WO2006102861A1

WO2006102861A1 - Scan microscope and phase contrast microscopy method

Info

Publication number: WO2006102861A1
Application number: PCT/DE2006/000397
Authority: WO
Inventors: Werner Knebel
Original assignee: Leica Microsystems Cms Gmbh
Priority date: 2005-04-01
Filing date: 2006-03-06
Publication date: 2006-10-05
Also published as: DE102005015294A1

Abstract

A scan microscope, especially a confocal scan microscope, comprises a light source (1) for producing an illumination light (2), a lens system (17, 21) for guiding the illumination light (2) to a sample plane of a sample (19), and a position detector (28) for measuring the lateral deflection, caused by the sample (19), of an illumination light passing through the sample (19). In order to allow for an especially high dissolution, at least one lens system (17, 21) is arranged on each side of the sample plane and at least two lens systems (17, 21) which are opposite each other in relation to the sample plane are directed towards each other in such a manner that the illumination light (2) guided through the lens (17, 21) towards the sample (19) can be collected by the opposite lens system (21, 17) and guided to the position detector (28) as the detection light. The invention also relates to a phase contrast microscopy method using the inventive scan microscope.

Description

SCAN MICROSCOPE AND A METHOD FOR PHASE CONTRAST MICROSCOPY

The present invention relates to a scanning microscope, in particular confocal scanning microscope, with a light source for generating an illumination light, with a lens for guiding the illumination light to a sample plane of a sample and a position detector for measuring the caused by the sample lateral deflection of an illuminated light passed through the sample. Furthermore, the invention relates to a method for phase contrast microscopy with such a scanning microscope.

US Pat. No. 4,745,270 discloses a scanning microscope in which a phase contrast with respect to a sample is examined by means of a position detector arranged in the beam path after the sample. In this case, an illumination light is guided via an objective to a sample plane of a sample and a lateral deflection of the illumination light which has passed through the sample is determined by means of the position detector.

Samples of living material, such as cells, cell cultures and tissue sections, leave the amplitude of the light virtually unchanged. It is difficult or even impossible to stain these samples, which is necessary for bright field observation, as this intervention would have profound changes to the object. In such preparations, local differences in the refractive index influence the phase position of the light wave and one speaks of phase objects. With optical contrasting methods, such as dark field, phase and interference contrast methods, phase objects can also be observed.

Various contrasting methods are customary, for example the phase contrast method according to Zernecke, the Hoffman modulation contrast method (see, for example, DE 39 26 199 A1) or the DODT contrast method, which is known from DE 195 14 358 C2.

In the DODT method, a segment aperture is arranged in a Fourier plane and a segment polarization optics in the Hoffman method. The biology preferred method is Differential Interference Contrast (DIC). According to Nomarski (G. Nomarski, J. Rad. Phys. 16 (1955) 9.) In the differential interference contrast method, the linearly polarized illumination light in front of the objective is transformed into two linearly polarized partial beams by means of a Wolfaston prism, the polarization directions being perpendicular to one another have, spatially split. The partial beams pass through the sample in slightly different ways to be united after the condenser by means of a second Wollaston prism. If the two partial beams have covered optical path lengths of different lengths, this is manifested after the beam combination in the polarization properties of the reunited light beam-as a rule elliptically polarized light-which can be detected with the aid of a polarizer as the analyzer.

The differential interference contrast method (DIC) is disadvantageous in birefringent samples due to the polarization-influencing effect of these samples is not or only very limited use, since among other things in the image analysis is indistinguishable, whether the signals on the sample birefringence or on a variation of the refractive index within the Sample are due. Likewise, the use of birefringent sample containers or birefringent slides, such as plastic, is not possible.

From T. Wilson, "Enhanced differential phase contrast imaging in scanning microscopy using a quadrant detector", Optics, 80, No. 4 (1987) 167-170, is known, the angular deflection of a focused laser beam as a function of the focus position with a quadrant detector to This method provides usable results only as long as the beam intensity remains largely constant during scanning of the sample, and the optical resolution properties of this method are much poorer for thick samples than for the differential interference contrast method that this method - as far as it has spread at all - can only be used for very thin samples. The present invention is therefore based on the object of specifying a scanning microscope and a method for phase contrast microscopy with such a scanning microscope, according to which a particularly high resolution is achieved with structurally simple means.

According to the invention, the above object is achieved by a scanning microscope having the features of patent claim 1. Thereafter, the scanning microscope of the aforementioned type is configured and further developed such that at least one objective is arranged on both sides of the sample plane and that at least two mutually opposite with respect to the sample plane lenses are directed towards each other, so that guided through an objective to the sample illumination light through the opposite lens can be captured and can be guided as a detection light to the position detector.

According to the invention, it has been recognized that the above object is achieved in a surprisingly simple manner by the guidance of the illumination light which has passed through the sample via a further objective to the position detector. In concrete terms, at least one objective is arranged on both sides of the sample plane for this purpose, wherein at least two lenses lying opposite one another with respect to the sample plane are directed toward one another. Illumination light guided through a lens to the sample can thus be picked up by the opposing objective and guided as a detection light to the position detector.

With the scanning microscope according to the invention, a particularly high resolution is achieved, regardless of the thickness of the sample, in particular in living biological specimens, and furthermore the examination of polarization-influencing - in particular birefringent - specimens is made possible. Consequently, a scanning microscope is specified with the scanning microscope according to the invention, according to which a particularly high resolution is achieved with structurally simple means.

A further improvement of the resolution can be achieved if the illuminating light is guided through two lenses which lie opposite one another and are directed towards one another with respect to the sample plane and are collected by the respectively opposite objective and guided as a detection light to the position detector becomes. In other words, in this case the illumination light is guided not only by an objective, but by two lenses which lie opposite one another with respect to the sample plane and which are directed towards one another. The sample is illuminated from two sides. The illumination light of the one lens that has passed through the sample is thereby picked up by the respectively opposite objective and guided as a detection light to the position detector. In this case, the position detector receives both the detection light which has been picked up by the one objective and the detection light which has been picked up by the opposing objective.

Furthermore, to ensure a particularly high resolution, a detection pinhole could be arranged in the beam path of the detection light in front of the position detector. With such a detection pinhole can be ensured that only detection light, which is modulated in the focus of the lenses by the sample passes through the detection pinhole and thus on the position detector. Both in the case of one-sided illumination of the sample and in the case of the alternative illumination of the sample on both sides, a very narrow focus point can be viewed. This advantage results in particular in a confocal operation of the scanning microscope. Finally, detection light portions that do not originate from the desired narrow focus area are filtered out by the detection pinhole.

With regard to a particularly simple realization of the illumination of the sample from two sides, a beam splitter could be arranged in the beam path of the illumination light in front of the objective or in front of the objectives. The beam splitter serves for splitting the illumination light and for combining the detection light.

For reliable guidance of the illumination light over the sample, a scanning device could be arranged in the beam path of the illumination light in front of the objective or the objectives. Such a scanning device can be realized by mirrors. In this case, the illumination light which has passed through the sample could be feasible as detection light via the scanning device and / or a further scanning device can be guided to the position detector. In a structurally particularly advantageous embodiment, the position detector could be an RDC detector - ret ractive differential contrast detector.

The position detector could comprise a plurality of individual detectors which have a substantially smaller optically effective detection area than the cross-sectional area of a detection light beam. In concrete terms, the individual detectors could be arranged in a row or in an array or in a matrix. In this case, a photodiode line or a photodiode array is suitable.

In a further embodiment, the position detector could comprise a photomultiplier. In this case, a multi-channel or multi-anode photomultiplier would be particularly advantageous. The photomultiplier could be formed in a further advantageous manner as a resistive anode photomultiplier. In a further variant, the position detector could have a microchannel PIate. When choosing the suitable photomultiplier, the respective application should be considered.

Furthermore, the position detector could have at least one CCD chip or a position-sensitive detector - PSD.

In a particularly preferred embodiment, the position detector could have a substantially continuous point resolution function. In this case, the position detector could advantageously be designed as a PSD position sensitive detector or device. A position sensitive detector - PSD - works much like a standard photodiode. The light falling on the active area generates a photocurrent that flows in the direction of the p and n areas. Unlike a photodiode, however, a PSD has multiple electrical contacts. This results in a division of the photocurrent under the contacts as a function of the position of the light spot. The position is determined by forming the current difference between two opposing contacts. A one-dimensional PSD allows continuous positioning of a light spot along an axis. Particularly advantageous is an embodiment variant in which the position detector has a two-dimensional PSD, which enables a continuous position determination along two axes - for example X and Y direction. In a particularly preferred embodiment with a two-dimensionally detecting position detector, the shear direction could be rotated by preferably electronically processing the signals of the first and second dimensions. In the simplest variant, by swapping the signals, a 90 ° rotation of the shear direction is achieved. It is also possible, by weighting the signals of the first and second dimensions, to achieve a rotation of the shear direction by an arbitrary angle.

In any case, the position detector could be coupled to an electronic processing device, wherein an AC coupling is particularly advantageous. In an advantageous embodiment, logarithmic current-voltage converters are provided for eliminating the image-impairing consequences of fluctuations in the light output of the illumination light, as occur, for example, when using gas lasers, in order to further process the signals of the position detector in the context of the processing device. For this purpose, the processing device could have at least one logarithmic current-voltage converter. Particularly in confocal fluorescence scanning microscopy, in which the light output of the illumination light must be varied in order to achieve sufficient fluorescence excitation, the use of logarithmic current-voltage converters is particularly advantageous, and in the variant in which the position detector has a PSD, advantageously after the current-voltage conversion, the difference of the signals with respect to the two measuring directions is generated. This variant is particularly advantageous for simultaneously recording a phase contrast image and at least one fluorescence image of the sample. In each case, a phase contrast image could be recorded by means of the position detector, wherein at the same time other observation techniques could be used, for example in the context of two-photon excitation, FRET or CARS.

The simultaneous recording of a fluorescence and a phase contrast image makes it possible, for example, to position micropipettes or micro-patch clamps visually in a fluorescence sample. In a preferred embodiment, the position detector is - as already mentioned - coupled to an electronic processing device. The coupling is preferably an AC coupling, whereby the requirement for the centering of the position detector with respect to the optical axis is considerably reduced, since in such an arrangement systematic lateral shelves do not manifest themselves in an offset current.

Preferably, the position detector is arranged in a plane conjugate to the sample plane or focal plane-Fourier plane. The focused illuminating light cone emerging from the microscope objective is deflected by the phase object in the focal plane of the objective, so that the divergent cone emanating from the focal plane has a rather tilted central axis with respect to the incident illumination light cone. The degree of tilt is a direct measure of the variation in refractive index at the location of the pixel to be observed. In a plane conjugate to the focal plane, this tilting of the central axis acts as a parallel displacement of the collimated illuminating light beam passing through the sample. It is particularly advantageous to position the position detector in such a conjugate plane to measure the generated parallel displacement there.

In a further advantageous embodiment, a DIC optics - differential interference contrast optics - could be arranged in the beam path.

In a further alternative embodiment, in each case a Wollaston prism could be arranged in the beam path in front of the objective or the objectives and after the sample.

In a specific embodiment, the scanning microscope could be a 4Pi microscope. As a result, a confocal transmission image can be generated. With the scanning microscope according to the invention can be generated from a phase information - such as refractive differences - an amplitude contrast without disturbing background information. In contrast to a conventional transmitted-light detector, the transmitted light can be collected again with the aid of the second objective, the raster process can be canceled again via the return path via the scanning mirror and thus the light can be focused onto the detection pinhole. Light that has been scattered, broken or bent in the sample is filtered out at the pinhole. This allows a completely new view of the transmission images, with only information from the focal plane being displayed. The blur from the other focal planes is suppressed by the detection pinhole.

According to the invention the above object is further achieved by a method for phase contrast microscopy with the features of claim 24. Thereafter, the method is configured such that in each case at least one objective is arranged on both sides of the sample plane and that at least two mutually opposite with respect to the sample plane lenses are directed towards each other, so that guided by a lens to the sample illumination light collected by the opposite lens and as Detection light is guided to the position detector.

In an advantageous embodiment of the method, the illumination light could be guided by two mutually opposite with respect to the sample plane and facing each other lenses and collected by the respective opposite lens and guided as a detection light to the position detector.

With the method according to the invention, a phase contrast image could be recorded by means of the position detector, wherein simultaneously a phase contrast image and at least one fluorescence image could be recorded.

With regard to the advantages of the method according to the invention, in order to avoid repetition, reference is made to the advantages of a corresponding scanning microscope described above for carrying out the method according to the invention.

There are now various possibilities for designing and developing the teaching of the present invention in an advantageous manner. This is on the one hand on the subordinate claims, on the other hand, to refer to the following explanation of a preferred embodiment of the teaching of the invention with reference to the drawing. In connection with the explanation of the preferred embodiment of the teaching according to the invention with reference to the drawing, generally preferred embodiments and developments of the teaching are explained. In the drawing show

Fig. 1 in a schematic representation of an embodiment of a scanning microscope according to the invention and

2 shows a further schematic representation of a processing device connected to the scanning microscope with a monitor.

Fig. 1 shows a scanning microscope according to the invention with a light source 1, which is designed as a multi-line laser. The scanning microscope is designed as a confocal scanning microscope. The light source 1 generates an illumination light 2, which is focused by an optical system 3 on a illumination pinhole 4. The illumination light 2 which has passed through the illumination pinhole 4 passes to a main beam splitter 5 and is directed by the latter via a further optical system 6 to a scanning device 7, which has a gimbal-mounted scanning mirror 8. The scanning device 7 directs the illumination light 2 through a scanning optical system 9, via a deflection mirror 12, through a tube optical system 14, via a deflection mirror 15 and through an objective 17 to a sample 19, which may be a brain section, for example. The pupil of the objective 17 is identified by the reference numeral 16.

Optics 10 and 11 are necessary for the adaptation of the 4Pi module to the confocal beam path, with a beam splitter 13 dividing the illumination light 2 in the direction of the objective 17 and / or 21. The 4Pi module, which can be flanged to the remaining components of the scanning microscope, is essentially formed by the beam splitter 13 and the components provided in the subsequent beam path, in particular optics 14 and 24 as well as objectives 17 and 21. The sample 19 is located on a slide 20 and is optically coupled to the lens 17 with a first immersion agent 18, which may be, for example, water, glycerol or oil. On the side facing away from the sample 19 of the slide 20, an objective 21 is arranged, which is optically coupled with a further immersion agent, which may be, for example, water, oil or glycerol.

The objective 21 collimates the illumination light beam 2 passed through the sample 19 in a plane 25 corresponding to the rear focal plane 22 of the objective 21 and in a further corresponding plane 29 via a deflection mirror 23 by means of a lens 24, the optics 11 and 10, the scanning optics 9, the optics 6 and another lens 27th

In the beam path to the lens 27, a position detector 28 is arranged, which is designed as a position-sensitive device (PSD). The rear focal plane 22 of the objective 21 and the plane 29 corresponding thereto are Fourier planes to the focal plane of the objective 17.

The position detector 28 measures the lateral deflection of the illumination light 2 caused by the sample 19, which shows at the location of the position detector 28 in a substantially parallel displacement of the collimated illumination light 2 passing through the sample 19. The position detector 28 detects the lateral deflection in two dimensions - X- and Y-direction -, wherein in the figure 1 for the sake of clarity, only the wiring is shown for a dimension.

With respect to the dimension shown, the Position Sensitive Device (PSD) has two contacts, each connected to a logarithmic current-to-voltage converter. The signals of the two logarithmic current-voltage converter are passed to a differential amplifier and forwarded the output signal generated by this to a PC for further image processing and display. The PC is not shown in FIG. 1 for the sake of clarity. The logarithmic current-voltage converters and the differential amplifier are part of an electronic processing device 30. For example, as a position-sensitive device, the model two L4SP from the company Sitek can be used.

The scanning microscope shown in Figure 1 can ultimately be operated in two ways. On the one hand, the illumination light 2 can only be guided through an objective 17 or 21 and, after its passage through the sample 19, can then be picked up by the other objective 21 or 17 and guided to the position detector 28. Alternatively, an illumination can be effected via both objectives 17 and 21 and then a corresponding collection of the detection light which has passed through the sample 19 through the corresponding objectives 21 and 17. In both cases, the detection light is processed by means of the position detector 28 and by means of the processing device 30.

The illuminating light 2 emanating from the light source 1 additionally comprises a wavelength component for the optical fluorescence excitation of the sample 19. The fluorescent light emanating from the specimen 19 passes through the objective 17 and / or the objective 21, the tube optics 14 and / or 24, the scanning optics 9 as well the further optics 11 and 10 via the scanning device 8 and the optics 6 to the main beam splitter 5. The fluorescent light passes through the main beam splitter 5 and the subsequent detection pinhole 26 and finally passes through another beam splitter 31 to a detector 32 which is designed as a photomultiplier or APD. The detector 32 generates electrical, proportional to the power of the fluorescent light signals, which are also given to further image processing and presentation to the PC, not shown.

With the scanning microscope shown, it is thus possible to simultaneously generate both a phase contrast image and a fluorescence image of the sample 19 in order to display them separately and also superimposed if necessary.

FIG. 2 shows a schematic representation of the lens 27, the position detector 28 and the processing device 30, wherein a control unit 33 and a monitor 34 are coupled to the processing device 30. A phase contrast image and / or a fluorescence image of the sample 19 can be viewed via the monitor 34. With regard to further advantageous embodiments of the teaching of the invention, reference is made to avoid repetition to the general part of the specification and to the appended claims.

Finally, it should be expressly understood that the embodiment described above is only for the purpose of discussion of the claimed teaching, but does not limit the embodiment.

Claims

Patent claims

1. Scanning microscope, in particular confocal scanning microscope, with a light source (1) for generating an illumination light (2), with a lens (17, 21) for guiding the illumination light (2) to a sample plane of a sample (19) and a position detector (28 ) for measuring the lateral deflection caused by the sample (19) of an illumination light (2) which has passed through the sample (19), characterized in that at least one objective (17, 21) is arranged on both sides of the sample plane and at least two mutually with respect to the sample plane opposite lenses (17, 21) are directed towards each other, so that by a lens (17, 21) to the sample (19) guided illumination light (2) by the opposite lens (21, 17) trappable and as a detection light to the position detector (28) is feasible.

2. Scanning microscope according to claim 1, characterized in that the illumination light (2) by two opposite each other with respect to the sample plane and facing each other lenses (17, 21) feasible and by the respective opposite objective (21, 17) trappable and as a detection light for Position detector (28) is feasible.

3. Scanning microscope according to claim 1 or 2, characterized in that in the beam path of the detection light in front of the position detector (28) a detection hole aperture (26) is arranged.

4. Scanning microscope according to one of claims 1 to 3, characterized in that in the beam path of the illumination light (2) in front of the lens (17, 21) or the lenses (17, 21), a beam splitter (13) is arranged.

5. Scanning microscope according to one of claims 1 to 4, characterized in that in the beam path of the illumination light (2) in front of the lens (17, 21) or the lenses (17, 21) a scanning device (7) is arranged.

6. Scanning microscope according to one of claims 1 to 5, characterized in that the through the sample (19) has passed illumination light (2) as Detekti- onslicht on the scanning device (7) and / or another scanning device to the position detector (28) can be guided ,

7. Scanning microscope according to one of claims 1 to 6, characterized in that the position detector (28) is an RDC detector - refractive differential contrast detector - is.

8. Scanning microscope according to one of claims 1 to 7, characterized in that the position detector (28) comprises a plurality of individual detectors having a substantially smaller optically effective detection area than the cross-sectional area of a detection light beam.

9. Scanning microscope according to claim 8, characterized in that the individual detectors are arranged in a row or in an array.

10. Scanning microscope according to one of claims 1 to 9, characterized in that the position detector (28) comprises a photomultiplier.

11. Scanning microscope according to claim 10, characterized in that the photomultiplier (28) is designed as a multi-anode photomultiplier.

12. A scanning microscope according to claim 10, characterized in that the photomultiplier (28) is designed as a resistive anode photomultiplier.

13. Scanning microscope according to one of claims 1 to 12, characterized in that the position detector (28) has a micro-Chanel plate.

14. Scanning microscope according to one of claims 1 to 13, characterized in that the position detector (28) has at least one CCD chip.

15. Scanning microscope according to one of claims 1 to 14, characterized in that the position detector has a continuous point resolution function.

16. Scanning microscope according to one of claims 1 to 15, characterized in that the position detector (28) to an electronic processing device (30) coupled - preferably AC coupled - is.

17. Scanning microscope according to claim 16, characterized in that the processing device (30) has at least one logarithmic current-voltage converter.

18. Scanning microscope according to one of claims 1 to 17, characterized in that the position detector (28) in a plane conjugate to the sample plane - Fourierbene - is arranged.

19. Scanning microscope according to one of claims 1 to 18, characterized in that by means of the position detector (28), a phase contrast image is receivable.

20. Scanning microscope according to one of claims 1 to 19, characterized in that simultaneously a phase contrast image and at least one fluorescence image are receivable.

21. Scanning microscope according to one of claims 1 to 20, characterized in that in the beam path, a DIC optics - Differential Interference Contrast optics - is arranged.

22. Scanning microscope according to one of claims 1 to 21, characterized in that in the beam path in front of the lens or the lenses and after the sample in each case a Wollaston prism is arranged.

23. Scanning microscope according to one of claims 1 to 22, characterized in that the scanning microscope is a 4Pi microscope.

24. A method for phase contrast microscopy with a scanning microscope, in particular confocal scanning microscope, according to one of claims 1 to 23, with a light source (1) for generating an illumination light (2), with a lens (17, 21) for guiding the illumination light (2) to a sample plane of a sample (19) and a position detector (28) for measuring the lateral deflection of the sample (19) caused by an illumination light (2) passing through the sample (19). , characterized in that at least one objective (17, 21) is arranged on both sides of the sample plane and that at least two lenses (17, 21) lying opposite each other with respect to the sample plane are directed toward one another, so that a lens (17, 21) to the sample (19) guided illumination light (2) by the opposite objective (21, 17) is collected and fed as detection light to the position detector (28).

25. Scanning microscope according to claim 24, characterized in that the illumination light (2) guided by two mutually relative to the sample plane and facing each other lenses (17, 21) and collected by the respective opposite objective (21, 17) and as a detection light for Position detector (28) is guided.

26. A scanning microscope according to claim 24 or 25, characterized in that by means of the position detector (28), a phase contrast image is taken.

27. Scanning microscope according to one of claims 24 to 26, characterized in that a phase contrast image and at least one fluorescence image are recorded simultaneously.