Multifocal scanning fluorescence microscope

Abstract

Scanning fluorescence microscopes with an observation beam path from a measurement volume to an image plane. A beam combiner is provided for coupling an illumination system and a diaphragm arranged in the image plane for slow composition of the image because of the sequential scanning and subject the sample to loading as a result of inefficient use of the excitation light. The microscope simultaneously detects fluorescence from different focal planes in each case quasi-confocally. The observation beam path between the beam combiner and the image plane has a first diffractive optics for splitting light beams into beam bundles along different orders of diffraction, imparting to the light beams a spherical phase that is different from the other orders of diffraction. A second diffractive optics is provided for the compensation of chromatic aberrations of the split beam bundles, and a collecting optics is provided for focusing split beam bundles into the image plane.

Claims

1. A confocal scanning fluorescence microscope comprising an optical system-having a microscopic observation beam path from a measurement volume to an image plane, a microscope objective having an optical axis, a beam combiner with an input for coupling an illumination system and a diaphragm arranged in the image plane, said optical system including a first diffractive optics for splitting light beams into beam bundles along different orders of diffraction in the observation beam path between the beam combiner and the image plane, said first diffractive optics configured to impart to the light beams of each order of diffraction a spherical phase that is different from the other orders of diffraction, in particular a respective integral multiple of a spherical phase, a second diffractive optics for the compensation of chromatic aberrations of the split beam bundles, a collecting optics for the focusing of the split beam bundles into the image plane, optics for extending along said optical axis of the objective a depth of focus of the microscope objective for the illumination system, said optics for extending the depth of focus being arranged outside the observation beam path between the illumination system and the beam combiner input for the coupling of the illumination system, and wherein the diffractive optics are formed such that midpoints of adjacent axial measurement volumes for a predetermined excitation wavelength, a predetermined numerical aperture of the microscope objective, a predetermined confocal diaphragm aperture size and a predetermined refractive index of an immersion medium are more than two optical section thicknesses of the microscope from one another.

2. The microscope as claimed in claim 1, wherein said diffractive optics being a two-dimensional phase grating, which splits wavefronts coming from the microscope objective into (2m+1).sup.2 orders of diffraction with m=1,2,3, . . . over two different spatial directions.

3. The microscope as claimed in claim 1, wherein said diaphragm has an aperture and the collecting optics focusing each of the split beam bundles of the various orders of diffraction onto said aperture.

4. The microscope as claimed in claim 1, wherein said diaphragm has for each of the split orders of diffraction, or at least for a subset of the split orders of diffraction, a respective aperture and the collecting optics focusing each of the split beam bundles of the various orders of diffraction onto the relevant aperture.

5. The microscope as claimed in claim 1, wherein said optical system further comprises at least one optics for producing a further image plane, with an arrangement of detectors in the further image plane.

6. The microscope as claimed in claim 1, wherein at least one spectrally dispersive element is arranged between the image plane and the further image plane in such a way that, for each of the split beam bundles at least of the orders of diffraction other than zero, different wavelengths are focused onto different locations of the further image plane, in particular with arrangement of the at least one dispersive element in a collimated beam path portion of the optics for producing the further image plane.

7. The microscope as claimed in claim 6, wherein the spectrally dispersive element is arranged in or at least approximately in a plane conjugate to the pupil plane of the microscope objective.

8. The microscope as claimed in claim 6, wherein the spectrally dispersive element is movably mounted for reversible removal from the observation beam path.

9. The microscope as claimed in claim 1, wherein the optics for producing an extended depth of focus comprises a cubic phase modulation mask or means for producing Bessel beams, in particular in a plane conjugate to the pupil plane of the microscope objective, and/or being designed for underfilling the pupil of the microscope objective, in particular by beam shaping, in particular to reduce a beam cross section of collimated light.

10. The microscope as claimed in claim 1, wherein the optics for producing an extended depth of focus produces an illumination volume of which the axial extent is at least five times its lateral extent.

11. The microscope as claimed in claim 1, wherein the first diffractive optics are arranged in or at least approximately in a plane conjugate to a pupil of the microscope objective.

12. The microscope as claimed in claim 1, wherein the optical system having an adjustable beam deflecting unit for sequentially scanning different measurement volumes and a transfer optics for imaging the deflecting unit into a pupil of the microscope objective located between the microscope objective and the beam combiner.

13. The microscope as claimed in claim 5, wherein the detectors are arranged for the spatial oversampling of the point spread function in at least one of the orders of diffraction, preferably in each order of diffraction.

14. The microscope as claimed in claim 1, wherein the optics for extending the depth of focus are formed such that all of the measurement volumes imaged into the image plane lie within the extended depth of focus.

15. The microscope as claimed in claim 1, wherein illumination light extends in a longitudinal direction parallel to the optical axis of the microscope objective.

16. The microscope as claimed in claim 1, wherein the optics for producing an extended depth of focus has a thickness producing an illumination volume of which the axial extent is at least ten times, its lateral extent and/or, for a predetermined excitation wavelength, a predetermined numerical aperture of the microscope objective, a predetermined confocal diaphragm aperture size and a predetermined refractive index of an immersion medium, corresponds to at least two optical section thicknesses of the microscope.

17. The microscope as claimed in claim 1, wherein the optics for producing an extended depth of focus has a thickness producing an illumination volume of which the axial extent is at least twenty times, its lateral extent and/or, for a predetermined excitation wavelength, a predetermined numerical aperture of the microscope objective, a predetermined confocal diaphragm aperture size and a predetermined refractive index of an immersion medium, corresponds to at least two optical section thicknesses of the microscope.

18. The microscope as claimed in claim 1, wherein the optics for producing an extended depth of focus produces an illumination volume whose extent, for a predetermined excitation wavelength, a predetermined numerical aperture of the microscope objective, a predetermined confocal diaphragm aperture size and a predetermined refractive index of an immersion medium, corresponds to at least two optical section thicknesses of the microscope.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the drawings:

(2) FIG. 1 shows a microscope for axial-multifocal detection,

(3) FIG. 2 shows cutouts from a first embodiment and a second embodiment of the optical system for the definition of an observation beam path and

(4) FIG. 3 shows cutouts from a third embodiment and a fourth embodiment of the optical system for the definition of an observation beam path.

DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

(5) In all of the drawings, parts that coincide bear the same reference signs.

(6) FIG. 1 schematically shows a scanning fluorescence microscope 1 in the form of a laser scanning microscope (LSM). Here, a laser as a light source 11 as an illumination system together with a microscope objective 2 defines an illumination beam path A, which contains a phase mask 10 and is combined (optically coupled) by a beam splitter 6, for example a dichroic beam splitter cube, with the observation beam path B. The phase mask 10 is arranged outside the observation beam path B. An optical transfer system 5 images the plane of the phase mask 10 onto a deflecting unit 4, which can deflect the excitation light beam in the x and y directions. A further optical transfer system 3 images the deflecting unit 4 into the pupil plane of the objective 2. The objective 2 focuses the laser beam into the sample P, the lateral position of the illumination volume depending on the deflecting angles that are set on the deflecting unit 4.

(7) The axial length of the illumination volume, that can be given for example by the full half-width of the axial intensity profile of its PSF, is established by the nature of the phase mask 10 and is significantly lengthened in the z direction with respect to that of the microscope objective 2 on its own (without the phase mask 10), whereas the lateral size of the illumination PSF in the x and y directions is approximately identical. For this purpose, the phase mask 10 is for example a cubic phase modulation mask. The extent of the illumination volume in the z direction is for example five times that in the x direction or in the y direction.

(8) The fluorescent light produced along the lengthened focal profile in the sample P is substantially collimated by the objective 2 and passes back through the optics described above to the beam splitter 6, which spectrally separates the observation beam path B from the illumination beam path A. The transfer optics 5 thereby transforms the intermediate image plane ZBE, in which there is information distributed in the axial direction, into a first grating plane GE1, which is the pupil plane of the transfer optics 5. Arranged in the first grating plane GE1 is a first diffractive optics 7 in the form of a two-dimensional DOE phase grating, which produces (2m+1).sup.2 orders of diffraction (here for example m=1) and thus splits the incident light into a corresponding number of beam bundles. The phase grating 7 imparts a constant spherical phase term on each order of diffraction (each of the beam bundles), whereby a refocusing of the intermediate image plane ZBE by the respective phase term takes place, advantageously in equidistant steps, in dependence on the wavefront curvature, and consequently in dependence on the distance of the fluorescence emission from the objective 2. Downstream of the first diffractive optics 7 there is a second diffractive optics 13, for example a grating or a DOE, in order to cancel out the spectral dispersion of the phase grating 7 in GE1. The light beam bundles, split into the orders of diffraction, refocused and color-corrected, are imaged by the lens 8 as a collecting optics onto the confocal diaphragm 15 in the image plane BE, behind which a detector matrix 9 is arranged. The detectors 9.sub.ik (for example i=1 . . . 128; k=1 . . . 128) of the matrix 9 are for example SPAD, but may also be CCD or CMOS sensors.

(9) A control unit 14 controls the deflecting unit 4 and the light source 11 and also the detectors 9.sub.ik. It is also designed for accepting and for example computationally correcting their measured values.

(10) In FIG. 2A, a cutout of the observation beam path B is schematically shown in detail. The lens designated by L1 is in this case for example part of the transfer optics 5. After the refocused splitting at the diffractive optics 7 in the first grating plane GE1 conjugate to the pupil plane of the objective 2, the refocused information is color-corrected by the second diffractive optics 13, in order to compensate for the spectral dispersion of the phase grating 7 in GE1, and is imaged by the collecting optics 8 in the plane L2 directly onto the pixelated sensor 9. Each order of diffraction consequently images a respective measurement volume from a different plane of the sample P sharply onto the detector matrix 9. Furthermore, the fluorescent light of each order of diffraction apart from the zeroth order is spectrally dispersed. The resultant light distribution on the detector matrix 9 is schematically indicated in the two Subfigs. 2A, 2B schematically alongside the beam path.

(11) Ideally, all non-relevant orders of diffraction of the second diffractive optics 13 are suppressed to the greatest extent in GE2. The imaging by the collecting optics L2 then has the effect that all of the subbeams of each wavelength and each plane of origin are focused onto one point. In this image plane BE (at the same time pinhole plane PHE), the sample light is then filtered quasi-confocally by means of a pinhole diaphragm 15 and out-of-focal light is separated from the fluorescent light originating from the measurement volume (focal plane) considered in the respective order of diffraction.

(12) The lens in the plane L3, which for example with the lens in the plane L4 forms a further transfer optics, collimates the light beams transmitted through the pinhole diaphragm 15 in the PHE and produces a further pupil plane GE3, in which all of the orders of diffraction again separate from one another. In this plane GE3 there may optionally be inserted a spectrally dispersing and order-separating element 12, in order on the one hand to image the different planes of the ZBE by means of the lens L4 onto different positions of the detector matrix 9 and on the other hand to disperse the spectral information among the detectors 9.sub.ik. The element 12 is for example a segmented prism, which spreads the orders of diffraction in relation to one another, the segment in the zeroth order of diffraction being a plane-parallel plate. As a result, the zeroth order is not spectrally resolved on the detector matrix 9. The eight other orders of diffraction can be detected in a spectrally resolved manner, because each beam bundle is dispersed among a relevant group of detectors 9.sub.ik (respective subset of all the detectors) on account of the spatial/spectral splitting.

(13) In FIG. 2B, a spectrally dispersing element 12, for example a prism or a diffraction grating, is additionally arranged in the beam path of the zeroth order, so that all the orders of diffraction are split spatially spectrally among a respective group of detectors 9.sub.ik. Thus, the spectral information can also be ascertained for the sample plane that is represented by the zeroth order. The spectrally dispersing element 12 is advantageously arranged such that it can be repositioned in the observation beam path B and can be reversibly removed again.

(14) The data measured by means of the detectors 9.sub.ik are already quasi-confocal in relation to the primary and secondary focal planes. Only the orientation of the dispersion has to be included in the calculation by means of a calibration. The calibration of the detection system takes place for example by an individual fluorescent bead being moved axially through the illumination focal region of the microscope 1, the element 12 being removed from the beam path of the zeroth order. In this case, the detectors 9.sub.1k measure under the zeroth order the chromatically undisturbed PSF of the optical system. On the basis of this PSF, the spectral dispersion of every other order of diffraction can be ascertained, since the identical PSF must be present in these other orders, just corrected by the respective (predetermined) phase term. With the then known PSF, the dispersion of the element 12 can finally also be calibrated.

(15) In FIG. 3A, a cutout from a further optical system for the definition of an observation beam path B is represented. As a difference from FIG. 2A, an order-separating element 16, for example segmented by different prisms, has been introduced into the grating plane GE3 between the correction grating in GE2 and the collecting optics L2. The sum of the spectral dispersions of the elements in GE2 and GE3 are equal and opposite to the spectral dispersion of the diffractive optics 7 in GE1. On account of the order-separating element 16, all of the orders of diffraction of the phase grating 7 are then imaged by the collecting optics L2 onto separate lateral positions of the PHE, after they have been spectrally corrected by the second diffractive optics 13. Correspondingly, a confocal diaphragm 15 with a matrix arrangement of (2m+1).sup.2 apertures is arranged in the confocal plane. Finally, the lens L3 images the PHE onto the detector matrix 9, which is arranged in a further image plane wBE. In the variant of FIG. 3A, the imaging takes place without spectral dispersion on the detectors 9.

(16) In FIG. 3B, a spectrally dispersing element 12, which is passed through by all the orders of diffraction, has been introduced in the pupil plane (PE) upstream of the sensor matrix 9. Accordingly, the orientation of the spectral light distribution on the sensor matrix is the same for all of the orders of diffraction of the phase grating. However, the spectral resolution may vary on account of the different angles of incidence. Ideally, the element 12 is reversibly positionable, so that it is possible to switch over between the arrangements in FIGS. 3A and 3B. This may in turn be used for the calibration of the spectral resolution.

(17) In alternative embodiments, the confocal diaphragm does not have (2m+1).sup.2 apertures, but for example only (2m+1) apertures.

(18) While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

LIST OF REFERENCE SIGNS

(19) 1 Microscope

(20) 2 Objective

(21) 3 Transfer optics

(22) 4 Deflecting unit

(23) 5 Transfer optics

(24) 6 Beam splitter

(25) 7 Diffractive optics

(26) 8 Collecting optics

(27) 9 Detector matrix

(28) 10 Phase modulation mask

(29) 11 Light source (illumination system)

(30) 12 Spectrally dispersing element

(31) 13 Diffractive optics

(32) 14 Control unit

(33) 15 Confocal diaphragm

(34) 16 Order-separating element

(35) A Illumination beam path

(36) B Observation beam path

(37) P Sample

(38) PHE Pinhole plane

(39) ZBE Intermediate image plane

(40) L1/2/3 Planes

(41) GE1/2/3 Planes

(42) BE Image plane

(43) PE Pupil plane

(44) wBE Further image plane