EP1678544A1

EP1678544A1 - Method for analysing a sample and microscope for evanescently illuminating the sample

Info

Publication number: EP1678544A1
Application number: EP04787190A
Authority: EP
Inventors: Andreas Hecker; Heinrich Ulrich; Werner Knebel; Kyra MÖLLMANN
Original assignee: Leica Microsystems Heidelberg GmbH; Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2003-09-25
Filing date: 2004-09-22
Publication date: 2006-07-12
Also published as: WO2005031427A1; US7633622B2; US20080151226A1

Abstract

The invention relates to a microscope (1) for evanescently illuminating (9) a sample, and to a method for sample analysis. According to the invention, a first evanescent field having a first penetration depth in the sample is produced, in addition to a second evanescent field having a second penetration depth in the sample (11), that is deeper than the first penetration depth. Said microscope comprises a sensor (43) that detects first detection light emitted from the part of the sample (11) illuminated by the first evanescent field, producing first detection light data therefrom, and detects second detection light emitted from the part of the sample (11) illuminated by the second evanescent field, producing second detection light data therefrom. The inventive microscope also comprises a processing module (45) for processing the first and second detection light data (37).

Description

Sample examination method and microscope with evanescent sample illumination

The invention relates to a method for microscopic examination of a sample.

The invention also relates to a microscope with evanescent sample illumination.

A microscope with evanescent illumination of a sample is known from US 2002/0097489 A1. The microscope contains a white light source, the light of which is coupled via a slit diaphragm through the microscope objective into the specimen slide for evanescent illumination. The illuminating light propagates in the slide by total internal reflection, the sample being illuminated only in the area of the evanescent field protruding from the slide. Microscopes of this type are known under the term TIRFM (Total Internal Reflection Fluorescent Microscope).

The z-resolution of TIRF microscopes is extremely good due to the evanescent field that only projects into the sample by approx. 100 nm.

DE 101 08 796 A1 discloses a high-aperture lens, in particular for TIRF applications. The lens consists of a first lens a positive refractive power, a second lens with a negative refractive power, the focal length ratio between the two lenses being in the range of - 0.4 and - 0.1 and the total refractive power being greater than zero. Furthermore, the lens contains two positive lenses, whose ratio diameter to the focal length is greater than 0.3 and less than 0.6. The objective also includes a negative lens and a converging lens, the negative lens facing the front group and the focal length ratio of the negative lens and the converging lens being between −0.5 and −2.

From DE 102 17 098 A1 an incident light arrangement for TIRF microscopy is known. The incident light illuminating arrangement contains an illuminating source that is polarized during operation

Outputs illuminating beam that propagates at an angle to the optical axis and a deflection device that deflects the illuminating beam and couples it into the lens parallel to the optical axis. It is provided in this incident light illumination arrangement that the illumination beam emitted by the illumination source has s and p polarization directions with a phase difference and the deflection device reflects the illumination beam x times, where x = (n x 180 ° -d) / 60 °. A microscope for TIRM (Total Internal Reflection Microscopy) is known from DE 101 43 481 A1. The microscope has a microscope housing and an objective. The illuminating light emanating from an illuminating device can be coupled in via an adapter which can be inserted into the microscope housing. From US 2004/0001253 A1 is a microscope with an optical lighting system that enables simple switching between evanescent lighting and reflection lighting. The

Illumination system includes a laser light source, the light of which is coupled into an optical fiber. A coupling optic is also provided, which focuses the light emerging from the fiber into a rear focal point of the microscope objective. The optical fiber can be displaced in a plane perpendicular to the optical axis of the microscope objective. DE 102 29 935 A1 discloses a device for coupling light into a microscope. Laser light is directed onto the specimen in the light field diaphragm plane by an optical fiber coupling designed as a slide. The invention is particularly suitable for the TIRF process.

The previously known techniques for evanescent sample illumination only allow the examination of the sample layers that are directly adjacent to the cover glass or directly to the slide.

It is an object of the present invention to provide a method for microscopic examination of a sample with evanescent sample illumination that is not limited to the sample layers that directly adjoin the cover glass or the slide, and also a 3-D examination of the sample allows. This task is solved by a method with the following steps: Illumination of the sample with a first evanescent field, the first evanscent field having a first depth of penetration into the sample, Detection of the first detection light which is illuminated by the one with the first evanscent field Part of the sample starts and generation of first detection light data, • illuminating the sample with a second evanescent field, the second evanscent field having a second depth of penetration into the sample which is greater than the first depth of penetration, • detecting a second detection light emitted by runs out of the part of the sample illuminated with the second evanscent field, and generate second detection light data, and • process the first and second detection light data. It is a further object of the present invention to provide a microscope which enables a 3-D examination of the sample with evanescent sample illumination.

This further object is achieved by a microscope, which is characterized in that a first evanscent field, which has a first depth of penetration into the sample, and a second evanscent field, which has a second depth of penetration into the sample, which is greater than the first depth of penetration , has, can be generated, and that at least one detector is provided, which detects the first detection light emanating from the part of the sample illuminated with the first evanscent field and generates first detection light data therefrom, and the second detection light that comes from that with the second evansescent Field illuminated part of the sample goes out, detected and second detection light data generated therefrom, and that a processing module is provided for processing the first and second detection light data.

The method according to the invention preferably comprises the further steps of illuminating the sample with one or more further evanescent fields of different penetration depth and detecting further detection light emanating from the part or parts of the sample illuminated with the further evansent field and generating of further detection light data. The processing then preferably comprises the first, second and further detection light data. According to the invention, a three-dimensional examination of the sample is consequently made possible by sequentially changing the penetration depth of the illuminating light. By increasing the penetration depth sequentially, additional sample layers are recorded. By - clearly speaking - "subtracting" the image data of the previous data - recorded at a lower penetration depth - from the data, which each comprise the additional sample layer, an exact assignment of individual image grid points and / or image objects to the layer depths can be made.

The first and second detection light data contain data According to the invention, data from image objects and / or from parts of image objects. The processing preferably includes assigning image objects to different layer depths (eg 20 nm - 40 nm, 40 nm - 60 nm, 60 nm - 80 nm ...) of the sample. This can be done with the help of a processing module.

In a special variant, the processing comprises the generation of a 3-D data stack, preferably with a processing module. This data stack or the data record generated by the processing module can preferably be represented as a three-dimensional image of the sample or a sample area - preferably on a display.

The microscope according to the invention preferably has a lens with a lens pupil, the first and / or the second and / or the further evanescent field being generated by an illuminating light beam which has a focus in the area of the lens pupil of the lens. In a preferred variant, the penetration depth of the first and / or the second and / or the further evanescent field can be set by adjusting the distance of the focus from the optical axis of the objective. For this purpose, an adjustment means can be provided with which the spatial position of the focus can be changed within the plane of the objective pupil. The setting means can comprise, for example, a beam deflection device with a plurality of rotating or tilting mirrors or with a gimbal-mounted mirror. The setting means can also be designed as an acousto-optical element or contain micromirrors. A displaceable optical fiber can also be used to adjust the spatial position of the focus of the illuminating light beam.

The angle with respect to the optical axis of the objective, at which the one intended for evanescent illumination of the sample

Illuminating light bundle leaves the lens depends on the spatial position of the focus in the lens pupil. The greater the distance between the respective focus and the optical axis, the greater the angle. Therefore, according to the invention, the distance of the focus from the optical axis of the lens to adjust the angle and thus to adjust the depth of penetration of the evanescent field into the sample.

In another advantageous variant, the penetration depth is set by adjusting the polarization of the illuminating light beam that generates the first and / or the second and / or the further evanescent field. To set the polarization of a phase plate, for example, a rotatable λ / 2 plate can be provided.

The microscope is advantageously calibrated - regardless of the method with which the penetration depth into the sample is set - so that reproducible examinations are made possible and quantitative statements about the nature of the sample - in particular about the arrangement of the individual sample components within the sample - are made can.

Detection is preferably carried out with at least one detector which comprises a camera and / or a CCD element and / or an EMCCD element and / or a multiband detector. Bandpass filters and / or edge filters are preferably arranged in front of the detector and are matched to the respective emission focal length of the fluorescence signal. A dispersive element can be provided for color selection, which generates a spectral splitting from which the wavelength components to be detected are masked out. The detector can also be designed as a color detector, for example as a color camera. It is also possible for a dispersive element to split the detection light over several detectors in order to achieve spectral detection. In one variant, the detector is designed as a point detector and the detection comprises a point-by-point scanning of the respectively illuminated part of the sample. For point-by-point scanning, a further adjustable beam deflection device can preferably be provided in the beam path of the detection light. In a very particularly preferred embodiment, the microscope comprises a scanning microscope; especially a confocal scanning microscope. The microscope according to the invention preferably has at least one multi-line light source and / or at least one broadband light source. The wavelengths or the wavelength of the first illuminating light beam and / or of the second illuminating light beam are preferably adjustable. According to the invention, it is possible for the first illuminating light beam and also the second illuminating light beam to contain light of one or more wavelengths, the wavelengths of the first illuminating light beam and the second illuminating light beam being able to differ from one another.

In a particularly preferred variant, the diameter of the illuminating light beam is adjustable. In particular, it is advantageous if the opening angle of the illuminating light beam bundle converging to form a focus lying in the objective pupil can be adjusted. By changing the opening angle when focusing in the objective pupil, the size of the surface that is illuminated evanescently changes.

The subject matter of the invention is shown schematically in the drawing and is described below with reference to the figures, elements having the same effect being provided with the same reference symbols. 1 shows a microscope according to the invention,

Fig. 2 shows another microscope according to the invention and

Fig. 3 is an illustration of the method according to the invention.

1 shows a microscope 1 according to the invention with an objective 3 and a light source 5, which is designed as a laser 7 and which generates an illuminating light beam 9. The illuminating light beam 9 emanating from the light source 5 serves for the evanescent illumination of a sample 11 which is attached to a specimen slide 13. The illuminating light beam 9 has a focus 19 represented by a point in the plane 15 of the objective pupil 17. In the beam path of the Microscope 1, several optical elements for beam guidance and beam shaping are arranged. For example, there are first optics 21, second optics 23 and optics 25 that generate a first intermediate image plane 27 and a second intermediate image plane 29. The spatial position of the focus 19 within the plane 15 of the objective pupil 17 can be changed with the aid of an adjusting means 31, which comprises an adjustable beam deflection device 33. The adjustable beam deflection device 33 includes a gimbaled rotating mirror, not shown. With the aid of the setting means 31, the distance of the focus 19 from the optical axis 35 of the objective 3 can be adjusted and thus the depth of penetration of the illuminating light beam into the sample 11 can be varied. The detection light 37 emanating from the sample 11 passes through the lens 3 and through the beam splitter 39, which directs the illuminating light beam 9 to the lens 3, to a detector 41 which comprises a CCD camera 43 and which generates detection light data. The beam splitter 39 is designed as a dichroic beam splitter and is designed such that light of the wavelength of the illuminating light beam is reflected while light of the wavelength of the detection light 37 can pass through.

First, a first distance between the focus of the illuminating light beam 9 and the optical axis 35 and thus a first penetration depth of a first evanescent field are selected. It happens

Detecting first detection light emanating from the part of the sample 11 illuminated with the first evanscent field, and that

Generation of first detection light data. The first detection light data are forwarded to a processing module 45. Then the distance between the focus of the illuminating light beam 9 and the optical axis 35 is increased and a second evanescent field is generated, the second

Depth of penetration into the sample is greater than the first depth of penetration. The second detection light is emitted, which emanates from the part of the sample 11 illuminated with the second evanscent field, and that

Generation of second detection light data. The second detection light data are also forwarded to the processing module 45. in the Processing module 45 uses the first and second detection light data to assign image objects to different layer depths of the sample and to generate a 3-D data stack which is shown as a three-dimensional image of the sample or the illuminated sample area on the display 47 of a PC 49.

FIG. 2 shows a microscope according to the invention in which the penetration depth of the first and / or the second and / or the further evanescent field is set by adjusting the polarization of the illuminating light beam. A λ / 2 plate 51 is rotatably arranged in the beam path of the illuminating light beam 9 with which the direction of polarization of the illuminating light beam 9 can be rotated. To monitor the set polarization, a beam splitter 53 is arranged in the further beam path of the illuminating light beam 9, which splits off a small part of the illuminating light beam 9 for polarization control as a measuring beam 55. The measuring beam 55 is split by a polarization beam splitter 57 into an s-polarized partial beam 63, which is detected by a first detector 59, and into a p-polarized partial beam 65, which is detected by a second detector 61. The polarization of the illuminating light beam 9 can be inferred from the ratio of the light powers measured with the first detector 59 and with the second detector 61. The rotary position λ / 2 plate 51 is set in accordance with the user specifications by means of a control circuit (not shown).

3 illustrates the method according to the invention. First, the sample is illuminated with a first evanescent field with a penetration depth of 20 nm, for example, and the first detection light for generating first detection light data 67 is detected. In this example, the first detection light data comprise 3 image objects. The sample is then illuminated with a second evanescent field with a penetration depth of 40 nm, for example, and the second detection light is detected to generate second detection light data 69. In this example, the second detection light data comprise 5 image objects, 3 of which are already from the first Detection light data are known. The sample is then illuminated with a further evanescent field with a penetration depth of 60 nm, for example, and the further detection light is detected to generate further detection light data 71. In this example, the further detection light data comprise 8 image objects, 5 of which are already known from the first detection light data and the second detection light data. The first, second and further detection light data are then processed 73. The processing includes an assignment of which image objects belong to first sample layer data 75 (0-20 nm), which second sample layer data 77 (20-40 nm) and which to third sample layer data 79 (40-60 nm). The data is stored as a 3-D data stack and can be displayed to the user, for example on a monitor.

The invention has been described in relation to a particular embodiment. However, it is understood that changes and modifications can be made without departing from the scope of the following claims.

LIST OF REFERENCE NUMBERS

1 microscope

3 lens

5 light source

7 lasers

9 illuminating light beams

11 rehearsal

13 slides

15 plane of the objective pupil 17

17 objective pupil

19 focus 1 first optics 3 second optics 5 optics 7 first intermediate image level 9 second intermediate image level 1 setting means 3 beam deflection device 5 optical axis 7 detection light 9 beam splitter 1 detector 3 CCD camera 5 processing module Display PC λy2 plate beam splitter measuring beam polarization beam splitter first detector second detector s-polarized partial beam p-polarized partial beam first detection light data second detection light data further detection light data processing first sample layer data second sample layer data third sample layer data

Claims

claims

1. A method for microscopic examination of a sample, characterized by the following steps: • Illuminating the sample with a first evanescent field, the first evanscent field having a first depth of penetration into the sample, • Detecting the first detection light from that with the first evanscent field illuminated part of the sample, and generating first detection light data, • illuminating the sample with a second evanescent field, the second evanscent field having a second depth of penetration into the sample that is greater than the first depth of penetration, • detecting a second detection light that starts from the part of the sample illuminated with the second evanscent field and generates second detection light data, and • processing the first and second detection light data.

2. The method according to claim 1, characterized by the further steps: • illuminating the sample with one or more further evanescent fields of different penetration depth and • detecting further detection light from the part or parts of the illuminated with the further evansentent field Sample runs out and generation of further detection light data, • processing of the first, second and further detection light data.

3. The method according to any one of claims 1 or 2, characterized in that the first and second detection light data from

Include picture objects and / or parts of picture objects and that the processing involves assigning picture objects to different layer depths the sample includes.

4. The method according to any one of claims 1 to 3, characterized in that the processing comprises generating a 3-D data stack,

5. The method according to any one of claims 1 to 4, characterized in that the illumination takes place through the objective of a microscope, the first and / or the second and / or the further evanescent field being generated by an illuminating light beam which is in the region of the objective pupil of the lens has a focus.

6. The method according to claim 6, characterized in that the

Penetration depth of the first and / or the second and / or the further evanescent field is set by adjusting the distance of the focus from the optical axis of the objective.

7. The method according to claim 6, characterized in that an adjusting means is provided with which the spatial position of the focus can be changed within the plane of the objective pupil.

8. The method according to claim 7, characterized in that the setting means comprises an adjustable beam deflection device arranged in the beam path of the illuminating light beam.

9. The method according to claim 6, characterized in that the

Penetration depth of the first and / or the second and / or the further evanescent field is set by adjusting the polarization of the illuminating light beam.

10. The method according to any one of claims 1 to 9, characterized in that the detection is carried out with at least one detector which comprises a camera and / or a CCD element and / or an EMCCD element.

11. The method according to any one of claims 1 to 9, characterized in that the detection is carried out by point-by-point scanning of the respectively illuminated part of the sample.

12. Microscope with evanescent sample illumination, characterized in that a first evanscent field, which has a first penetration depth into the sample, and a second evanscent field, which has a second penetration depth into the sample, which is greater than the first penetration depth, can be generated and that at least one detector is provided which detects the first detection light emanating from the part of the sample illuminated with the first evanscent field and generates first detection light data therefrom and the second detection light which emits from the part illuminated with the second evansent field Sample runs out, detected and second detection light data generated therefrom, and that a processing module is provided for processing the first and second detection light data.

13. Microscope according to claim 12, characterized in that further evanescent fields of different penetration depth can be generated and that the detector detects further detection light emanating from the part or parts of the sample illuminated with the further evansentent field and generates further detection light data that can be processed with the processing module.

14. Microscope according to one of claims 12 or 13, characterized in that the first and second detection light data from

Include image objects and / or parts of image objects and that the processing module assigns captured image objects to different layer depths of the sample.

15. Microscope according to one of claims 12 to 14, characterized in that the processing module generates a 3-D data stack.

16. Microscope according to one of claims 12 to 15, characterized in that the processing module generates a data record which can be represented as a three-dimensional image of the sample or a sample area - preferably on a display.

17. Microscope according to one of claims 12 to 16, characterized characterized in that the microscope has a lens with a lens pupil and that the first and / or the second and / or the further evanescent field is generated by an illuminating light beam which has a focus in the area of the lens pupil of the lens.

18. Microscope according to claim 17, characterized in that the

Penetration depth of the first and / or the second and / or the further evanescent field can be adjusted by adjusting the distance of the focus from the optical axis of the objective.

19. Microscope according to claim 18, characterized in that an adjusting means is provided with which the spatial position of the focus can be changed within the plane of the objective pupil.

20. Microscope according to claim 19, characterized in that the setting means comprises an adjustable beam deflection device arranged in the beam path of the illuminating light beam.

21. Microscope according to claim 17, characterized in that the

Penetration depth of the first and / or the second and / or the further evanescent field can be adjusted by adjusting the polarization of the illuminating light beam.

22. Microscope according to claim 21, characterized in that a phase plate, preferably a λ / 2 plate, is provided for adjusting the polarization.

23. Microscope according to one of claims 12 to 22, characterized in that the detector comprises a camera and / or a CCD element and / or an EMCCD element.

24. Microscope according to one of claims 12 to 23, characterized in that the detector is a point detector and the detection comprises a point-by-point scanning of the respectively illuminated part of the sample.

25. Microscope according to claim 24, characterized in that a further adjustable beam deflection device is provided in the beam path of the detection light.