WO2006120386A1 - An optical system for laser excitation and collection of fluorescence emissions - Google Patents

An optical system for laser excitation and collection of fluorescence emissions Download PDF

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Publication number
WO2006120386A1
WO2006120386A1 PCT/GB2006/001612 GB2006001612W WO2006120386A1 WO 2006120386 A1 WO2006120386 A1 WO 2006120386A1 GB 2006001612 W GB2006001612 W GB 2006001612W WO 2006120386 A1 WO2006120386 A1 WO 2006120386A1
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Prior art keywords
sample
optical system
excitation
light
focusing
Prior art date
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PCT/GB2006/001612
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French (fr)
Inventor
Stanley Desmond Smith
Original Assignee
Iti Scotland Ltd
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Publication of WO2006120386A1 publication Critical patent/WO2006120386A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0208—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/42—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/42—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect
    • G02B27/4205—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect having a diffractive optical element [DOE] contributing to image formation, e.g. whereby modulation transfer function MTF or optical aberrations are relevant
    • G02B27/4216—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect having a diffractive optical element [DOE] contributing to image formation, e.g. whereby modulation transfer function MTF or optical aberrations are relevant correcting geometrical aberrations
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/42—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect
    • G02B27/4272—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect having plural diffractive elements positioned sequentially along the optical path
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy

Definitions

  • the present invention relates to a system for focusing, controlling and collecting light beams for both excitation and emission of fluorescence from a sample, and in particular a biological sample.
  • Figure 1 shows a typical optical system for excitation and collection of fluorescence emissions from a sample in a plate reader 10.
  • This has a diode laser 12 for emitting light at a wavelength suitable for stimulating sample fluorescence, an interference filter 14 in front of the laser 12 for removing unwanted wavelengths, a dichroic beam splitter 16 for directing filtered light from the laser 12 towards a well 18 of the plate reader 10, and a focusing lens 20 for focusing light into the sample well 18, and so onto a sample contained therein.
  • Fluorescent light emitted by the sample, at a shifted wavelength, in response to excitation by the laser 12 follows a collection path to a detector 22.
  • the focusing lens 20 On the collection path is the focusing lens 20, the dichroic beam splitter 16 and a collector arrangement 24.
  • the beam splitter 16 is transparent at the wavelength of the fluoresced light so that such light passes through it and along the collection path onto the detector 22.
  • the collector 24 has in sequence an interference filter 26 and a collection lens 30 for focusing the fluoresced light onto the detector 22.
  • the fluorescent emission from the sample must be distinguishable from both the laser excitation and any stray auto fluorescence from the biological materials and importantly background fluorescence from the various optical components, particularly the focusing and collection lenses 20 and 30 respectively.
  • background fluorescence can be minimised by choice of material.
  • fused silica lenses emit less background fluorescence than corresponding glass lenses, as shown in Figure 2.
  • Another option is to make the optical components as thin as possible. However, this is not practical for some applications, such as applications that require high spatial resolution. This can cause problems where the amount of light emitted by a sample is very low, as the real fluoresced signal may be swamped and so indistinguishable from background fluorescence from the optical components.
  • an optical system for exciting a sample using light transmitted along an excitation path and collecting fluoresced light emitted from that sample along a collection path, the system having focusing means for focusing excitation light onto the sample, and a collector for collecting light emitted from the sample and directing it towards a detector, wherein the focusing means comprises a diffractive optical element.
  • This focusing diffractive optical element consists of a surface relief multi-level structure in the form of steps of concentric rings of (say) fused silica, the steps typically having a height of a fraction of a wavelength.
  • the diffractive optical element may lie on both the excitation and collection paths.
  • diffractive optical elements can be made very thin.
  • the thickness of a diffractive optical element for use in the present invention could be of the order of a few hundred microns, instead of 5- 10mm, as would be required for a conventional lens. This corresponds to a reduction of volume of material of up to one hundred times. In practice, this means that the background fluorescence generated by the focusing element is significantly less than would be the case if a conventional lens were used. For some applications this is highly advantageous.
  • Diffractive optical elements have been used in fluorescence microscopes and spectrometers before. Examples are described in WO00/40935, WO00/58715, WO01/63260, WO03/029768, WO2005/017598, and WO2005/0063282.
  • a diffractive optical element essentially as the objective lens for focusing light onto a sample. Instead, these elements are more conventionally used merely for beam shaping or conditioning.
  • the collector may include a diffractive optical element for focusing fluoresced light towards the detector. By using a second diffractive element, again in place of a conventional lens, further reductions in background fluorescence can be achieved.
  • the system may include a light source.
  • the light source may be a single wavelength light source, such a laser. Alternatively, the light source may provide a range of optical wavelengths.
  • Spectral selection means may be positioned on the optical path between the variable output light source and the focusing diffractive optical element. Varying the spectral selection means would enable the sample fluorescence to be measured as a function of input wavelength/frequency.
  • the spectral selection means may comprise a diffraction grating or a variable wavelength filter, such as a circular wedge filter or a linear wedge filter.
  • Spectral selection means may also be positioned on the optical path in front of the detector. Varying the output would enable the spectral selection of the measured emission.
  • the spectral selection means may comprise a diffraction grating or a variable wavelength filter, such as a circular or linear wedge filter.
  • the system may include a support or carriage for holding a sample plate reader having a plurality of sample wells.
  • the support or carriage may be movable relative to the illuminating beam so that measurements can be taken from each well.
  • the system may include beam-splitting means for splitting the illuminating beam into a plurality of beams, each at a location corresponding to a location of one of the wells of the plate reader. In this way, measurements can be taken in multiple wells simultaneously.
  • the system may be operable to measure the fluorescence lifetime and/or intensity and/or polarization.
  • the lifetime and/or intensity and/or polarization may be measured as a function of wavelength.
  • time correlated single photon counting may be used, which gives the maximum possible sensitivity.
  • Figure 3 is a representation of a confocal microscope scheme for measuring fluorescence emissions from a sample at a single excitation wavelength/frequency;
  • Figure 4 is a schematic representation of a diffractive optical element for use in the microscope of Figure 3;
  • Figure 5 is a modified version of the confocal microscope scheme of Figure 3, and
  • Figure 6 is schematic representation of a fluorescence spectroscope for measuring fluorescence emissions from a sample as a function of excitation frequency/wavelength.
  • Figure 3 shows a microscope 32 for single wavelength excitation and collection of fluorescence emissions from a sample in a plate reader.
  • the microscope of Figure 3 is essentially the same as that of Figure 1 except that the conventional convex objective lens of Figure 1 is replaced by a diffractive optical element 34 that is configured to focus the excitation light that passes through it onto the sample.
  • An example of the diffractive optical element 34 is shown in Figure 4.
  • This consists of a surface relief multi-level structure in the form of steps of concentric rings, the steps typically having a height of a fraction of a wavelength of the excitation light.
  • These elements can be made relatively thin, which reduces the effects of background fluorescence and allows the use of materials other than fused silica. This could be advantageous at certain wavelengths.
  • the specific design of the element would depend on various factors, such as the excitation wavelength and the spot size required in the sample plane. A skilled person would understand and be able to design such an element using techniques well known in the art, such as described in WO97/01171.
  • Figure 5 shows a modified version 36 of the confocal microscope scheme of Figure 3.
  • the conventional convex lens 30 that is used in the collection system of Figure 3 is replaced with a second diffractive optical element 38.
  • This element 38 is designed to focus the fluoresced light onto the detector. Again, because diffractive optical elements can be made relatively thin, this reduces background-fluorescence.
  • Figure 6 shows a spectrometer 40. This is similar to the system of Figure 3, but rather than having a monochromatic source, such as a laser, this includes a source 42 that is able to provide a range of optical wavelengths, such as a white light source. At the output of the source is a spectral selection arrangement 44, which allows the excitation wavelength to be selectively varied.
  • the spectral selection arrangement 44 could be a diffraction grating or a variable output filter, such as a circular wedge filter or a linear filter.
  • a similar modification could be made to the system of Figure 5.
  • the optical systems of Figures 3, 5 and 6 can be set-up to measure various features of light fluoresced from a sample, such as the fluorescence lifetime and/or intensity and/or polarization.
  • the lifetime and/or intensity and/or polarization may be measured as a function of wavelength.
  • time con-elated single photon counting may be used. Techniques for doing this are well known in the art and so will not be described in detail.
  • Using a diffractive optical element as the objective lens for focusing excitation light onto a sample reduces significantly the effects of background-fluorescence. This means that even very low sample emission levels can be detected. This is advantageous when only small amounts of sample are available, such as is often the case for biological material.
  • the diffractive optical element described above functions solely to focus light onto a sample
  • it could be designed to simultaneously provide additional useful beam manipulations, e.g. spectral filtering, beam splitting/shaping, and generation of multiple fan-out beams capable of interrogating multiple wells.
  • the diffractive optical element could be designed to modify the beam into a uniform illumination matched to the dimensions of the sample or sample holder, such as a plate reader sample well or a well in a mirco- array.
  • the system is likely to include a plate reader support or carriage that is movable relative to the illuminating beam so that measurements can be taken from each well.
  • the system may include beam-splitting means (which may or may not be incorporated into the excitation diffractive optical element) for splitting the illuminating beam into a plurality of beams, each at a location corresponding to a location of one of the wells of the plate reader. In this way, measurements can be taken in multiple wells simultaneously.

Abstract

An optical system, such as a microscope or spectroscope, for stimulating a sample and collecting fluoresced light emitted from that sample. The system has a focusing element for focusing light onto the sample, and a collector for collecting light emitted from the sample and directing it towards a detector. To reduce the effects of background fluorescence, the focusing element is a diffractive optical element.

Description

An Optical System for Laser Excitation and Collection of Fluorescence Emissions
The present invention relates to a system for focusing, controlling and collecting light beams for both excitation and emission of fluorescence from a sample, and in particular a biological sample.
Figure 1 shows a typical optical system for excitation and collection of fluorescence emissions from a sample in a plate reader 10. This has a diode laser 12 for emitting light at a wavelength suitable for stimulating sample fluorescence, an interference filter 14 in front of the laser 12 for removing unwanted wavelengths, a dichroic beam splitter 16 for directing filtered light from the laser 12 towards a well 18 of the plate reader 10, and a focusing lens 20 for focusing light into the sample well 18, and so onto a sample contained therein. Fluorescent light emitted by the sample, at a shifted wavelength, in response to excitation by the laser 12 follows a collection path to a detector 22. On the collection path is the focusing lens 20, the dichroic beam splitter 16 and a collector arrangement 24. The beam splitter 16 is transparent at the wavelength of the fluoresced light so that such light passes through it and along the collection path onto the detector 22. The collector 24 has in sequence an interference filter 26 and a collection lens 30 for focusing the fluoresced light onto the detector 22.
To ensure that measurements taken using the system of Figure 1 are meaningful, the fluorescent emission from the sample must be distinguishable from both the laser excitation and any stray auto fluorescence from the biological materials and importantly background fluorescence from the various optical components, particularly the focusing and collection lenses 20 and 30 respectively. In some circumstances, background fluorescence can be minimised by choice of material. For example, fused silica lenses emit less background fluorescence than corresponding glass lenses, as shown in Figure 2. Another option is to make the optical components as thin as possible. However, this is not practical for some applications, such as applications that require high spatial resolution. This can cause problems where the amount of light emitted by a sample is very low, as the real fluoresced signal may be swamped and so indistinguishable from background fluorescence from the optical components.
According to the present invention, there is provided an optical system for exciting a sample using light transmitted along an excitation path and collecting fluoresced light emitted from that sample along a collection path, the system having focusing means for focusing excitation light onto the sample, and a collector for collecting light emitted from the sample and directing it towards a detector, wherein the focusing means comprises a diffractive optical element. This focusing diffractive optical element consists of a surface relief multi-level structure in the form of steps of concentric rings of (say) fused silica, the steps typically having a height of a fraction of a wavelength. The diffractive optical element may lie on both the excitation and collection paths.
By using a diffractive optical element to focus light onto the sample, background fluorescence can be reduced. This is because diffractive optical elements can be made very thin. For example, the thickness of a diffractive optical element for use in the present invention could be of the order of a few hundred microns, instead of 5- 10mm, as would be required for a conventional lens. This corresponds to a reduction of volume of material of up to one hundred times. In practice, this means that the background fluorescence generated by the focusing element is significantly less than would be the case if a conventional lens were used. For some applications this is highly advantageous.
Diffractive optical elements have been used in fluorescence microscopes and spectrometers before. Examples are described in WO00/40935, WO00/58715, WO01/63260, WO03/029768, WO2005/017598, and WO2005/0063282. However, none of the known systems use a diffractive optical element essentially as the objective lens for focusing light onto a sample. Instead, these elements are more conventionally used merely for beam shaping or conditioning. The collector may include a diffractive optical element for focusing fluoresced light towards the detector. By using a second diffractive element, again in place of a conventional lens, further reductions in background fluorescence can be achieved.
The system may include a light source. The light source may be a single wavelength light source, such a laser. Alternatively, the light source may provide a range of optical wavelengths.
Spectral selection means may be positioned on the optical path between the variable output light source and the focusing diffractive optical element. Varying the spectral selection means would enable the sample fluorescence to be measured as a function of input wavelength/frequency. The spectral selection means may comprise a diffraction grating or a variable wavelength filter, such as a circular wedge filter or a linear wedge filter.
Spectral selection means may also be positioned on the optical path in front of the detector. Varying the output would enable the spectral selection of the measured emission. The spectral selection means may comprise a diffraction grating or a variable wavelength filter, such as a circular or linear wedge filter.
The system may include a support or carriage for holding a sample plate reader having a plurality of sample wells. The support or carriage may be movable relative to the illuminating beam so that measurements can be taken from each well. Alternatively, the system may include beam-splitting means for splitting the illuminating beam into a plurality of beams, each at a location corresponding to a location of one of the wells of the plate reader. In this way, measurements can be taken in multiple wells simultaneously.
The system may be operable to measure the fluorescence lifetime and/or intensity and/or polarization. When a variable wavelength excitation source and filtered detector are used, the lifetime and/or intensity and/or polarization may be measured as a function of wavelength. For fluorescence lifetime measurements, time correlated single photon counting may be used, which gives the maximum possible sensitivity.
Various aspects of the invention will now be described by way of example only and with reference to the accompanying drawings, of which:
Figure 3 is a representation of a confocal microscope scheme for measuring fluorescence emissions from a sample at a single excitation wavelength/frequency;
Figure 4 is a schematic representation of a diffractive optical element for use in the microscope of Figure 3; Figure 5 is a modified version of the confocal microscope scheme of Figure 3, and
Figure 6 is schematic representation of a fluorescence spectroscope for measuring fluorescence emissions from a sample as a function of excitation frequency/wavelength.
Figure 3 shows a microscope 32 for single wavelength excitation and collection of fluorescence emissions from a sample in a plate reader. The microscope of Figure 3 is essentially the same as that of Figure 1 except that the conventional convex objective lens of Figure 1 is replaced by a diffractive optical element 34 that is configured to focus the excitation light that passes through it onto the sample. An example of the diffractive optical element 34 is shown in Figure 4. This consists of a surface relief multi-level structure in the form of steps of concentric rings, the steps typically having a height of a fraction of a wavelength of the excitation light. These elements can be made relatively thin, which reduces the effects of background fluorescence and allows the use of materials other than fused silica. This could be advantageous at certain wavelengths.
The specific design of the element would depend on various factors, such as the excitation wavelength and the spot size required in the sample plane. A skilled person would understand and be able to design such an element using techniques well known in the art, such as described in WO97/01171.
Figure 5 shows a modified version 36 of the confocal microscope scheme of Figure 3. Here, the conventional convex lens 30 that is used in the collection system of Figure 3 is replaced with a second diffractive optical element 38. This element 38 is designed to focus the fluoresced light onto the detector. Again, because diffractive optical elements can be made relatively thin, this reduces background-fluorescence.
Figure 6 shows a spectrometer 40. This is similar to the system of Figure 3, but rather than having a monochromatic source, such as a laser, this includes a source 42 that is able to provide a range of optical wavelengths, such as a white light source. At the output of the source is a spectral selection arrangement 44, which allows the excitation wavelength to be selectively varied. The spectral selection arrangement 44 could be a diffraction grating or a variable output filter, such as a circular wedge filter or a linear filter. Of course, a similar modification could be made to the system of Figure 5.
The optical systems of Figures 3, 5 and 6 can be set-up to measure various features of light fluoresced from a sample, such as the fluorescence lifetime and/or intensity and/or polarization. When a variable wavelength excitation source is used, the lifetime and/or intensity and/or polarization may be measured as a function of wavelength. For fluorescence lifetime measurements, time con-elated single photon counting may be used. Techniques for doing this are well known in the art and so will not be described in detail.
Using a diffractive optical element as the objective lens for focusing excitation light onto a sample reduces significantly the effects of background-fluorescence. This means that even very low sample emission levels can be detected. This is advantageous when only small amounts of sample are available, such as is often the case for biological material.
A skilled person will appreciate that variations of the disclosed arrangements are possible without departing from the invention. For example, whilst the diffractive optical element described above functions solely to focus light onto a sample, it will be appreciated that it could be designed to simultaneously provide additional useful beam manipulations, e.g. spectral filtering, beam splitting/shaping, and generation of multiple fan-out beams capable of interrogating multiple wells. Also, the diffractive optical element could be designed to modify the beam into a uniform illumination matched to the dimensions of the sample or sample holder, such as a plate reader sample well or a well in a mirco- array.
Although a single excitation diffractive optical element is described with reference to Figure 3, 5 and 6, a plurality of such elements could be used, provided the overall function of these is to focus light that passes through them onto pre-determined sample area. Furthermore, although the invention is described with reference to the illumination of a single well of a microscope/spectroscope plate reader, in practice the system is likely to include a plate reader support or carriage that is movable relative to the illuminating beam so that measurements can be taken from each well. Alternatively, the system may include beam-splitting means (which may or may not be incorporated into the excitation diffractive optical element) for splitting the illuminating beam into a plurality of beams, each at a location corresponding to a location of one of the wells of the plate reader. In this way, measurements can be taken in multiple wells simultaneously. Accordingly, the above description of a specific embodiment is made by way of example only and not for the purposes of limitations. It will be clear to the skilled person that minor modifications may be made without significant changes to the operation described.

Claims

Claims
1. An optical system for exciting a sample using light transmitted along an excitation path and collecting fluoresced light emitted from that sample along a collection path, the system having focusing means for focusing excitation light onto the sample and a detector for detecting light fluoresced from the sample, wherein the focusing means comprises a diffractive optical element.
2. An optical system as claimed in claim 1 comprising a collector for collecting light emitted from the sample and directing it towards the detector.
3. An optical system as claimed in claim 1 or claim 2 wherein the collector includes a diffractive optical element for focusing fluoresced light towards the detector.
4. An optical system as claimed in any of the preceding claims wherein the diffractive element lies on both the excitation and collection optical paths.
5. An optical system as claimed in any of the preceding claims including a light source.
6. An optical system as claimed in claim 5 wherein the light source is a single wavelength light source.
7. An optical system as claimed in claim 5 wherein the light source is operable to provide a range of optical wavelengths.
8. An optical system as claimed in claim 7 wherein a variable frequency filter is positioned on the optical path between the light source and the focusing diffractive optical element.
9. An optical system as claimed in claim 8 wherein the variable filter is a circular wedge filter or a linear wedge filter.
10. An optical system as claimed in any of the preceding claims wherein the focusing means are positioned on the excitation path between the light source and the sample, so that the excitation light has to pass through the diffractive optical element.
11. An optical system as claimed in any of the preceding claims wherein the excitation diffraction optical element is operable to modify the excitation beam to match the dimensions of a target, such as sample holder, and in particular, a plate reader well.
PCT/GB2006/001612 2005-05-13 2006-05-04 An optical system for laser excitation and collection of fluorescence emissions WO2006120386A1 (en)

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Publication number Priority date Publication date Assignee Title
EP3654076A1 (en) * 2018-11-15 2020-05-20 FRAUNHOFER-GESELLSCHAFT zur Förderung der angewandten Forschung e.V. Diffractive optical element, confocal microscope and method for designing a diffractive optical element
WO2020099538A1 (en) * 2018-11-15 2020-05-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Diffractive optical element, confocal microscope and method for designing a diffractive optical element

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