Light microscope and method for image recording using a light microscope

Abstract

The invention relates to a light microscope comprising a polychromatic light source for emitting illumination light in the direction of a sample, focussing means for focussing illumination light onto the sample, wherein the focussing means, for generating a depth resolution, have a longitudinal chromatic aberration, and a detection device, which comprises a two-dimensional array of detector elements, for detecting sample light coming from the sample. According to the invention, the light microscope is characterized in that, for detecting both confocal portions and non-confocal portions of the sample light, a beam path from the sample to the detection device is free of elements for completely masking out non-confocal portions. In addition, the invention relates to a method for image recording using a light microscope.

Claims

1. A light microscope, comprising a polychromatic light source for emitting illuminating light in the direction of a specimen, focusing means for focusing illuminating light onto the specimen, wherein the focusing means have a longitudinal chromatic aberration to produce a depth resolution, a structured element arranged between the light source and the focusing means to produce structured illuminating light from the illuminating light emitted by the light source, and a detection unit which comprises a two-dimensional array of detector elements for detecting specimen light coming from the specimen, wherein for the detection of both confocal portions and also non-confocal portions of the specimen light, an optical path from the specimen to the detection unit is free of elements for complete filtering-out of non-confocal portions, wherein specimen light coming from the specimen is guided through the structured element onto the detection unit, wherein structures of the structured element are sufficiently large for the passage of non-confocal portions of the specimen light, wherein the structures of the structured element are larger than one Airy disc, and the optical path from the specimen to the detection unit is free of elements that have structures for leading specimen light to the detection unit which are smaller than one Airy disc to obtain both confocal information and wide-field information through a single measurement or a single measurement sequence, whereby confocal and wide-field information are obtained simultaneously or one after another without mechanical movements of components of the light microscope to switch between obtaining confocal and wide-field information.

2. The light microscope according to claim 1, wherein the structured element comprises a Nipkow disc, a microlens array, a grating, a Fresnel bi-prism or an element for generating a speckle pattern.

3. The light microscope according to claim 1, wherein the focusing means comprise at least one of: a light refractive or a light diffractive microlens array to produce a wavelength-dependent focal position.

4. The light microscope according to claim 1, wherein a wavelength selection unit is present to select a variably adjustable spectral range of the illuminating light in order to focus via the focusing means illuminating light onto different positions along an optical axis of the light microscope, and electronic control means are present to select a spectral range by means of the wavelength selection unit.

5. The light microscope according to claim 4, wherein the wavelength selection unit comprises at least one of: a prism, a grating, a colour filter or an acousto-optic tunable filter.

6. The light microscope according to one of claim 1, wherein a deflection means is present for selective guiding of illuminating light onto a first or a second light path, the structured element is mirrored on one side, wherein illuminating light of the first light path is guided onto the mirrored side of the structured element and is reflected at the structured element in the direction of the specimen, and wherein illuminating light of the second light path is guided onto a different side of the structured element and transmitted by the structured element in the direction of the specimen, and wherein the transmitted illuminating light and the reflected illuminating light produce images of the structured element, phase displaced with respect to each other, on the specimen.

7. The light microscope according to claim 6, wherein the deflection means comprises at least one of: a switchable mirror, an acousto-optic modulator, an acousto-optic deflector, an electro-optic modulator or a switching unit based upon polarization direction.

8. The light microscope according to claim 1, wherein positioning means for at least one of: displacing or rotating the structured element are provided and electronic control means are provided which are adapted, with the detection unit, to record images of the specimen at different positions of the structured element and to calculate a specimen image from these images.

9. The light microscope according to claim 1, wherein the detection unit is a spectrally resolving detection unit.

10. The light microscope according to claim 9, wherein spectral filtering means are provided to produce the spectral resolution of the detection unit, or the detection unit comprises an interferometer.

11. A method for recording images with a light microscope, comprising: transmitting illuminating light with a polychromatic light source in the direction of a specimen, guiding the illuminating light to a structured element to produce structured illuminating light, focusing the structured illuminating light onto the specimen with focusing means, wherein a depth resolution is achieved through a longitudinal chromatic aberration of the focusing means, guiding specimen light coming from the specimen through the structured element to a detection unit which comprises a two-dimensional array of detector elements, guiding both confocal portions and also non-confocal portions of the specimen light onto the detection unit, wherein structures of the structured element are sufficiently large for the passage of non-confocal portions of the specimen light, wherein the structures of the structured element are larger than one Airy disc, and wherein both confocal information and wide-field information are obtained through a single measurement or a single measurement sequence simultaneously or one after another without mechanical movements of components of the light microscope to switch between obtaining confocal and wide-field information, by providing the optical path from the specimen to the detection unit to be free of elements that have structures for leading specimen light to the detection unit which are smaller than one Airy disc.

12. The method according to claim 11, wherein for the determination of a height profile of a specimen, images of the specimen are recorded which differ in the detected wavelength range of the specimen light, a modulation contrast is determined for image points of each image, the image point with the greatest modulation contrast is selected from those image points that have the same position in the different images, and height information of the specimen is assigned to the selected image point in dependence upon the associated wavelength range, wherein respective height information has been previously stored for each of the different wavelength ranges.

13. The method according to claim 11, wherein the detection unit is arranged in a plane which is conjugated with the specimen plane, for the production of a confocal image, the detector elements of the detection unit are used as a digital diaphragm, wherein for the purpose of creating a confocal image those detector elements are used onto which specimen regions lying in the focus of the illuminating light are sharply imaged.

14. The method according to claim 11, wherein with an interferometer, a part of the specimen light is guided onto a reference path with adjustable length and subsequently guided back onto the specimen, wherein a constructive interference on a surface of the specimen between illuminating light and specimen light guided back depends upon a height profile of the specimen and also upon the length of the reference path, the length of the reference path is varied and the length, at which a maximum signal is received with the detection unit, is selected, the height profile of the specimen is concluded from the selected length of the reference path with the aid of previously stored values.

15. The method according to claim 11, wherein for the improvement of the spectral resolution, a wavelength selection is realized both via spectral filtering means of the detection unit and also via a wavelength selection unit at the light source.

16. The method according to claim 11, wherein a first image of the specimen is recorded, wherein a structured element is located in the optical path to produce a structured illumination, a second image is recorded for the examination of specimen regions that were not illuminated with the structured illumination when recording the first image, wherein the structured element is not located in the optical path, and a specimen image is calculated from the first and the second image.

17. The method according to claim 11, wherein a polarization encoding of at least one of: illuminating light or specimen light is realized for improving a measurement resolution in a specimen plane lying transversely to an optical axis, wherein regions lying one beside the other in the specimen plane are irradiated with illuminating light of different polarizations, or wherein a position-dependent specimen light polarization is realized, wherein a certain polarization is imprinted on specimen light in dependence upon its position transversely to the optical axis, wherein specimen light is detected separately for different polarizations, and wherein position information within the specimen plane is assigned to the detected specimen light in dependence upon its polarization.

18. A light microscope, comprising: a polychromatic light source for emitting illuminating light in the direction of a specimen, focusing means for focusing illuminating light onto the specimen, wherein the focusing means have a longitudinal chromatic aberration to produce a depth resolution, a structured element arranged between the light source and the focusing means to produce structured illuminating light from the illuminating light emitted by the light source, and a detection unit which comprises a two-dimensional array of detector elements for detecting specimen light coming from the specimen, wherein for the detection of both confocal portions and also non-confocal portions of the specimen light, an optical path from the specimen to the detection unit is free of elements for complete filtering-out of non-confocal portions, the structured element is partially mirrored on one side to split incoming specimen light into a confocal beam path formed by specimen light transmitted at the structured element and including only confocal light portions and a non-confocal beam path formed by specimen light reflected at the structured element and including at least non-confocal light portions, the detection unit has a first and a second detector, the first detector is arranged in the confocal beam path and the second detector is arranged in the non-confocal beam path.

Description

(1) Further features and advantages of the invention are described below by reference to the attached schematic drawing, in which:

(2) FIG. 1 shows a schematic illustration of a first embodiment of a light microscope according to the invention;

(3) FIG. 2 shows a schematic illustration of a second embodiment of a light microscope according to the invention, and

(4) FIG. 3 shows a schematic illustration of a part of a further embodiment of a light microscope according to the invention.

(5) The same components and those having the same effect are generally identified in the figures with the same reference signs.

(6) FIG. 1 shows an embodiment of a light microscope 100 according to the invention. Essential components of the light microscope 100 are: a polychromatic light source 10 to emit illuminating light 15, a structured element 30, with which a spatial structure is imprinted on the illuminating light 15, and focussing means 50 to focus the illuminating light 15 onto a specimen 60. Furthermore the light microscope 100 comprises a detection unit 80, with which specimen light 16 emitted by the specimen 60 in the direction of the focussing means 50 is detected. The specimen light 16 can in particular be illuminating light 15 which is reflected and/or scattered at the specimen 16. In principle the specimen light 16 can, however, also be fluorescent or phosphorescent light which is irradiated as a consequence of the absorption of illuminating light through the specimen.

(7) The light source 10 can comprise for example one or more lasers, a halogen lamp or a diode. The irradiated illuminating light 15 can have a comparatively broad wavelength range and is guided with optical imaging means 12, for example with one or more lenses, to a wavelength selection unit 20. The wavelength selection unit 20 can comprise an AOTF or one or more prisms, gratings or colour filters. The wavelength selection unit 20 can be operated with electronic control means 90 for sequential selection of narrow-band wavelength ranges of the illuminating light 15.

(8) The portion of the illuminating light 15 selected with the wavelength selection unit 20 is then guided to a deflection means 25. This can for example have a galvanometer mirror or another switchable mirror. Alternatively, the deflection means can also comprise an acousto-optic deflector. The electronic control means 90 are adapted to carry out at least two different adjustments of the deflection means 25, wherein the illuminating light 15 is electively deflected onto a first light path 17 or a second light path 18.

(9) On the first light path 17, the illuminating light 15 is guided onto a mirrored side of the structured element 30. An optical fibre 27 is used for this purpose in the embodiment shown. The illuminating light 15 is in-coupled via a lens 26 into the optical fibre 27, and illuminating light leaving the optical fibre 27 is guided via a further lens 28 onto a beam splitter 29, for example a semi-transparent mirror, and thus further onto the mirrored side of the structured element 30.

(10) On the second light path 18, the illuminating light is in-coupled with a lens 21 into an optical fibre 22 and is guided at the other end of the optical fibre with a lens 23 onto another side of the structured element 30 which is not mirrored.

(11) Instead of optical fibres, the illuminating light 15 can, however, also be guided on the two light paths 17, 18 solely with mirrors and/or lenses. In addition, polarisation filter means can also be present to polarise the illuminating light 15.

(12) The illuminating light 15 can be transmitted on the second light path 18 through the structured element 30, wherein a structure is imprinted on the illuminating light 15, with which it can also be described as structured illuminating light.

(13) The structured element 30 is located in a field plane which is conjugated with a specimen plane so that the structured element 30 is sharply imaged at the specimen plane.

(14) The structured element 30 can be for example a one-dimensional or two-dimensional grating structure. If the two light paths 17, 18 are sequentially used, two phases of the structured element 30 can be imaged one after the other at the specimen location. In order to image further phases of the structured element 30 on the specimen 60, the electronic control means 90 are adapted to displace the structured element 30 in the direction of the double arrow, that is to say: transversely to an optical axis of the light microscope 100, and/or to rotate the structured element 30. By using the two light paths 17, 18, the number of positions of the structured element 30 required for the measurement can advantageously be reduced.

(15) By means of the beam splitter 29, illuminating light 15 of the first light path 17 which is reflected at the structured element and illuminating light 15 of the second light path 18 which is transmitted at the structured element 30 are guided on the same optical path to the specimen 60.

(16) The illuminating light 15 hereby travels through further optical imaging means 31 and also through a beam splitter 40. Illuminating light 15 travelling towards the specimen 60 and specimen light 16 coming from the specimen 60 are separated with the beam splitter 40. For this, the beam splitter 40 can be designed as a polarisation beam splitter 40. In this case, means for changing the polarisation direction of light, for example a /4 plate 41, are present between the polarisation beam splitter 40 and the specimen 60. Through the /4 plate, the polarisation direction of the illuminating light 15 is rotated on the way to the specimen 60 and the polarisation direction of the specimen light 16 travelling back is rotated once again. The polarisation directions of the illuminating light 15 and the specimen light 16 thereby differ by 90 at the beam splitter, so that efficient separation is possible.

(17) Focussing means 50 are present to focus the illuminating light 15 onto the specimen 60. These comprise here a diffractive element 48 which has a longitudinal chromatic aberration, and also an objective 49. Alternatively, the diffractive element 48 can also be a component of the objective 49. On account of the longitudinal chromatic aberration the focal position of the illuminating light 15 along the optical axis depends upon the wavelength of the illuminating light 15. FIG. 1 shows three different focuses 51 for different wavelengths of the specimen light 16.

(18) By evaluating light of different wavelengths separately, different depths, that is to say: different sections along the optical axis, can be examined. A height profile of the specimen 60 can thereby be created. In the case of a partially transparent specimen, an examination within the specimen can also be realised at different depths.

(19) Specimen light 16 emitted back by the specimen 60 is guided at the polarisation beam splitter 40 into a detection channel, in which it passes via a polarisation filter 42 and optical imaging means 43 onto the detection unit 80. Stray light with a different polarisation direction can be filtered out by the polarisation filter 42.

(20) In order to examine different depths in the specimen 60, tuning of the wavelength of the illuminating light 15 can be carried out, in particular by means of the wavelength selection unit 20, and/or with spectral filtering means at the detection unit 80. To this end, the electronic control means 90 are adapted to tune the wavelength of the light to be detected with the wavelength selection unit 20 and/or the spectral filtering means at the detection unit 80 in order to achieve scanning of the specimen in z-direction without displacement of the specimen 60 being necessary.

(21) Sequentially changing the wavelength of both the illuminating light guided onto the specimen and also the specimen light guided with the filter means onto the detection unit 80 can be advantageous if an adjustable bandwidth of wavelength ranges of the light source cannot be realised with the desired spectral resolution and/or if a spectral resolution of the detection unit, for example using a limited number of colour filters, does not lead to the desired resolution. In this case, an increased spectral resolution can be achieved by a certain wavelength range being selected at the light source in addition with a wavelength filtration being realised at the detection unit. This embodiment can also be advantageous when the imaging of different grating phases is realised through a colour encoding.

(22) A height profile of the specimen 60 can be determined by a respective modulation contrast being determined for the different wavelengths for image points which are adjacent in an xy-plane, thus transversely to the optical axis. The modulation contrast is thereby most marked at the wavelength, of which the z-focus position coincides with the specimen surface. A z-focus position is stored in a value table for each wavelength such that a position of the specimen surface can be determined in z-direction from the determined wavelengths.

(23) The height profile of the specimen 60 can also be determined in a different way than with the modulation contrast. A cross-sectional image of the specimen can be calculated for each wavelength from the plurality of images for different grating phases of the structured element 30. A determination of the height profile can then be realised by selecting, from image points that correspond to each other in their position in the different cross-sectional images, the image point with the greatest intensity or the greatest contrast.

(24) The detection unit 80 comprises a smart pixel array detector in order to take into calculation the images for different grating phases. In order to reduce the measurement time it can additionally be provided that the grating phases are imaged onto the specimen through a polarisation encoding of the illuminating light.

(25) In addition or alternatively to the displacement of the structured element 30, a manipulation of the structured illuminating light 15 in a pupil plane of the objective 50 can also be realised. A grating of the structured element 30 is represented according to the Fourier transformation in the pupil plane essentially as a point pattern which corresponds to the individual orders of diffraction of the grating. Deflection means, for example an electro-optic modulator, an acousto-optic deflector or a galvanometer mirror, can be arranged in the pupil plane. An angular modulation of the illuminating light which corresponds in the pupil plane to a point pattern can hereby be realised, with which it is likewise possible to switch between different grating positions without displacement or rotation of the structured element 30 being necessary.

(26) Alternatively, the structured element 30 can also be formed with a two-dimensional pinhole array instead of the grating. In this case, only the light path 18 is used. The detector elements of the detection unit 80 can thereby be used as a digital pinhole, whereby only the regions of the different images of the specimen sharply imaged onto the detection elements are used to create a confocal image.

(27) A further embodiment of a light microscope 100 according to the invention is shown schematically in FIG. 2. Once again, illuminating light 15 of the light source 10 is guided onto a structured element 30, for which optical imaging means 32 as well as the components 12, 25, 90, 21, 22, 23 and 40 are used which have already been described in relation to FIG. 1.

(28) Illuminating light 15 transmitted through the structured element 30 is guided, here also, via a /4 plate and focussing means 50 onto the specimen 60.

(29) Unlike FIG. 1, however, the beam splitter 40 is not located here between the structured element 30 and the specimen 60, but instead between the light source 10 and the structured element 30. As a result, specimen light 16 leaving the specimen travels through the focussing means 50 and is imaged sharply onto the structured element 30 via optical imaging means 53. The structured element 30 is rotated through the electronic control means 90 and respective images of the specimen are recorded with the detection unit 80 at different rotation positions. A side of the structured element 30 which faces the specimen 60 is mirrored. Specimen light 16 is hereby partially reflected and partially transmitted at the structured element 30.

(30) The transmitted specimen light 16 is spatially separated from the illuminating light 15 with the beam splitter 40 and is detected with a first detector 81 of the detection unit 80. The structuring of the structured element 30 as a pinhole can hereby serve for confocal imaging. Specimen light 16 which is transmitted at the structured element 30 thereby has solely confocal portions. Non-confocal light portions are filtered out at the structured element 30. The detector 81 can thereby record a confocal image of the specimen 60.

(31) Illuminating light 16 which is reflected at the structured element 30 has on the other hand both confocal and also non-confocal portions. The reflected specimen light 16 is then imaged via optical imaging means 53, a deflection mirror 54, a polarisation filter 55 and via further optical imaging means 56 onto a second detector 82 of the detection unit 80. The second detector 82 thereby records a wide-field image of the specimen 60 which contains image information from different depths and not only from the focal position of the illuminating light 15.

(32) A depth resolution is also realised in this embodiment via the wavelength-dependent focal position 51 of the illuminating light 15.

(33) In the example shown, the two detectors 81 and 82 are formed through separate camera chips. Alternatively, however, a single camera chip can also be used, wherein the two detectors 81 and 82 represent different regions of the camera chip.

(34) The depth resolution, thus the resolution along the optical axis at the specimen 60, can be increased by measuring with better spectral resolution. An interferometer 70 is present for this purpose according to a further embodiment of a light microscope 100 according to the invention, which is shown in a cut-out in FIG. 3. By way of an optional extension, the interferometer 70 is also shown in FIGS. 1 and 2 between the focussing means 50 and the structured element 30.

(35) The interferometer 70 is designed here as a Linnik interferometer. It comprises a beam splitter 71, with which a proportion of the specimen light 16 is guided onto a reference path 72. On the reference path 72 there is a, preferably achromatic, objective 74 and also a mirror 75 which is arranged in the focus of the objective 74 and radiates specimen light 16 leaving the objective 74 back thereto. Optionally, the mirror 75 can also be moved via the electronic control means 90. By displacing the mirror 75 the length of the reference path 72 and hence the path difference required for the spectral interferometry can be adjusted.

(36) Furthermore a /4 plate 73 can be present between the beam splitter 71 and the objective 74 in order to rotate the polarisation direction of the specimen light 16.

(37) Illuminating light 15 which is transmitted at the beam splitter 71 travels, as described in relation to FIG. 1, via a /4 plate 41 and the focussing means 50 onto the specimen 60.

(38) Further information going beyond the chromatic information can advantageously hereby be obtained which can be used in relation to a height evaluation of the specimen. Indeed, the path difference which is to be adjusted with the interferometer 70 for a specified wavelength depends upon the distance of the specimen surface and hence upon the height profile of the specimen. The position of the mirror 75, with which a path difference for constructive interference at the specimen surface is adjusted, can likewise be used to determine the height profile.

(39) The detected light should hereby be measured with a spectral resolution in the order of 0.1 nm, which is not possible solely with a simple colour camera as a detection unit. It is thus preferable to use a tunable light source together with a spectrally resolving detection unit.

(40) Much information for a microscopic specimen can be obtained with the light microscope according to the invention. In particular, a confocal image and also a wide-field image of the specimen can thereby be recorded without the need to reorganise or remove components from the light microscope. Scanning of the specimen through mechanical displacement of components can be mostly omitted. Different xy-points of the specimen can thus be examined using structured illuminating light, whereby no repositioning of components is required or, at most, an adjustment of the structured element is necessary. A z-resolution is produced with the aid of a longitudinal chromatic aberration, wherein the z-resolution depends upon the measurement accuracy of the wavelength of the light to be detected. The wavelength can be determined particularly exactly in variants, in which the light source is tunable and the detection unit is additionally spectrally resolving.

LIST OF REFERENCE SIGNS

(41) 10 Light source 12 Optical imaging means, lens 15 Illuminating light 16 Specimen light 17 First light path 18 Second light path 20 Wavelength selection unit 21 Lens 22 Optical fibre 23 Lens 25 Deflection means 26 Lens 27 Optical fibre 28 Lens 29 Semi-transparent mirror 30 Structured element 31 Optical imaging means, lens 32 Optical imaging means, lens 40 Beam splitter, polarisation beam splitter 41 /4 plate 42 Polarisation filter 43 Optical imaging means, lens 48 Diffractive element with longitudinal chromatic aberration 49 Objective 50 Focussing means 51 Focus of the illuminating light 53 Optical imaging means, lens 54 Deflection mirror 55 Polarisation filter 56 Optical imaging means, lens 60 Specimen 70 Interferometer 71 Beam splitter 72 Reference path 73 /4 plate 74 Achromatic objective 75 Mirror 80 Detection unit 90 Electronic control means 100 Light microscope