Device for examining a sample comprising a microscope

Abstract

Device for examining a sample (30), comprising a microscope, with a light source (22) that generates an illumination light beam (23), a scan unit (24) that deflects the illumination light beam (23) directed onto the sample (30) via a lens and thereby moves a focus area of ​​the illumination light beam (23) within a predetermined scan field on or in the sample (30), with a first detector arrangement (38) comprising two or more photosensitive semiconductor elements (50) which detects a first detection light beam (40) emanating from the sample (30) in the direction of illumination and not deflected by the scanning unit (24) and wherein the device is characterized that the device comprises at least a second detector arrangement (36) comprising two or more photosensitive semiconductor elements (50) and detecting a second detection light beam (31) emanating from the sample (30) in the opposite direction to the illumination direction and not deflected by the scanning unit (24), that the two or more semiconductor elements (50) of the first and second detector arrangement (36, 38) are each connected in parallel and each is connected to an evaluation unit via a common output line, wherein the semiconductor elements (50) each have an avalanche photodiode which is operated in Geiger mode above its breakdown voltage.

Description

[0001] The invention relates to a device for examining a sample, comprising a microscope with a light source, a scanning unit, and a detector. The light source generates an illuminating light beam. The scanning unit deflects the illuminating light beam directed onto the sample via an objective lens, thereby moving a focal region of the illuminating light beam within a predetermined scan field on or in the sample. The detector detects a detection light beam emanating from the sample and not deflected by the scanning unit.

[0002] When optically scanning a sample with a scanning microscope, a selected point-like or line-like region of the sample is optically scanned at a predetermined time. For this purpose, focusing optics, in particular an objective lens, of the microscope focuses the illuminating light beam onto the selected region of the sample and detects the portion of light reflected from the selected region, for example due to fluorescence effects in the sample. The acquired image data is saved. Subsequently, further point-like or line-like regions are examined, preferably adjacent to one another and forming, for example, a line or a surface. In this way, a large region of the sample can be optically scanned point by point and line by line. Furthermore, scanning in a direction parallel to the illuminating light beam is also possible, so that an overall three-dimensional region, in particular a volume, can be scanned.The data from the individual points are then combined using a data processing system to create an overall image of the examined area of ​​the sample.

[0003] Confocal scanning microscopes regularly employ detectors commonly referred to as NDDs (non-descanned detectors). These detectors are designed to detect light beams that hit the detectors without being returned via the scanning unit. Unlike the illumination light beams, they are therefore not deflected by a scanning unit of the scanning microscope. In conventional microscopes, NDDs are mounted on both the objective and condenser sides. In the case of the objective-side arrangement, they are also referred to as RLDs, and in the case of the condenser-side arrangement, they are also referred to as TLDs. The detected events are evaluated for each detector individually or for all detectors combined. Photomultipliers or EMCCDs (electron multiplying CCDs) are used as photosensitive components in conventional NDDs.These are preferred over other photosensitive components because their light-sensitive area can be designed relatively large. This is necessary because the scanning movement of the illumination light beam also causes the detection light beam to move at least slightly on the photosensitive surface of the detector. This requires a minimum size of the photosensitive component's light-sensitive area of ​​typically 3 mm × 3 mm to be able to direct light from all scanning positions onto the light-sensitive area. The detection light is, in particular, fluorescent light emitted by the sample.

[0004] To detect the detection beams returned by the scanner, avalanche photodiodes (APDs) are used as light detectors. These are significantly more sensitive than photomultipliers. However, the maximum size of the light-sensitive area of ​​such an avalanche photodiode is only approximately 50 µm × 50 µm for known avalanche photodiodes.

[0005] Document DE 102 53 609 A1 discloses a scanning microscope with a detector arranged in a detection beam path for receiving detection light emanating from a sample. The detector has an optical shutter between the sample and the detector, with which the detection beam path can be blocked. A control means is provided for controlling the shutter. The detection light beam path can be automatically blocked outside of the scanning process and when the detection light power is too high.

[0006] Document US 6,134,002 A discloses a confocal scanning microscope for rapidly capturing spectrally resolved images. The confocal scanning microscope includes optics for simultaneously capturing at least two points along a scanning pattern on a sample plane.

[0007] Document DE 10 2007 024 074 A1 discloses a light detector for use in a line-scanning microscope. The light detector comprises a line array of avalanche semiconductor detectors and an electronic control circuit for operating the avalanche semiconductor detectors in a Geiger mode with internal charge amplification and / or in a linear mode.

[0008] It is an object of the present invention to provide a device for examining a sample comprising a microscope, which makes it possible to detect detection beams that are not returned via a scanning unit with a high sensitivity.

[0009] The problem is solved by the features of patent claim 1. Advantageous embodiments are specified in the subclaims.

[0010] The invention is characterized in that a detector arrangement comprising two or more photosensitive semiconductor elements is arranged as the detector. The use of multiple semiconductor elements for a detector, in particular for the detector arrangement, makes it possible to cover a large area with the light-sensitive surface of the photosensitive semiconductor elements and thus to detect the moving detection light beams in any scanning position. Furthermore, the use of the photosensitive semiconductor elements enables high detector sensitivity, in particular high quantum efficiency, and high temporal resolution of the detected events. Furthermore, compared to known non-confocal microscopes, which use, among other things, PMT tubes as detectors, the size of the microscope, in particular of the detector arrangement, can be kept particularly small.This is particularly important when multiple detectors are to be used in one device, for example to detect different spectral ranges.

[0011] The detector arrangement can also be referred to as a detector array. The detector arrangement with the multiple photosensitive semiconductor elements can be manufactured as a single component and incorporated into the device. Furthermore, the device can also comprise multiple detector arrangements, each having two or more photosensitive semiconductor elements. One of the semiconductor elements is, for example, a photodiode, in particular an avalanche photodiode (APD). Semiconductor diodes, in particular avalanche photodiodes in Geiger mode, release an avalanche of electrons of almost the same size for each detected photon. This eliminates the noise source associated with photomultiplier tubes (PMTs) or amplified CCDs, which results from the varying number of released electrons. A further advantage of APDs over conventional PMTs is that they function perfectly even in a strong magnetic field.Furthermore, the temporal resolution of APDs is better than that of PMTs (e.g., FLIM). Thus, APDs are more suitable for single-photon measurements (e.g., FCS) than PMTs.

[0012] The quantum efficiency can be further increased by arranging a lens along the detection beam path upstream of, in particular directly upstream of, each semiconductor element. This lens focuses the detection light beam onto the corresponding semiconductor element, in particular the photosensitive region of the corresponding semiconductor element. This allows detection light, which without the lenses would strike a light-insensitive region of one or more semiconductor elements, for example, between two of the semiconductor elements, to be easily focused onto the photosensitive region of one of the adjacent semiconductor elements. This ensures that no detection light beams are lost. The individual lenses can also be combined as a unit and / or referred to as a microlens array.

[0013] Reducing the dead time between two detectable events and increasing the dynamic range of the detector array is preferably achieved by connecting at least two of the semiconductor elements in parallel. Furthermore, an output of the detector array can be assigned to several semiconductor elements arranged in parallel or to parallel rows of semiconductor elements. This contributes to optimizing the structure of the detector array.

[0014] In an advantageous embodiment, quantum efficiency is further increased by arranging light-insensitive structures on the semiconductor elements facing away from the detection light beam. This special arrangement of the light-insensitive structures is often referred to as "back-thinned" or "back-illuminated technology." This technology enables a photon detection probability of over 80% per semiconductor element.

[0015] Due to the high photon detection probability and favorable noise behavior, the sensitivity of the detector array is particularly high compared to known detectors for non-confocal scanning microscopes. Furthermore, the detector array has a particularly good signal-to-noise ratio at a given illuminance.

[0016] The detector arrangement with the photosensitive semiconductor elements can also be used in a confocal microscope with a scanning stage.

[0017] Embodiments of the invention are explained in more detail below with reference to schematic drawings.

[0018] They show: Fig. 1 a scanning microscope, Fig. 2 a circuit diagram of a first embodiment of a detector arrangement, Fig. 3 a circuit diagram of a second embodiment of the detector arrangement, Fig. 4 is a plan view of a third embodiment of the detector arrangement, and Fig. 5 a side view of the third embodiment of the detector arrangement.

[0019] Elements of the same design or function are marked with the same reference symbols throughout the figures.

[0020] Fig. 1 shows a scanning microscope 20 having at least one light source 22. The light source 22 generates an illumination light beam 23, which is focused onto a sample 30 via a scanning unit 24 and a beam splitter 26 with the aid of a first objective 28. A first detection beam 31 emanating from the sample in the illumination direction is directed via a second objective 32 and a mirror 34 onto a first detector array 36. A second detection beam 40 emanating from the sample 30 opposite to the illumination direction is directed via the beam splitter 26 onto a second detector array 38. The detection beams 31 and 40 are not deflected by the scanning unit 24. The first and second detector arrays 36, 38 are therefore also referred to as "non-descanned detectors" (NDD). The scanning microscope 20 can also be referred to as a device comprising a microscope for examining the sample 30.In addition to the detection beams 31 and 40, further detection beams can be generated, which are returned via the scanning unit 24 and detected by one or more detectors (not shown). Thus, the scanning microscope 20 can be a confocal scanning microscope.

[0021] The first objective lens 28 focuses the illumination light beam 23 onto a focal area on or within the sample 30. The focal area is point-shaped. Alternatively, the focal area can be linear, for example, when using a line scanner. When scanning the sample 30, the focal area is moved in the x- and y-directions within a plane perpendicular to the illumination light beam 23. Preferably, a closed scan field is optically scanned. In addition, the focal area can also be moved in the z-direction perpendicular to the plane.

[0022] The detection beams 31, 40 emanating from the sample 30 arise essentially from fluorescence effects that occur as a result of the illumination of the sample 30. Special effects, such as two- or multi-photon effects, can also be observed. Microscopy methods in which the devices according to the invention are applicable, or effects that occur during this process and can be observed with the assembly 20, include, in particular, FLIM (Fluorescence Lifetime Imaging), FRAP (Fluorescence Recovery After Photobleaching), FRET (Fluorescence Resonance Energy Transfer), and FCS (Fluorescence Correlation Spectroscopy).

[0023] Since the illumination light beam 23 is deflected by the scanning unit 24, but the detection light beams 31 and 40 are not confocally returned and therefore their beam path is not corrected by the scanning unit 24, the detection light beams 31 and 40 are also moved on a surface of the first and second detector arrays 36, 38. In order to reliably detect the detection light beams 31, 40 in each scanning position of the scanning unit 24, each of the detector arrays 36, 38 has two or more photosensitive semiconductor elements 50, as shown in Fig. 2. Thus, the photosensitive semiconductor elements 50 form the detector arrays 36, 38, which can each also be referred to as a sensor array or detector array.

[0024] Avalanche photodiodes (APDs) are used as semiconductor elements 50. These are operated in Geiger mode above their breakdown voltage. For evaluation, all individual pulses are summed analogously, so that the total signal is proportional to the number of incident photons. Alternatively or additionally, the individual pulses can be evaluated directly and counted in the measurement interval. By using multiple avalanche photodiodes, the disadvantage that arises from using the avalanche photodiode in Geiger mode, namely the longer dead time, can be compensated. Alternatively, the avalanche photodiodes can be operated in avalanche mode below their breakdown voltage. Furthermore, analog summation and direct evaluation can be combined, as in Fig. 3 shown.

[0025] Fig. Figure 2 shows a possible circuit according to which the individual semiconductor elements 50 of a detector arrangement 36, 38 can be electrically connected to one another. The semiconductor elements 50 are connected in series, each with a series resistor 58, but in parallel. In particular, the illustrated embodiment of the detector arrangement 36, 38 has a first row 52 and a second row 54 of semiconductor elements 50. The parallel-connected semiconductor elements 50 are connected via a single output line 60 to an amplifier 62, which amplifies the signals from the semiconductor elements 50 for a common evaluation unit 64. The evaluation unit 64 sums the individual detection events in an analog manner. After each detected event, one of the semiconductor elements 50 has a short dead time during which no new photon 56 can be detected.The use of several semiconductor elements 50 connected in parallel reduces the dead time for the entire detector arrangement 36, 38, since each of the semiconductor elements 50 can detect a photon 56 while, for example, a neighboring semiconductor element 50 is currently in its dead time.

[0026] The dead time can be further reduced if a module (not shown) is provided for resetting the semiconductor elements 50, wherein the capacitance of the semiconductor elements 50 can be passively reset using the module. Alternatively or additionally, a separate circuit can be provided that actively resets the capacitance of the semiconductor elements 50.

[0027] Fig. Figure 3 shows an alternative circuit of the semiconductor elements 50, in which two of the semiconductor elements 50 are connected in parallel and each connected to the evaluation unit 64 via an output line 60. This circuit allows the individual signals of the two parallel-connected semiconductor elements 50 to be detected as a pair, independently of the other pairs of semiconductor elements 50. The detection events can then be summed analogously and evaluated in pairs.

[0028] The Fig. 2 and Fig. The embodiments shown in Figure 3 can be easily combined with one another by detecting any number of parallel-connected semiconductor elements 50 together via a common output 60. This allows the chip structure, in particular the number of outputs 60, to be easily specified.

[0029] The Fig. 4 and Fig.5 show an embodiment in plan view and side view, respectively, in which the quantum yield of the detector arrangements 36, 38 is particularly high. The sides of the semiconductor elements 50 facing the detection beams 31, 40 have the largest possible, most closed photosensitive region 70. In other words, the fill factor of the photosensitive surface is as large as possible. This is achieved, on the one hand, by ensuring that the photosensitive semiconductor elements 50 are connected to one another with as few gaps as possible. On the other hand, semiconductor elements 50 are used that are based on back-thinned technology, in which light-insensitive structures 74 of the semiconductor elements 50, for example contacts, are arranged on the side of the corresponding semiconductor element 50 facing away from the detection beams 31, 40.The quantum yield is further increased by arranging a lens 72 in front of each light-sensitive region 70 of a semiconductor element 50. The lens 72 focuses the light from the detection light beams 31, 40 onto the light-sensitive region 70 of each semiconductor element 50. The individual lenses 72 can also be combined as a unit and referred to as a lens arrangement or a microlens array.

[0030] Dark noise from one or both detector arrays 36, 38 is kept particularly low by thermally coupling the corresponding detector array 36, 38 to a cooling device and thereby cooling it. The cooling device can, for example, comprise Peltier elements, in particular multi-stage Peltier elements. Furthermore, the detector arrays 36, 38 can optionally be arranged in a protective atmosphere. The protective atmosphere prevents the formation of condensate on the photosensitive regions 70 of the semiconductor elements 50. This is particularly advantageous for detecting small signals that, without cooling or a protective atmosphere, are only slightly or not at all distinguishable from the dark noise.

[0031] Preferably, the detection light beams 31, 40 are coupled into a light guide (not shown) before being detected. The use of a liquid light guide is particularly advantageous because, firstly, it has relatively good transmission properties and, secondly, it can be manufactured with a relatively large diameter. The large diameter makes it easier to reliably project even highly deflected detection beams 31, 40 onto the cross-section of the corresponding light guide, thus ensuring that they are projected onto one of the detector arrays 36, 38 with virtually no impact on the detection quality.

[0032] The invention is not limited to the specified embodiments. For example, other light-sensitive components can also be used as semiconductor elements 50 for the detector assemblies 36, 38. Furthermore, only one or more detector assemblies 36, 38 can be arranged. For example, different detector assemblies 36, 38 can be provided for detecting different spectral ranges of the detection beams 31, 40. Furthermore, instead of the scanning unit 24, the scanning microscope can comprise a scanning stage on which the sample 30 is arranged and which moves the sample 30 under the illumination light beam 23 directed onto the sample 30, thereby moving a focus region of the illumination light beam 23 on or in the sample 30 within the predetermined scanning field. List of reference symbols: 20 scanning microscope 22 Light source 23 Illumination light beam 24 scanning unit 26 beam splitters 28 first lens 30 samples 31 first detection beam 32 second lens 34 mirrors 36 first detector arrangement 38 second detector arrangement 40 second detection beam 50 semiconductor elements 52 first row 54 second row 56 avalanche photodiode 58 Series resistor 60 Exit 62 amplifiers 64 Evaluation unit 70 light-sensitive area 72 lens 75 light-insensitive area

Claims

[1] Device for examining a sample (30), comprising a microscope, with a light source (22) which generates an illuminating light beam (23), a scanning unit (24) which deflects the illuminating light beam (23) directed onto the sample (30) via an objective and thereby moves a focus area of the illuminating light beam (23) within a predetermined scanning field on or in the sample (30), with a first detector arrangement (38) comprising two or more photosensitive semiconductor elements (50) and detecting a first detection light beam (40) emanating from the sample (30) in the illumination direction and not deflected by the scanning unit (24), and wherein the device is characterized, that the device comprises at least one second detector arrangement (36) which comprises two or more photosensitive semiconductor elements (50) and which detects a second detection light beam (31) emanating from the sample (30) opposite to the illumination direction and not deflected by the scanning unit (24), that the two or more semiconductor elements (50) of the first and second detector arrangements (36, 38) are each connected in parallel and are each connected to an evaluation unit via a common output line, wherein the semiconductor elements (50) each have an avalanche photodiode which is operated in Geiger mode above its breakdown voltage. [2] Device according to claim 1, wherein a lens (72) is arranged along the detection beam path (31, 40) in front of each semiconductor element (50), which lens focuses the detection light beam (31, 40) onto the corresponding semiconductor element (50). [3] Device according to one of the preceding claims, in which light-insensitive structures (74) of the semiconductor elements (50) are arranged on a side of the semiconductor elements (50) facing away from the detection light beam (31, 40). [4] Device according to one of the preceding claims, in which individual signals of the parallel-connected semiconductor elements (50) can be detected as a pair independently of another pair of semiconductor elements (50), wherein detection events can be summed up analogously and evaluated in pairs. [5] Device according to one of the preceding claims, wherein the detector arrangement (36, 38) is arranged in a protective atmosphere and / or is thermally coupled to a cooling unit. [6] Device according to one of the preceding claims, in which the detection beam path (31, 40) runs via a light guide and / or a liquid light guide. [7] Device according to one of the preceding claims, comprising a module for resetting the semiconductor elements (50) which passively or actively resets the capacitance of the semiconductor elements (50).

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