阅读说明：本技术 具有tirfm能力的显微镜及其操作方法 (TIRFM-capable microscope and method of operating same ) 是由 M·舒费尔 K·C·舒曼 J-R·克鲁格 F·怀恩豪森 宫薗侑也 D·克鲁格 于 2021-03-22 设计创作，主要内容包括：具有TIRFM能力的显微镜及其操作方法。具有TIRFM能力的显微镜包括用于产生和发射非相干激发光的第一光源。第一投影透镜系统将激发光投射到空间滤波器装置上,该空间滤波器装置位于与物镜的后焦平面(BFP)共轭的平面(cBFP)中,并利用二维图案对激发光进行空间滤波。物镜包括物镜透镜,该物镜透镜被设计和布置成将激发光引导到样本上并接收来自样本的荧光,其中,对于物镜的数值孔径NA-(Obj)和样本的折射率n-(spec),NA-(Obj)>n-(spec)适用。第一控制单元激活空间滤波器装置以选择或产生各种二维图案,并且选择或调整二维(特别是环形)图案的位置、形状和/或尺寸,从而产生对样本的TIRF照明。(A microscope with TIRFM capability and method of operation thereof. A TIRFM-capable microscope includes a first light source for generating and emitting incoherent excitation light. The first projection lens system projects the excitation light onto a spatial filter arrangement located in a plane (cBFP) conjugate to a Back Focal Plane (BFP) of the objective lens and spatially filters the excitation light with a two-dimensional pattern. The objective lens comprises an objective lens designed and arranged to direct excitation light onto the sample and to receive fluorescence light from the sample, wherein the numerical aperture NA for the objective lens Obj And refractive index n of the sample spec ，NA Obj >n spec The method is applicable. The first control unit activates the spatial filter device to select or generate various two-dimensional patterns and to select or adjust the position, shape and/or size of the two-dimensional, in particular annular, patterns, thereby generating TIRF illumination of the sample.)

1. A TIRFM-capable microscope, the TIRFM-capable microscope comprising:

a first light source (201, 301, 501a, 601) designed to generate incoherent excitation light and to emit the incoherent excitation light onto a first light path comprising in sequence a first projection lens system (202, 302, 502a, 602), a first spatial filter device (203, 303, 504, 603), a second projection lens system (204, 304, 306, 505, 604, 606) and an objective lens (206, 308, 507, 608),

wherein the first projection lens system (202, 302, 502a, 602) is designed to project the excitation light onto the first spatial filter device (203, 303, 504, 603) and the first spatial filter device (203, 303, 504, 603) is designed to spatially filter the excitation light with a two-dimensional pattern,

wherein the first spatial filter arrangement (203, 303, 504, 603) is in a first configuration in a plane (cBFP) conjugate to a Back Focal Plane (BFP) of the objective lens (206, 308, 507, 608),

wherein the objective lens (206, 308, 507, 608) comprises an objective lens designed and arranged to direct the excitation light onto a sample and to receive fluorescence light from the sample, wherein the numerical aperture NA for the objective lens (206, 308, 507, 608) is_ObjAnd the refractive index n of the sample_spec，NA_Obj>n_specIs applicable to

Wherein a first control unit is included and designed to activate the first spatial filter device (203, 303, 504, 603) to select or generate various two-dimensional patterns and to select or adjust the position, shape and/or size of the two-dimensional patterns such that TIRF illumination of the sample is generated.

2. TIRFM-capable microscope according to claim 1, characterized in that the control unit is designed to activate the first spatial filter device (203, 303, 504, 603) to provide a circular pattern and a circular pattern as two-dimensional patterns for switching between TIRF illumination and non-TIRF illumination.

3. The TIRFM-capable microscope of claim 2, wherein an outer radius of the annular pattern is less than or equal to a maximum of an extension of the back focal plane of the objective lens (206, 308, 507, 608), and an inner radius of the annular pattern is greater than or equal to a critical radius of the Back Focal Plane (BFP), the critical radius corresponding to a critical angle of total internal reflection in a Focal Plane (FP) of the objective lens (206, 308, 507, 608).

4. TIRFM-capable microscope according to claim 1, characterized in that a second spatial filter device (305, 605) is arranged in the first light path in a plane (cFP) conjugate to the Focal Plane (FP) of the objective (308, 608), and that the second spatial filter device (305, 605) is designed to spatially filter the excitation light with a plurality of different two-dimensional patterns, and that the first or second control unit is designed to activate the second spatial filter device (305, 605) to select or generate a series of two-dimensional patterns.

5. TIRFM-capable microscope according to claim 1, characterized in that the first spatial filter means (203, 303, 504, 603) is designed as aperture varying means with a plurality of circular and ring apertures.

6. TIRFM-capable microscope according to claim 5, characterized in that the first spatial filter means (203, 303, 504, 603) is designed as a rotatable aperture ring (603).

7. TIRFM-capable microscope according to claim 5, characterized in that the first spatial filter means (203, 303, 504, 603) are designed as a programmable spatial light modulator (203, 303, 504).

8. TIRFM-capable microscope according to claim 7, characterized in that the first spatial filter means (203, 303, 504, 603) is designed as a spatially transmissive light modulator (203, 303) or a spatially reflective light modulator (504).

9. TIRFM-capable microscope according to claim 8, characterized in that the second spatial filter means (305, 605) is designed as a programmable spatially transmissive light modulator or a spatially reflective light modulator.

10. The TIRFM-capable microscope according to claim 1, characterized in that at least one of the first projection lens system (202, 302, 502a, 602), the second projection lens system (204, 304, 306, 505, 604, 606) and the first spatial filter device (203, 303, 504, 603) is movably arranged, completely or partly along the first optical path, such that the first spatial filter device (203, 303, 504, 603) can be brought from the first configuration into a second configuration in which the first spatial filter device (203, 303, 504, 603) is arranged in a plane conjugate to a Focal Plane (FP) of the objective lens (206, 308, 507, 608).

11. TIRFM-capable microscope according to claim 1, characterized in that a third projection lens system is comprised and designed and arranged to conjugate the first spatial filter device (203, 303, 504, 603) with the Focal Plane (FP) of the objective lens (206, 308, 507, 608), wherein a switching optical unit is arranged between the first spatial filter device (203, 303, 504, 603) and the objective lens (206, 308, 507, 608), which switching optical unit is designed to switch the optical path between the second projection lens system (204, 304, 306, 505, 604, 606) and the third projection lens system.

12. The TIRFM-capable microscope according to claim 1, wherein a second incoherent light source (501b) is comprised and this second incoherent light source (501b) is designed to generate and emit incoherent excitation light, wherein the first light source (501a) and the second light source (501b) are each oriented towards the first spatial filter device (504) designed as a digital micromirror device, wherein the pivotable micromirror element of the digital micromirror device (504) directs excitation light from the first light source (501a) into the first light path in a first pivoting position and directs excitation light from the second light source (501b) into the first light path in a second pivoting position.

13. The TIRFM-capable microscope of claim 1, wherein at least one of the first light source (201, 301, 501a, 601) and the second light source (501b) comprises an LED or a plurality of LEDs, a combination of a bulb and a light conductor, or a combination of a laser light source and a dynamic diffuser.

14. Method of operating a TIRFM-capable microscope according to any of claims 1 to 13, characterized in that the center of the Back Focal Plane (BFP) of the objective lens (206, 308, 507) is found using a search pattern sequence of two-dimensional patterns on the first spatial filter device (203, 303, 504), each search pattern of the search pattern sequence having a single small pixel cluster for conveying excitation light on the first light path, which pixel cluster is moved on a search path within the search pattern sequence, wherein the position where the luminosity of the fluorescence light returned from the objective lens (206, 308, 507) has a maximum is recorded and the center of the circle in which a number of luminosity maxima have been found is determined as the center of the Back Focal Plane (BFP) of the objective lens (206, 308, 507).

15. The method of claim 14, wherein the search path is straight.

16. A method according to claim 14, characterized in that a plurality of search paths extend at various angles from the edge to the center of the first spatial filter means (203, 303, 504).

17. Method according to claim 14, characterized in that after an initial rough determination of the center of the Back Focal Plane (BFP) of the objective lens (206, 308, 507, 608) at least one of the following is made: running a sequence of search patterns with increasing finesse and running a control search pattern, wherein the found circles are scanned radially from different directions to determine the photometric maximum with the greatest sharpness and thus to determine the center accurately.

Technical Field

The invention relates to a TIRFM-capable microscope and a method of operating the same.

Background

Total Internal Reflection Fluorescence Microscopy (TIRFM), microscopy with TIRF illumination, involves the use of evanescent fields to excite fluorescence of a specimen or sample. To generate the evanescent field, the light is totally reflected inside a reflective element (e.g. a cover glass) at the interface with the sample. This makes use of the fact that: when the angle of incidence theta is calculated from normal to interface₁Exceeding the critical angle theta_c＝arcsin(n₂/n₁) At a smaller angle of incidence and with a lower refractive index n₂At the interface of the medium (2) and having a higher refractive index n₁Is totally reflected.

In this way, an optical field is formed in the sample on the glass beyond the interface, which dissipates exponentially perpendicular to the interface and has a typical penetration depth for visible light of 100-200 nm. If fluorescent molecules that can absorb light of the radiation wavelength are located in this region, they are excited to emit fluorescence. This fluorescence is known as Total Internal Reflection Fluorescence (TIRF). TIRF results in a well-confined area of generated fluorescence near the glass; the observed layer was only 100-200nm thin. This achieves a significantly better resolution along the optical axis than is the case with ordinary fluorescence microscopy or confocal microscopy.

Most TIRFM-capable microscopes (TIRF microscopy capable) use one objective for illumination and collection of fluorescence. For this purpose, the objective must be suitable for illuminating the sample at an angle exceeding the critical angle. Therefore, the numerical aperture NA of the objective lens must be larger than the refractive index n of the thinner medium₂This means that

NA＝n₁sinθ>n₂。

The position of the back focal plane where the light passes through the objective lens determines the angle of the focal plane where the light passes through the objective lens, according to the following equation

r＝f*NA＝f*n₁ sinθ，

Where r describes the radial distance of the through position of the ray from the central ray path of the optical system in the back focal plane of the objective lens and f describes the focal length or focal length of the objective lens. To generate an evanescent light field, the angle of the light in the focal plane must exceed a critical angle, and the distance of the beam from the center of the back focal plane must correspondingly exceed a critical radius r_c. Therefore, a microscope with TIRFM capability is typically designed as a retro-reflective microscope with an objective lens immersed in oil and a very high Numerical Aperture (NA) of up to 1.45 or more. This high numerical aperture allows for a flat illumination angle, wherein the excitation light is coupled at the edge of the objective lens such that the excitation light contacts the interface with the sample at a flat total reflection angle.

There are many possibilities to prevent light from also hitting the interface at angles smaller than the critical angle, which means that the light is not totally reflected at such angles. By using an incoherent light source, such as an LED or a light bulb, a circular aperture hiding a central portion of the light can be used at a point in the ray path that is conjugate to the Back Focal Plane (BFP) of the objective lens. Such an arrangement is known, for example, from US 6,597,499B 2. However, the use of such an aperture results in most of the light emitted by the light source being unused, which is further magnified due to the fact that the phase space (etendue) spanned by the microscope optics fits poorly with the etendue spanned by the light source, so that in some cases only about 1% of the emitted light is available to start excitation.

Another solution to avoid this problem is to use a laser light source in a so-called laser TIRFM, which is coupled into the light path of the microscope such that the light generated by the laser light source reaches the interface with the sample almost completely. However, this solution is more expensive and has the following disadvantages optically: light has only a very narrow spectrum and diffraction and interference effects that may damage the image occur due to the coherence of the laser light.

Disclosure of Invention

Based on this prior art, it was an object of the present invention to provide a microscope with TIRFM capability and a method of operating the same, which allow a high degree of measurement flexibility.

This object is achieved by a microscope with TIRFM capability comprising: a first light source designed to generate incoherent excitation light and emit the incoherent excitation light onto a first light path, the first optical path includes a first projection lens system, a first spatial filter device, a second projection lens system, and an objective lens in that order, wherein the first projection lens system is designed to project the excitation light onto the first spatial filter device, and said first spatial filter means is designed to spatially filter said excitation light with a two-dimensional pattern, wherein the first spatial filter arrangement is in a plane (cBFP) conjugate to a Back Focal Plane (BFP) of the objective lens in a first configuration, wherein the objective lens comprises an objective lens designed and arranged to direct the excitation light onto a sample and to receive fluorescence light from the sample, wherein the numerical aperture NA for the objective lens is determined._ObjAnd the refractive index n of the sample_spec，NA_Obj>n_specAnd wherein a first control unit is included and designed to activate the first spatial filter device to select or generate various two-dimensional patterns and to select or adjust the position, shape and/or size of the two-dimensional (in particular annular) patterns such that TIRF illumination of the sample is generated.

With a microscope with TIRFM capability according to the invention, a variant is further developed in which the objective is used both for collection and for illumination. Instead of a fixed circular aperture, a variable spatial filter arrangement is used. With the incoherent light used according to the invention, TIRF illumination of the sample according to the invention is achieved by selecting or creating an annular two-dimensional pattern, wherein the light in the path of the light rays remains only within the annular aperture thus created or selected, while the light in the central part of the pattern and outside the ring is blocked. By adjusting the size of the annular pattern, the depth of penetration of the evanescent field into the sample can be controlled, and so on.

The two-dimensional pattern that makes TIRF illumination possible does not have to be annular. It is sufficient to exclude the central part up to the critical radius. Outside this central portion beyond the critical radius, any pattern will result in TIRF illumination. However, the annular pattern ensures a relatively high luminosity and uniformity of illumination.

In an embodiment, the control unit is designed to activate the first spatial filter device to provide the annular pattern and the circular pattern as a two-dimensional pattern for switching between TIRF illumination and non-TIRF illumination. Using a circular pattern, where the excitation light is also transmitted within the critical radius, a microscope with TIRFM capability can also be used as an epi-fluorescence microscope with so-called epi-illumination, which passes through the objective lens like TIRF illumination. However, the epi-excitation light does not undergo total reflection; instead, it penetrates completely through the interface into the sample. The latter is thus illuminated throughout its thickness and excited to fluoresce. Thus, by selecting layers near the interface, a higher light output is obtained at the expense of spatial resolution.

Preferably, the outer radius of the annular pattern is smaller than or equal to the maximum of the extension of the back focal plane of the objective lens, and the inner radius of the annular pattern is larger than or equal to the critical radius of the back focal plane, which corresponds to the critical angle for total internal reflection in the focal plane of the objective lens. The limitation of the outer radius of the annular pattern ensures that the portion of the light that is allowed to pass through remains in the light path of the microscope and thus is not disturbed by light scattering, while the limitation of the inner radius of the annular pattern ensures that the critical radius does not fall below. By adjusting the inner radius, the penetration depth into the sample can also be controlled.

In an embodiment of the TIRFM-capable microscope, a second spatial filter device is arranged in the first light path in a plane (cFP) conjugate to the Focal Plane (FP) of the objective lens and designed to spatially filter the excitation light with a plurality of different two-dimensional patterns, and the first or second control unit is designed to activate the second spatial filter device to select or generate a series of two-dimensional patterns. The second spatial filter arrangement is arranged in a plane (cFP) conjugate to the focal plane of the objective lens such that the two-dimensional pattern of the second spatial filter arrangement results in a corresponding spatial distribution of the illumination light at the focal position, i.e. in the specimen or specimen. Combining the first and second filter means together makes TIRF microscopy with structured illumination (total internal reflection fluorescence structured illumination microscopy, TIRF-SIM) possible. In this case, the first spatial filter means impresses an annular pattern on the excitation light in a plane conjugate to the back focal plane of the objective lens to produce TIRF illumination, while the second spatial filter means impresses a sequence of structured patterns on the excitation light in a plane conjugate to the focal plane of the objective lens and in this way determines the location in the specimen illuminated by the excitation light. TIRF microscope with structured illumination (TIRF-SIM) is also a TIRFM capable microscope.

In a suitable embodiment of the variable spatial filter device, the first spatial filter device is designed as an aperture-changing device, in particular as a rotatable aperture ring, having a plurality of circular and annular apertures, or as a programmable spatial light modulator, in particular as a spatially transmissive light modulator or a spatially reflective light modulator, wherein in particular the second spatial filter device is designed as a programmable spatially transmissive or reflective light modulator. In one embodiment, the programmable Spatial Light Modulator (SLM) is an LCD matrix operating in transmission, whose individual pixels can be switched back and forth between a light transmissive state and a light opaque state (transmissive SLM), and in an alternative embodiment, the programmable Spatial Light Modulator (SLM) is a digital micromirror device (DMD or reflective SLM) having an array or matrix of pivotable micromirror elements. Both transmissive and reflective SLMs can be activated in a targeted manner such that they produce a specific two-dimensional pattern, in particular a ring, for example, with an adjustable inner radius and an adjustable outer radius. The fact that the spatial light modulator is freely programmable allows a TIRFM capable microscope to be flexibly constructed and operated with a variety of settings.

Embodiments of the microscope with TIRFM capability provide the possibility to switch between TIRF illumination and position selective epi illumination, because the first projection lens system is movably arranged, completely or partly, along the first optical path such that the first spatial filter means can reversibly go from the first configuration into the second configuration, the second projection lens system is movably arranged, completely or partly, along the first optical path such that the first spatial filter means can reversibly go from the first configuration into the second configuration, and/or the first spatial filter means is movably arranged, along the first optical path, such that the first spatial filter means can reversibly go from the first configuration into the second configuration, in this second configuration, the first spatial filter arrangement is arranged in a plane conjugate to the focal plane of the objective lens. In this way, TIRF filtering in a plane conjugate to the back focal plane of the objective lens is eliminated, thereby automatically setting the epi-illumination, which is spatially filtered by the first spatial filter arrangement in the second configuration.

In an embodiment, an alternative or additional possibility of switching between TIRF illumination and position-selective epi-illumination is given if a third projection lens system is included, which is designed and arranged to conjugate the first spatial filter device with the focal plane of the objective lens, wherein a switching optical unit is arranged between the first spatial filter device and the objective lens, which switching optical unit is designed to switch the optical path between the second projection lens system and the third projection lens system.

The possibility of illumination with two different light sources (e.g. with different chromatograms) is given in the following embodiments: the first spatial filter device is designed as a Digital Micromirror Device (DMD) when comprising a second incoherent light source designed to generate and emit incoherent excitation light, wherein the first light source and the second light source are each oriented towards the first spatial filter device designed as a digital micromirror device, wherein the pivotable micromirror element of the digital micromirror device guides excitation light from the first light source into the first optical path in a first pivot position and guides excitation light from the second light source into the first optical path in a second pivot position. In this manner, by activating the DMD, the light sources may be selected and switched between. In a further development, a light absorber is provided for each of the two light sources, which light absorber receives and absorbs a portion of the light from the light source which is not transmitted into the light path of the microscope.

In an embodiment, the first light source and/or the second light source comprises one LED or a plurality of LEDs, a combination of a bulb and a light conductor, or a combination of a laser light source and a dynamic diffuser. A dynamic diffuser is a diffuser that moves at high frequency (e.g., in the ultrasonic range) and thus destroys the coherence of the laser light. Thus, no interference effects and diffraction effects occur anymore, which may otherwise occur to a considerable extent, in particular, on the micromirror elements of the DMD.

The object of the invention is also achieved by a method according to the invention for operating a microscope with TIRFM capability as described previously, wherein the center of the back focal plane of the objective lens is found using a sequence of search patterns of a two-dimensional pattern on the first spatial filter device, each search pattern of the sequence of search patterns having a single small pixel cluster for delivering excitation light to the first light path, which pixel cluster moves within the sequence of search patterns over a (in particular straight) search path, wherein the positions at which the luminosity of the fluorescence light returned from the objective lens has a maximum are recorded, and the center of the circle in which a plurality of luminosity maxima have been found is determined as the center of the back focal plane of the objective lens

With this method, centering is possible, since the photometric maximum is all located on a circle corresponding to the critical angle for total reflection. The centering of the annular aperture pattern on this center already ensures an effective centering of the excitation light with respect to the spatial arrangement of the optical system of the microscope.

The plurality of search paths preferably extend at different angles from the edge to the centre of the first spatial filter means. If the optical system is centered close to the optimum centering, these radial search paths intersect the circle of critical radius substantially perpendicularly, so that the searched photometric maximum is shown particularly clearly in the photometric curve. Skewing the search path trajectory results in a broadening of the maxima, resulting in less accurate centering.

In order to optimize the centering in all cases, in an embodiment of the method, after an initial rough determination of the center of the back-focal plane of the objective lens, a sequence of search patterns with increasing fineness is run and/or a control search pattern is run, wherein the found circles are scanned radially from different directions to determine the photometric maximum with the greatest sharpness, so that the center is determined exactly.

In different embodiments, a TIRFM capable microscope according to the present invention may be switched between TIRF illumination and non-TIRF illumination, operated in TIRF-SIM mode, or operated in a switchable manner between a plurality of different light sources. It may also be arranged to combine these features and, by switching between the light sources, operate one of the plurality of light sources in TIRF and the other light source in epi-illumination, with a transition of the first spatial filter arrangement from a first configuration in the cffp plane to a second configuration in the cFP plane. In this way, for example, an overview mode with broadband epi-illumination may be switchably combined with a detail mode with narrowband TIRF illumination.

Other features of the invention will be apparent from the description of embodiments according to the invention and from the claims and the accompanying drawings. Embodiments in accordance with the present invention may implement individual features or a combination of features.

Within the scope of the present invention, features specified by "particularly" or "preferably" are understood as optional features.

Drawings

The invention is described below on the basis of exemplary embodiments and with reference to the accompanying drawings without limiting the general idea of the invention, whereby reference is made explicitly to the drawings for all details according to the invention which are not explained in more detail in the text. In the drawings:

figure 1 shows a schematic principle view of an optical device in the case of TIRF illumination,

figure 2 shows a schematic view of the optical components of a TIRFM capable microscope in a first embodiment,

figure 3 shows a schematic diagram of a second embodiment of an optical assembly with a TIRF-SIM capable microscope,

figure 4 shows a schematic diagram of the centering principle of a microscope with TIRFM capability,

figure 5 shows a schematic view of the optical components of a TIRFM capable microscope in a third embodiment,

figure 6 shows a schematic view of the optical components of a TIRFM capable microscope in a fourth embodiment,

FIG. 7 shows an example of luminosity measurement using a search path, an

Fig. 8 shows a luminance curve corresponding to the search path of fig. 7.

In the figures, identical or similar elements and/or components have in each case the same reference numerals, so that a renewed presentation is omitted in each case.

List of reference numerals

101 objective lens

102 light beam

103 collimated light beam

104 light beam

105 collimated light beam

201 light source

202 first projection lens system

203 first spatial filter arrangement

204 second projection lens system

205 deflection unit

206 objective lens

207 tubular lens

208 detector

301 light source

302 first projection lens system

303 first spatial filter device

304 front part of the second projection lens system

305 second spatial filter arrangement

306 rear portion of the second projection lens system

307 deflection unit

308 objective lens

309 tubular lens

310 detector

401 first spatial filter arrangement

402 searching for a cluster of pixels on a path

501a first light source

501b second light source

502a first projection lens system

502b third projection lens System

503a first light absorber

503b second light absorber

504 first spatial filter device

505 second projection lens system

506 deflection unit

507 objective lens

508 tubular lens

509 detector

601 light source

602 first projection lens system

603 aperture changing device

604 front part of the second projection lens system

605 second spatial filter device

606 rear of the second projection lens system

607 deflection unit

608 Objective lens

609 tubular lens

610 detector

BFP back focal plane

FP focal plane

cBFP plane conjugated with back focal plane

cFP focal plane conjugate plane

radius r

I luminosity

Detailed Description

Fig. 1 shows a schematic principle view of an optical device in the case of TIRF illumination. The objective 101 of the microscope, which may have one or more optical lenses and a large numerical aperture to enable TIRF microscopy, is shown as a black box. The propagation direction of the excitation light from the light source (not shown) is from bottom to top. The objective lens 101 has a focal plane FP and a back focal plane BFP, which are defined such that parallel light beams striking the objective lens 101 from the rear (bottom of fig. 1) are focused in the focal plane FP, and in the other direction, parallel light beams striking the objective lens 101 from the outside (top of fig. 1) are focused in the back focal plane BFP.

Two light beams 102, 104 are shown in fig. 1, which pass through the back focal plane BFP at two different points from a light source (not shown). Beam 102 passes through the center of the BFP while beam 104 passes at peripheral points. Both beams 102, 104 comprise light rays that pass through the two points at different angles, i.e. they are not collimated. The optical properties of the objective lens 101 are such that the rays of the central light beam 102 are collimated to form a light beam 103 exiting perpendicularly from the objective lens 101. The peripheral beam 103 is also collimated, but exits as beam 105 at an angle θ (r), depending on the distance or radius of the beam from the center of the BFP. This dependence explains why in the case of TIRF illumination, where total reflection should occur, only those rays of the BFP are allowed to pass which are located at a distance from the centre of the BFP which exceeds the critical radius.

In a practical TIRFM optical unit, this principle is then modified so that the aperture of the TIRF illumination is not placed directly in the back focal plane BFP of the objective lens 101, but in a plane cBFP conjugate to the BFP. Placing an appropriately sized annular aperture in the BFP has the same effect as filtering directly in the BFP, since the BFP is mapped into the BFP of objective lens 101 by subsequent optical elements in the ray path.

Fig. 2 shows a schematic diagram of the optical system of a TIRFM-capable microscope in a first embodiment. Incoherent light source 201 generates excitation light that is transmitted via a ray path to objective lens 206, which as shown in FIG. 1 has a front focal plane FP and a back focal plane BFP. The ray path comprises a first projection lens system 202 and a second projection lens system 204 and a deflection unit 205, which is configured, for example, as a dichroic mirror, by means of which deflection unit 205 excitation light is at least partially guided from the light source 201 into the objective 206. In a plane cfbp conjugate to the back focal plane BFP of the objective lens 206 between the first projection lens system 202 and the second projection lens system 204, a first filter device 203 is arranged, which first filter device 203 spatially filters the excitation light and is designed to be able to provide various two-dimensional patterns for spatial filtering. The activation is performed by a control unit (not shown). In this case the first filter means 203 can be designed as a transmissive SLM, i.e. with the aid of a programmable LCD matrix, the matrix points of which can be switched between a transparent state and a non-transparent state. The transmissive SLM may be activated to e.g. create a circular aperture with an inner diameter equal to or larger than the critical radius of total reflection from the objective lens 206. This arrangement produces TIRF illumination.

The fluorescence light excited in the sample arranged in the focal plane FP of the objective lens 206 is returned through the objective lens 206 to the TIRFM-capable microscope. After passing through the deflection unit 205 and the tube lens 207, it is incident on a detector 208, which detector 208 detects fluorescence and converts it into an analyzable electric signal. The deflection unit may be a beam splitter or a dichroic mirror with a transmission characteristic having an edge between the wavelength of the excitation light and the wavelength of the fluorescence light such that the excitation light is almost completely deflected and the fluorescence light is almost completely transmitted to the detector 208.

Fig. 3 shows a second exemplary embodiment of a TIRFM-capable microscope, which represents a variation of the first exemplary embodiment. Incoherent excitation light is generated by a light source 301 and is guided via a first projection lens system 302, a second projection lens system 304, 306 and a deflection unit 307 onto the focal plane FP of an objective lens 308, while, on the other hand, fluorescence light passes through the objective lens 308, the deflection unit 307 and a tubular lens 309 to be incident on a detector 310. However, in contrast to the first exemplary embodiment, the second projection lens system 304, 306 is divided into two parts, wherein between the first part 304 and the second part 306 of the second projection lens system 304, 306 a plane cFP is present which is conjugate to the focal plane FP of the objective lens 308. In the cFP, a second spatial filter means 305 is arranged, which is also programmable.

Since the first spatial filter means 303 is arranged in the cBFP, a circular aperture may be generated there, thereby generating TIRF illumination. The second spatial filter means in the cFP select which region of the focal plane FP is illuminated. In this way, TIRF microscopy with structured illumination (TIRF-SIM) was established.

An exemplary application of the operation of a TIRFM capable microscope is schematically illustrated in fig. 4. On the left side of fig. 4, the surface of a Digital Micromirror Device (DMD) is shown as an example of a spatial filter device 401, which spatial filter device 401 is arranged in a plane cffp conjugated to the back focal plane BFP of the objective lens. Instead of a DMD, a transmissive SLM may also be used. Overlaid above this plane is a circle corresponding to the critical angle of total reflection at the sample position in front of the objective lens and centered at the center of the cBFP. Also shown are pixel clusters 402 that are arranged on three search paths that extend from the edge to the center of the dmd at 120 ° angles to each other and are arranged partially outside and partially inside the critical radius. The pixel cluster 402 is marked with a different texture, which can also be found in the intensity curve shown on the right side of fig. 4. It can be recognized here that the intensity or luminosity I of the excited fluorescence within the critical radius r has only slightly varying amounts. Near the critical radius (or, correspondingly, the critical angle), the luminosity rises sharply, then falls sharply outside the critical radius and eventually disappears.

Therefore, the location of the critical angle center can be determined by measuring the maximum value of the luminosity of the fluorescence with respect to various search paths and calculating the center of a circle passing through a pixel cluster having the maximum luminosity.

For this pair, structured illumination according to the second exemplary embodiment of fig. 3 is not required; in contrast, the structuring is carried out in a plane conjugated to the back focal plane BFP of the objective lens. Each pixel cluster allows a non-collimated beam to pass, which, when passing through the focal plane FP of the objective lens, corresponds to a collimated beam passing through the interface between the sample holder and the sample at a certain angle and in a certain direction (see fig. 1), and is either fully reflected or not depending on the pass angle.

In fig. 5, the optical assembly of a TIRFM capable microscope in a third embodiment is schematically shown. In this exemplary embodiment, the microscope comprises two different incoherent light sources 501a, 501b, which can produce different spectra and are each oriented via their own first projection lens system 502a, 502b towards a first spatial filter arrangement 504 designed as a digital micromirror device arranged in a plane cBFP conjugate to the back focal plane BFP of the objective 507. The other components 505, 506, 507, 508, 509 correspond to the components 204, 205, 206, 207, 208 of the first exemplary embodiment. With regard to their function, reference is made to the description of the first and second exemplary embodiments.

The following describes a switching process between the first light source 501a and the second light source 501 b. The micro-mirror elements of the first spatial filter arrangement 504 may be pivoted between a first position and a second position. When excitation light from the first light source 501a is used, the micromirror elements needed to produce TIRF illumination are pivoted into a first position where the excitation light from the first light source is reflected to the second projection lens system. The remaining micro-mirror elements are pivoted into a second position and excitation light from first light source 501a is transmitted to radiation absorber 503a (beam dump). This contributes to a very high contrast. The second light source 501b is also assigned a radiation absorber 503 b. The light sources 501a, 501b and the radiation absorbers 503a, 503b are arranged symmetrically with respect to the further ray path, so that to switch from one light source to another, the pivoting positions of all micromirror elements have to be simply reversed and the first light source 501a has to be switched off and the second light source 501b has to be switched on.

For epi-illumination, both light sources 501a, 501b may also be kept on, and for switching, all micro-mirror elements of the first spatial filter device 504 are placed in either the first or second pivot positions.

Fig. 6 schematically shows an optical assembly of a TIRFM capable microscope in a fourth embodiment. The light source 601, the first projection lens system 602, the two-part second projection lens system 604, 606, the deflection unit 607, the objective lens 608, the tubular lens 609 and the detector 610 as well as the second spatial filter arrangement 605 in a plane cFP conjugate to the focal plane FP of the objective lens 608 between the two parts 604, 606 of the second projection lens system correspond to the arrangement and components of the second embodiment in fig. 3. Instead of a programmable transmissive SLM, in a fourth embodiment, an aperture varying means 603, for example in the form of an aperture ring, is provided, which provides a plurality of different aperture shapes and sizes. They may contain annular and circular apertures of various sizes, so that both epi-illumination and TIRF-illumination may be set for different critical angle values.

Fig. 7 and 8 show examples of centering according to the method according to the invention. This requires an arrangement according to the first, second or third embodiment, i.e. a programmable spatial filter arrangement at the location of the cBFP plane. In fig. 7, the x-y position on the surface of the spatial filter device (in this case a reflective SLM or DMD) is shown. Six search paths are shown, each at a distance of 60 ° from one another, with each extending from the edge along a straight line towards the center of the DMD at x-0 mm and y-0 mm. Along each of these search paths, a small circular area is cleared to relay the excitation light, and the excitation light is blocked in the rest of the surface. A small clearance area is scanned along six search paths and the luminosity of the returned fluorescence is measured. The measured luminosity is plotted as brightness, where black indicates a disappearing signal and white indicates a strong signal. In fig. 8, the corresponding trajectories along the respective search paths in the radial direction from the center of the DMD are plotted against the distance from the center.

It turns out that photometric maxima are found on each search path, but the distance of the corresponding location with a photometric maximum from the center depends on the angle of the search path. The maxima lie on a circle delineating the critical radius and the centre of the back focal plane. These values may be used to determine the inner radius of a suitable annular pattern for TIRF illumination for neutralization. A second search may be performed such that the search path extends towards the centre of the circle found in this way. This has the advantage that the maxima are better defined than in fig. 8 and that the respective maxima are as sharp as the maxima of the curve denoted by 105 ° in fig. 8. This achieves the best resolution and centering.

All the specified features, both individually and in combination, including the features taken alone from the drawings and the individual features disclosed in connection with the other features, are deemed essential to the invention. The embodiments according to the present invention may be realized by a single feature or by a combination of features.