regions per cell nucleus; in the case of low-stringent conditions, additional centromer regions are marked due to additional binding regions, so that the number of calibration regions increases. This can be very advantageous (IV) The measurement methods described can also be carried out in combination with axial tomography. For this purpose, the biological micro-object, for example a cell nucleus, in which the measurement structures to be located are already marked with fluorochromes and which also already contain calibration targets (for preparation see Example 1), arranged on a glass fiber or in the microcapillary With the axial tomograph, the object is rotated step by step through a defined angle with automatic refocusing if necessary. A complete 2D or 3D image stack is recorded for each angular step

The rotation takes place in such a way that a distance between two measurement structures or calibration targets (ie between their fluorescence intensity focal points) becomes maximum. The maximum measured distance corresponds to the actual distance

If you are only interested in the distances between the measurement structures or calibration targets, i.e. not in their absolute spatial arrangement, you can now proceed from one of the known measurement structures or calibration targets to maximize and determine the distance to a third measurement structure or a third calibration target the distances between the measurement structures or calibration targets are greater than the full width at half maximum of the main maximum of the point image function, so a single spectral signature is sufficient; if the distances are smaller, the measurement structures or calibration targets must be differentiated by means of a multispectral signature. The focal points (maxima) of the signals are used for localization if the investigated measurement structures have a diameter that is smaller than the half-width of the main maximum of the effective point image function, all diffraction images of the measurement structures or calibration targets are determined by a sharp point image function, so that ate the maxima can be determined optimally

If you are interested in the absolute arrangement of the measurement structures or calibration targets in space, the respective focal points (so-called "bary centers") must be precisely determined. Repeating the entire measurement procedure and statistical evaluation several times can result in the absolute localization of the measurement structures or calibration targets, ie the angle measurement, be improved Instead of performing the calibration and the distance measurement between the structures to be located, ie measurement structures, in the same biological object as described above, one can also carry out the calibration independently of the measurement structures on biological objects of the same type. In this method variant, the distinction between the fluorescence signals is Calibration targets and those of the measurement structures easier Using the values of the optical offset determined with the calibration targets, calibration curves can be created for the distance measurements between the measurement structures. Such calibration curves provide, for example, information about the spectral offset as a function of the refractive index and absorption of the immersion medium used, the optics used, Filters and detection units, the evaluation algorithms used, the biological objects used, the location of the measurement structures or calibration targets in them, etc. The use of the information from These special calibration curves for distance measurement according to the invention are particularly suitable in cases in which a greater precision tolerance is permitted

EXAMPLE 3: Examination and representation of three-dimensionally extended objects by means of multidimensional wave field microscopy, calibration method according to the invention and simultaneous image acquisition

A 3-D data record is usually obtained from a biological micro-object by sequential registration, e.g. in confocal laser scanning microscopy by point-by-point or line-by-line scanning of the 3D object volume; a second method is based on the registration of the fluorescence emission from the object plane by positioning a detector array in the to the object plane conjugated intermediate image plane, the position of this intermediate image plane is usually fixed, in order to obtain SD information about the biological object, it is moved sequentially through the object plane conjugated to the fixed intermediate image plane, each time a 2D image data record of the fluorescence emission in question is registered, and / or the object is rotated by means of axial tomographic methods through different angles of rotation, each time 2D data sets of the conjugate to the fixed intermediate image plane Object level can be registered

This sequential registration of object points, object lines or object planes has various disadvantages.For example, bleaching the registration of the 3D data record can lead to a shift in the 3D focal points of the fluorescence distribution of marked object points of a certain spectral signature determined according to the invention. Another disadvantage is that the 3D data acquisition is not fast enough to ensure that even with non-permanently stable, ie moving objects, in particular with in vivo markings such as fluorescence-labeled chromosome regions in nuclei of living cells, which move under physiological conditions at speeds of up to a few nm / sec ( medium shift) can ensure a satisfactory image quality

In order to obtain a satisfactory image quality even with moving objects, the present invention provides for a simultaneous recording of the 3D data record of a fluorescence-marked object.For this purpose, the fluorescent light emitted by the object of a given spectral signature is split into N partial beams by optical elements, e.g. splitter mirrors and mapped onto N detector arrays, which are located in N different intermediate image planes that are conjugated to N different object planes.A simple estimate based on the mapping equation shows that the distances between the intermediate image planes (detector planes) with simultaneous registration of an object area with 10 μm axial extension and a lens of conventional image width and high numerical aperture, for example, have to be varied by an area in the order of magnitude <20 cm (depending on the lens used). With N additional intermediate optics, those for high-precision distance measurements in r N object planes to be registered are also mapped to different areas of the same, suitably dimensioned detector array (or to L <N detector arrays). In this case, a specific section of the L detector array (s) corresponds to one of the N conjugated object planes. For example, a small object area becomes a few μm extension for measurement in wave field microscopy initially positioned so roughly that its center of gravity lies approximately in the center of the observation volume of the microscope objective used for registration given by the detection point image function for a specific (intermediate) image plane B ₀ , The fluorescent light emanating from the 3D object is separated according to its spectral signatures and split into N partial beams, which are imaged on N detector arrays (eg 8 x 8, 16 x 16 or 64 x 64 pixel size), the positioning of which registers the fluorescence emission from N conjugated object planes around the maximum of the detection point image function (based on the image plane B ₀ ) is permitted.For example, with simultaneous recording of objects with a distance of 20 nm each, according to the above assumptions, a shift of the conjugated intermediate image planes by a few 100 μm (in close to the maximum of the point image function) or correspondingly small optical individual corrections are to be carried out with simultaneous detection of the relevant object sections on a single (or L <N) detector array (s) of corresponding pixel numbers With a division of the fluorescence emission, for example, into N = 20 partial beams same intensity the number of photons counted by each of the N = 20 detector array (s) (or - excerpts) per unit of time is reduced by approximately the same factor an increase in the registration time by a factor of N (e.g. N = 20) can be eliminated In this case, the simultaneous registration of the object takes approximately the same time as with sequential registration. For objects with bleaching behavior, however, the advantage of simultaneous three-dimensional registration is one for all targets (one given spectral signature) of the observation volume similar (or similar) bleaching behavior, shifts in the center of gravity of the fluorescence emission image caused by fading are thereby reduced. For objects with a temporally dynamic structure (eg marked cells in viv o) if the acquisition time is reduced by a factor of N compared to sequential registration (eg 1 sec instead of 20 sec), the localization accuracy of the individual object points is estimated to be reduced by the factor \ N (for N = 20 z B 4 5)

For example, with a 3D localization accuracy in the wave field microscope of approx. ± 3 nm, obtained with sequential acquisition times of 1 sec per image plane, under the conditions mentioned with simultaneous registration (1 sec), the localization accuracy is reduced to an estimated ± 4 5 • 3 nm « 14 nm with simulta- ner recording of 20 object planes under otherwise identical conditions With an assumed (for example) object movement (mean shift) of 5 nm / sec, the actual localization inaccuracy would be considerably great with sequential recording with a total registration time of 20 seconds even without taking bleaching effects into account here the described simultaneous registration of object planes not only with respect to an optical axis, but also with regard to two and three orthogonal optical axes

If necessary, this simultaneous image recording according to the invention can be easily combined with a conventional sequential image recording

EXAMPLE 4: DNA sequencing using multidimensional wave field microscopy

Using known methods, such as the polymerase chain reaction, all complementary sub-sequences of the DNA sequence to be analyzed are produced. The sub-sequences all begin at the same nucleotide of the sequence to be analyzed. The fragments to be analyzed are all marked at the 3 'end with a reference fluorochrome marker α at the other end , the 5 'end, or at defined intermediate positions, they are each marked with a fluorochrome marker a, g, c, t of different spectral signature, depending on whether the nucleotide contains the base adenine (marker a), guamn (marker g), cytosine ( Marker c) or thymine (marker t) carries

All types of the fluorochrome markers used differ in their spectral signature in such a way that the fluorochrome markers α, a, g, c, t (if necessary, even more) can be detected separately from one another; according to the invention, a specific fluorochrome marker can be used by one to many fluorochrome molecules of the same or different ones Type are formed, the length and composition of the fluorochrome markers being selected according to the invention such that distance measurements between the centers of gravity of the intensity distributions of the initial fluorochrome marker α and terminal fluorescence markers (either a, or g, or c, or t, or, if appropriate, further bases) is possible with the method of multidimensional wave field microscopy, ie linker molecules for fluorescent markers, for example be shorter than 1/2 nucleotide diameter According to the invention, the use of longer linker molecules is also possible, provided that they have a configuration of such a high stiffness that the distance variation caused by them is sufficiently small, for example <1/2 nucleotide diameter

These sub-sequences marked in this way fully represent the DNA sequence to be analyzed. The fluorescent markers a, g, c, t or the reference fluorochrome α can each contain one or more fluorochrome molecules. The DNA sub-sequences are all fixed on suitable supports so that they are present as a linear sequence

The fluorochrome-marked DNA pieces are aligned linearly with the aid of DNA combining techniques. In contrast to the conventional "one-dimensional" wave field microscope, in the multi-dimensional wave field microscope according to the invention an additional precise alignment of the DNA strand in the direction of the optical axis of the system is no longer necessary. The DNA Sequences are preferably applied to a square glass fiber, the refractive index of which differs minimally from that of the surrounding medium, the alignment taking place at a certain angle, in particular orthogonal to the axis of the glass fiber, and the mean distance between the centers of gravity of the DNA sequences is greater than the maximum full width at half maximum of the point image functions used to register the fluorescence signals. Another method binds the 3 'end to a microsphere with the spectral signature α and then stretches the DNA thread with the tool of optical tweezers, the "tweezer laser" must be selected in its wavelength

After linearization or orientation of the DNA sequences, the preparation prepared in this way is fixed and the molecular movement is reduced, for example by lowering the temperature. Alternatively, the DNA ends can also be embedded in a crystalline solid structure. For the purposes of calibration, further, in particular, were used for calibration purposes polychromatic micro-objects placed on the glass fiber, the DNA slide or in the fixation solid of the DNA ends. The calibration objects additionally contain a spectral signature that is not a, t, c or g. The linearization of the DNA strand can be omitted if all nucleotides are at the syn thesis of the DNA complementary strand to be analyzed are suitably fluorescence-labeled

The fixed DNA sequences are introduced into a multidimensional wave field microscope, the linear DNA sub-sequences being oriented to the standing wave fronts in such a way that an exact distance measurement (accuracy <1 "10 ^{" 10} m) between oc and a or g, c or t - after determining the intensity barary centers and calibrating the imaging properties - is possible

The measurement is carried out in in-situ calibration using the calibration method according to the invention. The signals of the fluorochrome markers are recorded spectrally separated from one another in the wave field microscope with suitably adapted step sizes (preferably in a digital manner). The DNA base sequence of the Determine the DNA piece to be analyzed

Instead of a spectral separation or in addition to it, fluorescence lifetime parameters can also be analyzed. In a first phase of the evaluation, a rough determination of the focal points of the fluorochrome marker signals is carried out and, based on this information, the signals belonging to a DNA sequence according to the above-mentioned distance criteria signals belonging to another DNA sequence are separated, in a second phase of the evaluation the spectrally separated, modulated signals of the fluorochrome markers are analyzed with the aid of suitably adapted functions, from this the distances e.g. the focal points of the initial and terminal fluorochrome marker signals are analyzed a DNA sequence is determined from one another with molecular precision, the measurements made on the calibration objects being used to correct distance aberrations, for example chromatic shifts, in a third phase of the evaluation di e The distances of the fluorochrome marker signals corresponding to the lengths of the DNA sequences are sorted according to the length by the type of the terminal fluorochrome marker (e.g. B a, g, c, t), the arrangement achieved in this way corresponds to the pattern achieved in a conventional method, the then the desired sequence information can be obtained by known methods. Macromolecules with a linear sequence or known ordered structure are used analogously, the number and type of fluorochrome markers depending on the number and type of molecular building blocks

If the measurement of individual DNA strands necessitates their alignment in the direction of the optical axis, the procedure according to the invention can be followed as follows. The known end of the DNA chain is marked with a chemical "marker" in addition to a fluorescent marker of the spectral signature α. The rest of the DNA - Chain preparation, in particular the labeling of the terminal fluorescent markers a, c, g and t (stop nucleus) is carried out as described at the beginning. The terminal bases and / or the markers of the stop nucleotide and possibly further bases of the DNA chains to be aligned carry an electrical charge (e.g. negative)

The alignment of the DNA chains can take place before or during microscopy, first the method of alignment before microscopy is described

The prepared DNA chains are applied in solution in a buffer of low ionic strength to a specially coated slide (or cover glass, which will also be referred to below as a slide), which bind ("glue") the chemically labeled 3 'end of each DNA chain ) An immobilizing component is now added to the solution, which causes the DNA chains in solution to harden after a certain time. The slide is covered and sealed with an (uncoated) cover glass, the distance from the slide to the cover glass being covered with a suitable spacer "(eg a thin membrane) can be set to a well-defined value. The slide sealed in this way is exposed to a suitable homogeneous, static electrical field which aligns the electrically charged bases. The polarity of the electrical field (for example that of a capacitor) must be applied in this way be that with negatively (positively) charged bases the cathode (anode) is on the coated slide (with the "glued" 3 'ends) and the anode (cathode) is on the uncoated cover glass. The electric field lines are perpendicular to the slide surface and the strength of the electric field is chosen to be high enough to be a Align the DNA chains in the solution After the DNA chains aligned in this way have been immobilized in the intermediate space between slide and cover glass, they are recorded or measured with the multidimensional wave field microscope as described above

If the orientation of the DNA chains is to take place during the microscopic image, the electrical field described above is constructed according to the invention by the following method.

The coated slide is an electrically conductive mirror of sufficient flatness. The solution of the DNA chains (low ionic strength) is suitably viscous and no immobilizing components are added to the solution. Here too, a cover glass is applied (if necessary using a suitable spacer) and sealed. The wave field is now generated with only one lens in conjunction with the mirror, the excitation light emerging from the lens being reflected by the mirror and a one-dimensional wave field (parallel to the focal plane or to the mirror surface) being formed. The application of a positive (negative) voltage when using negatively (positively) charged bases also brings about an alignment of the DNA chains perpendicular to the surface of the mirror, i.e. along the optical axis of the microscope and which can now be sequenced as described above In this process, the strength of the electric field must be large enough to cause the alignment of the DNA chains; thermal movements of the molecules can e.g. can be reduced by lowering the temperature.

The same procedure can be used with any other linearized macromolecule.

EXAMPLE 5: Multi-dimensional type H wave field microscope with lateral spatially modulated fluorescence excitation

The multi-dimensional wave field microscope type II comprises in the spatial directions x a real illumination source for coherent light beams, a beam splitter for coupling out partial beams as a virtual illumination source, and a first lens that is assigned to these two illumination sources In this first objective, the light beams or light wave trains of the illumination sources are coupled in such a way that they generate or have two spaced apart focal points on the rear focal plane facing away from the object space (this plane is also called "back focal plane") and in the space between the two focal planes run at a certain angle to each other and interfere with a one-dimensional standing wave field

If you focus a light beam (light wave train) with a suitable distance to the optical axis in this focal plane (the optical axis is perpendicular to the focal plane and runs through its center), a parallel bundle of light with flat wavefronts leaves the lens in the object space, namely under one Specific angle to the optical axis This angle can be set variably, depending on the distance from the optical axis and the light rays are coupled into the lens and what type of lens it is

If the second light beam (light wave train) is coupled into the same lens at such an angle to the first and to the optical axis that its focus point on the rear focal plane lies diametrically opposite the focus point of the first light beam, i.e. focus point 1 - optical axis - focus point 2 form a line on the rear focal plane, a second parallel bundle of light with plane wavefronts is created in the space between the two focal planes, which runs at a certain angle to the first and interferes with it in the object space to form a one-dimensional standing wave field with stripes of maximum light intensity

The first lens has a second lens with a mirror image at a distance from it, so that these two lenses are on two opposite sides of the three-dimensional object space.This second lens is assigned a third and a fourth (real or virtual) illumination source for coherent light beams in such a way that As described for the first lens, the light beams from both illumination sources can focus on the rear focal plane of this second lens, ie, facing away from the object space, in the space between the two focal planes of this second lens to form a one-dimensional standing wave field. can ferieren, and in the object space with the one-dimensional standing wave field of the first lens to interference so that a three-dimensional wave field arises with points of maximum intensity that continue in three-dimensional space

For the generation of a two-dimensional wave field (i.e. points of maximum intensity in one plane), the structure described is modified to the extent that either the first and second lenses are used, but one is only combined with an illumination source, or only a single lens is used and combines this with a third illumination source, whose coherent light rays are coupled to the light rays of the other two illumination sources in this single objective in such a way that the corresponding three focus points on the focal plane form an isosceles triangle, through the center of which the optical axis runs. All three leave the object space The microscope lens beams light at the same angle to the optical axis but each in a different direction

This variant of the type II wave field microscope according to the invention for generating a multidimensional wave field microscope using a single objective can also be used for generating a three-dimensional wave field. For this purpose, four light beams are directed into the same objective and in such a way that the corresponding four focal points in the rear focal plane are equilateral Form a square

Claims

Expectations

Wave field microscope with an illumination or excitation system, which comprises an illumination source, a first lens and a second lens or a reflector, the first lens and second lens or reflector being arranged in relation to one another in such a way that they are suitable for generating a one-dimensional standing wave field, with a Object space, which comprises holding and maneuvering device (s) for an object, and with a detection system, which comprises an objective, an eyepiece and a detector, characterized in that the lighting or excitation system in two or all three spatial directions at least one real or virtual Illumination source for coherent light beams and at least one reflector or beam splitter for coupling out partial beams or a further illumination source for coherent light beams, each of which is assigned at least one lens and which are each suitable for generating light wave trains, wherein the light wave trains of one illumination source are aligned antiparallel or at variably adjustable angles to the light wave trains of the reflector or the other illumination source, such that the light wave trains emitted by the one illumination source interfere with those of the reflector or the other illumination source to form a standing wave field with flat wave fronts , and that the detection system comprises at least one detection lens suitable for epifluorescent detection and / or at least one suitable for scanning point detection, preferably a high numerical aperture, which is arranged with its optical axis perpendicular to the wave fronts of one of the interfering wave fields, which also includes a lens of the Excitation system can be identical, the detection lens suitable for epifluorescent detection being preceded by a flat (two-dimensional) detector, for example a camera, and the point to be rasterized Detection suitable detection lens at least one fixed confocal detection ring diaphragm and / or pinhole and / or at least one fixed detection slot and a point detector, in particular a photomultiplier, a photodiode or a diode array is arranged Wave field microscope according to claim 1, characterized in that a lens with a high numerical aperture is assigned a lens with a low numerical aperture or a reflector in at least one spatial direction, and either two lenses with a low numerical aperture or a lens with a low numerical aperture are assigned in one or both other spatial directions Aperture and a reflector are assigned to each other.

A wave field microscope with an illumination or excitation system, which comprises an illumination source and a lens, which are arranged with respect to one another in such a way that they are suitable for generating a standing wave field, with an object space which comprises holding and maneuvering device (s) for an object, and with a detection system comprising an objective, an eyepiece and a detector, characterized in that the illumination or excitation system has at least one real or virtual illumination source for coherent light beams and at least one beam splitter for coupling out at least one partial beam in at least one of the three spatial directions , to which a common lens is assigned, into which the light beams or light wave trains of the illumination source (s) and the beam splitter (s) can be coupled in such a way that they have two focal planes spaced apart on the rear focal plane (facing away from the object space) and in which room run between the two focal planes at a variably adjustable angle to one another and interfere with a one-dimensional standing wave field, and that the detection system comprises at least one detection lens suitable for epifluorescent detection and / or at least one detection lens suitable for scanning point detection, preferably a high numerical aperture, which can also be used with a lens of the Excitation system can be identical, the detection objective suitable for epifluorescent detection being preceded by a flat (two-dimensional) detector, for example a camera, and the detection objective suitable for scanning point detection being preceded by at least one fixed confocal detection ring aperture and / or pinhole aperture and / or at least one fixed detection slit and a point detector, in particular a photomultiplier, a photodiode or a diode array, is arranged downstream Wave field microscope according to claim 3, characterized in that the illumination or excitation system in the same or one or both other spatial direction (s) each has at least one further real or virtual illumination source for coherent light beams and / or at least one beam splitter for coupling out at least one partial beam, which is each assigned a further lens through which the light beam (light wave train) is directed into the object space and aligned in such a way that it can be combined with the light beam from the same or from the other two spatial direction (s) or from interfere with this one- or two-dimensional wave field to a two- or three-dimensional wave field.

Wave field microscope according to one of claims 1 to 4, characterized in that the object space comprises an object holder, in or on which the object with the measurement structures and / or optionally calibration target (s) can be rotated in the wave field about one or two orthogonal axes is mounted, with a rotatability through 360 degrees (2π) being preferred for at least one axis.

Wave field microscope according to one of claims 1 to 5, characterized in that the illumination source (s) generating the multidimensional wave field and / or the reflector (s) and / or the beam splitter and / or the objective (s) and thus the multidimensional one The wave field is / are rotatable about one or two orthogonal axes.

Wave field microscope according to one of claims 1 to 6, characterized in that a scanner mirror is provided in the detection system and is arranged such that it is suitable for imaging the lateral object areas with the desired, preferably maximum, fluorescence intensity. Wave field microscope according to one of claims 1 to 7, characterized in that the illumination system in at least one of the three spatial directions a real illumination source for the two or multi-photon excitation and in one or both other spatial direction (s) a real and / or virtual illumination source for the two - or multi - photon excitation, and that the standing wave fields (WFi, WF ₂ ,, WF,) generated with it differ from each other different wavelengths (λi, λ ₂ λ,) and distances (di, d ₂ ,, d,) between the respective wave maxima or Have wave minima of di = λi / 2n cosθi or d ₂ = λ ₂ / 2n cosθ ₂ or d, = λ 2n cosθ, (with n = refractive index in the object space, θi, θ ₂ , θ, = crossing angle of the light wave train of the wavelength λi, λ, λ, with the optical axis), and wherein the wave fields WFι, WF ₂ W, are aligned with each other in such a way that at least a maximum of two or all standing waves at the same point (namely the Or t of a multi-photon excitation)

Wave field microscope according to one of claims 1 to 8, characterized in that an arrangement of illumination source, lens and an electrically conductive mirror, which is suitable for generating a one-dimensional, electrical wave field, is provided relative to the object support preservation, in such a way that the in measurement structures and / or calibration targets located on the object can be aligned by applying the electrical field - before or during the microscopic measurement process

Wave field microscopy method for DNA sequencing using a wave field microscope according to one of claims 1 to 9, characterized by the following process steps, all complementary sub-sequences of the DNA sequence to be analyzed are produced in such a way that all sub-sequences begin on the same nucleotide of the sequence to be analyzed, the ones to be analyzed Fragments are all at the 3 'end with a reference fluorochrome marker α and at the 5' end and / or at defined intermediate points with a fluorochrome marker a, g, c, or t - depending on whether the nucleotide is the base adenine (Marker a), guanine (marker g), cytosine (marker c) or thymine (marker t) carries - marked, wherein the fluorochrome markers a, g, c, t and α have different spectral signature, and each contain one or more fluorochrome molecules , The labeled DNA sub-sequences are fixed on supports in such a way that they are present as a linear sequence and introduced into a multidimensional wave field microscope, the linear DNA sub-sequences being oriented in relation to the standing wave fronts in such a way that an exact distance measurement (accuracy <1 <10 ^"10 m) between α and a or g, c or t after determining the intensity bary centers and calibrating the imaging properties can be carried out by recording the signals of the fluorochrome markers step by step, spectrally separated from each other, and from the distances between the fluorescent markers and their spectral signature DNA base sequence of the DNA piece to be analyzed is determined

Calibration method for multidimensional wave field microscopy according to one of Claims 1 to 10, in which the object structures to be examined or located are located before, during or after the preparation of the object in question on or in an object holder, in particular object carrier plates, object carrier fibers, object carrier capillaries or object carrier fluids - is the same as measuring structures - are marked with fluorescent dyes of different or the same spectral signature, whereby at least those measuring structures to be located whose distance from one another is less than the half-value width of the main maximum of the effective point image function are marked with fluorescent dyes of different spectral signature, using the same fluorescent dyes to define calibration targets Large and spatial arrangement are marked, the fluorescent calibration targets either together with the objects or measurement structures or separately on or in the / an object be prepared

Measurement structures and calibration targets are examined microscopically under the same conditions, simultaneously or one after the other, and in each case two defined calibration targets with different spectral signatures taking into account the wavelength-dependent imaging and localization. behavior of the respective optical system are measured, the measured values determined - equal to actual values - are compared with the known actual distance values - equal to target values - and a correction value from the difference between the actual values and target values - is equal to the calibration value - Is determined with which the offset caused by the optical system in the detection of different emission loci, in particular the measurement structures, is corrected, characterized in that the biological object with the fluorochrome-marked measurement structures and / or fluorochrome-labeled calibration target (s) sequentially or simultaneously with individual ones (Separate), standing in two or three spatial directions orthogonal to each other and interfering to a two- or three-dimensional wave field interfering with each other, the fluorochromes are excited to fluorescence emission, that to detect the fluorescence intensity a camera and / or one or more two-dimensional arrangement (s) of individual detectors, each with a circular, ring-shaped or slit-shaped diaphragm, or an arrangement of several circular, ring-shaped or slit-shaped diaphragms, is used that either the object with the measuring structures and / or calibration target (s) or the one- or two-dimensional wave field or both is rotated step by step around one or two axes running orthogonally to one another during the measurement process, the fluorochrome-marked measurement structures and / or calibration targets being sequential or simultaneous with one or two individual, orthogonal to one another standing wave fields are illuminated