DE102006017841A1 - Laser scanning microscope with main beam splitter for the spatial separation of illumination and detection radiation - Google Patents

Abstract

A laser scanning microscope is provided with an illuminating beam source (5) and a detection beam path (2), which conducts (3) and / or backscattered radiation excited in a sample along an optical axis (OA) to a detector device (4) and In which a beam splitter (13) is provided, via which the illuminating radiation emitted by the illuminating beam source (5) is directed onto the sample (3) in an illuminating beam path (B), the illuminating radiation specularly reflected on the sample (3) cannot pass to the detector device (4) and is arranged in a pupil of the illuminating beam path (B) and partially mirrored, the beam splitter (13) having a beam splitter surface (22) lying in the detection beam path (2), which has at least three points (23a) d) is mirrored for the illuminating radiation which is on the beam splitter surface (22) on a circle around the penetration point (25) of the optical Axis (OA) lie, and the illuminating beam source (5) generates a number of partial beams (Sa-d) corresponding to the number of points and focuses them on the three points (23a-d) of the beam splitter (13) in such a way that in the sample (3rd ) an interference pattern (28) is formed in the form of illumination spots (26) distributed periodically over the sample (3).

Description

The The invention relates to a laser scanning microscope with a Illumination beam source and a detection beam path, which in a sample of excited and / or backscattered radiation along one optical axis leads to a detector device and in the one Beam splitter is provided over the emitted from the illumination beam source illumination radiation directed in an illumination beam path to the sample, wherein the beam splitter on the specimen reflects reflected illumination radiation does not let pass to the detector device and in a pupil of the Illuminating beam path is arranged and partially mirrored

For laser scanning microscopes, such as in the DE 19702753A1 As described above, detecting a sample by scanning a confocal image, the examination of biological preparations by fluorescence microscopy is a widespread application. In this case, fluorescence is excited in a sample, whereby compared to the excitation radiation spectrally Stokes-shifted fluorescence radiation is produced, which is then detected confocal with the laser scanning microscope. Instead of confocal detection, far-field detection can also be performed, for example in the case of multi-photon excitation.

Basically In a laser scanning microscope, the object is that the illumination radiation, in the mentioned Case of fluorescence microscopy is excitation radiation, from to Detecting radiation must be separated, since usually the illumination radiation and the detection radiation over a common objective are, i. the illumination radiation is incident on the lens, with the also the detection radiation is passed to the detector. It is therefore commonplace the illumination radiation over couple in a beam splitter which causes back reflection on the sample Illumination radiation not or to the least possible proportion to Detector happens.

In In fluorescence microscopy one makes the Stokes-related wavelength shift between Utilizing detection and excitation radiation and used for the beam splitter suitable dichroic elements, which is why the beam splitter and the Term natural color divider or main color divider. Distinguish yourself However, illumination and detection radiation is not spectrally helpful this approach does not continue. Also, the selectivity of a limited dichroic beam splitter, which either leads to that in the detection beam path after the beam splitter still reflected back to the sample excitation radiation remains or that the Detection radiation is unnecessarily attenuated when passing through the beam splitter.

A spectrally independent approach is the DE 10257237A1 in front. The laser scanning microscope of the type mentioned therein uses the fact that radiation coming from the sample is usually emitted incoherently, ie not directionally, on the sample, whereas specular or illumination radiation reflecting in the mirror direction is coupled into the detection beam path in a directed manner. The DE 10257237A1 Therefore proposes to insert in a pupil of the illumination beam path, a beam splitter, which is transparent at the piercing point of the optical axis and otherwise mirrored. At the same time, the illumination radiation is focused precisely on this transparent spot. As a result, this results in a surface illumination of the sample and at the same time specularly reflected illumination radiation arrives on the specimen again on the mirror surface of the beam splitter and is thus reflected from the subsequent part of the detection beam path. In contrast, the detection radiation generated diffusely in the sample is not focused, fills the entire pupil and is almost completely transmitted. The efficiency of this beam splitter to be understood as the coupling-out degree of the illumination radiation is therefore given only by the area ratio of the pupil to the transparent spot. It can be well over 90% with a suitable focusing of the illumination radiation. The so by the DE 10257237A1 reached beam splitter is spectrally insensitive and allows a high yield of detection radiation.

As already mentioned, laser scanning microscopy is particularly suitable for examining biological samples. The amount of time it takes to take an image is naturally a significant factor in biological samples, especially when examining living samples or analyzing fast-paced processes. It is therefore constant efforts in laser scanning microscopy to increase the image acquisition speed. For this purpose, microscope systems have recently been described which do not sample a sample with a dot-like light spot (ie, not with a confocal dot scanner), but use a line-shaped illumination and scan (ie, a confocal slit). Again, the DE 10257237A1 a suitable beam splitter, in which then a line-shaped area in the form of a narrow rectangle is mirrored on the beam splitter.

While line-scanning systems achieve high image acquisition speed, depth-of-field resolution is one Point-scanning system is reduced, as it comes in principle in the confocal imaging with the slit diaphragm to some crosstalk along the slit diaphragm direction.

Of the The invention is therefore based on the object of a laser scanning microscope of the type mentioned in such a way that a rapid image acquisition possible is without the depth resolution limitation associated with a slit diaphragm.

These Task is according to the invention with a Laser scanning microscope with an illumination beam source and a detection beam path that excite in a sample and / or backscattered Radiation along an optical axis leads to a detector device and in a beam splitter is provided over that of the illumination beam source emitted illumination radiation in an illumination beam path directed to the sample, with the beam splitter on the sample mirroring reflected illumination radiation not to the detector device lets happen and to arranged in a pupil of the illumination beam path as well partially mirrored, solved, in which the beam splitter has a beam splitter surface lying in the detection beam path, which is mirrored at three points, which on the beam splitter surface on a circle around the puncture point the optical axis, and in which the illumination beam source generated a number of points corresponding number of partial beams and focused on the points of the beam splitter such that in the Sample an interference pattern in the form of illumination spots distributed periodically over the sample arises.

The inventive solution can be Naturally also invert by the beam splitter the detection radiation reflecting and mirroring reflected light radiation happen leaves. To is provided, a laser scanning microscope with an illumination beam source and a detection beam path that is excited and / or backscattered in a sample Radiation along an optical axis leads to a detector device and in a beam splitter is provided over that of the illumination beam source emitted illumination radiation in an illumination beam path directed to the sample, with the beam splitter on the sample mirroring reflected illumination radiation not to the detector device lets happen and to arranged in a pupil of the illumination beam path as well is partially mirrored, wherein the beam splitter one in the detection beam path lying reflective beam splitter surface, at least at three points for the illumination radiation is not mirrored, which on the Beam splitter surface on a circle around the puncture point the optical axis, and the illumination beam source one of Score corresponding number of sub-beams generated and this focused on the three points of the beam splitter such that in the Sample an interference pattern in the form of illumination spots distributed periodically over the sample arises.

The Invention achieves highly efficient dot group generation by means of a simple construction.

According to the invention Beam splitter formed so that the Sample with a point group in the form of one generated by interference Pattern is illuminated. This allows a parallel illumination of several Points and also a parallel scanning of these lit simultaneously Points when the detector device to a multipoint detection the illuminated spots. In the result you get against one Single-point illumination multiplied by the number of points scanning speed, without that depth resolution would be impaired. The Illumination (in fluorescence microscopy, excitation) of the sample with the regular dot group pattern takes place according to the invention under utilization an inner-overtone effect, so that the number of illumination beams, which provides the illumination source is much lower, as the numbers of illumination spots in the regular pattern. The interference effect is needed only at least three reflective dots / transmitting holes on Beam splitter on which the illumination radiation is focused so that one at least three illumination beams coupled to the beam splitter. These Number of illumination beams (in this description, the Term "ray" synonymous with a corresponding beam used) can be easily produce. For example, become at a output beam only two plate elements needed. It is therefore preferred that the Illumination beam source a laser emitting a laser beam and a dish device, which the laser beam in the at least divides three partial beams and by means of an optical device on the points / holes focused, has.

The Number of reflective or transmitting points is not limited to three. It can Also, more points are used, which are symmetrical on the circle or along the circle as possible equally distributed or equidistant should lie, but must be disjoint. At four points, these can e.g. lie at the corners of a square, in the center of which the puncture point located.

The microscope according to the invention with the beam splitter which effects the illumination by utilizing the interference achieves, as already mentioned, a high scanning speed by parallelization of the illumination and possibly detection. At the same time, in an advantageous embodiment the demands on the scanning mechanism required for scanning are drastically reduced because it is only necessary to perform a shift within a period of the periodic dot group pattern for scanning the image area. The scanning device, if it is designed, for example, as an optical scanner acting in the beam path, then only has to achieve a comparatively small deflection angle. This matches the scanning speed as well as simplifications of apparatus. It is therefore to be preferred in a development that in the illumination direction of the beam splitter downstream of a scanning device is provided which causes a shift of the dot group pattern on the sample within a period of the dot group pattern. In addition to a beam deflecting scanning device can of course also a movement of the sample, for example by a so-called table scanner, are used.

The interference-related generation of the illumination point group pattern allows the brightness of the light spots to be compared with those surrounding the light spots Dark areas easy to adjust, namely by the intensity each diagonally opposite focused sub-beams is varied. It is therefore in one Development of the invention provides suitable means for variation, which are arranged upstream of the beam splitter in the illumination direction, and for example, in the form of adjustable attenuation elements are formed.

The Number of bright spots in the dot group pattern depends entirely on the illuminated area on the sample when in the illumination beam path essentially an image of the pupil in which the beam splitter is arranged on the sample. The illuminated area of the Sample and thus the number of sample points can then easily by Means for zooming the image between the beam splitter and sample effect. In other words, means of change the image field size detected by the optical image varies automatically the number of lighting points on the Probe caused by the interference.

A parallel detection of the simultaneously illuminated spots on the Sample increased, As already mentioned, the image acquisition speed. Especially easy can such parallel scanning can be achieved when the detection beam path in a matrix detector ends, the optional one more on the illumination pattern coordinated Pinholemaske can be arranged. Of course you can also the spatially resolving Elements of the matrix detector assume the function of the pinhole mask.

The reflective elements of the beam splitter must reflect only for illumination radiation, not for Detection radiation. The beam splitter can also be dichroic be formed.

Of the Point distance in the dot group pattern can be increased by the distance of the foci the beam splitter, so over the circle radius, to be adjusted.

The The invention will be described below with reference to the drawings for example, even closer explained. In the drawings shows:

1 a schematic representation of a laser scanning microscope,

2 an alternative embodiment of an illumination source for the microscope of 1 .

3 a plan view of a beam splitter of the microscope of 1 and

4 one from the microscope 1 caused illumination pattern on the sample.

1 shows a laser scanning microscope 1 in a schematic representation. The solid lines represent the illumination beam path B; the dashed lines the detection beam path 2 , The laser scanning microscope forms a sample 3 scanning a detector 4 from, the sample by means of a lighting source 5 is illuminated.

The detection beam path 2 indicates the sample 3 to the detector 4 along an optical axis OA a lens 6 as well as a tube lens 7 on, which is a scan lens 8a follows, after which a scanner 9 is provided. After a relay optics 8b . 10 as well as a Pinholeoptik 11 the radiation happens to be a beam splitter 13 and arrives at a detector 4 , which is designed here as a matrix detector, and a filter 12 upstream of blocking the last portions of the illumination radiation. Of course, the detection beam path 2 be designed differently, for example, you can on the filter 12 as well as in the construction of the 2 between the elements 8a and 7 intended intermediate image, which is symbolized by a dashed line, dispense.

Im in 1 shown microscope 1 are the pupil planes where the scanner is 9 and the beam splitter still to be explained 13 lie, to each other and to the rear focal plane of the lens 6 that is between the elements 6 and 7 is drawn as a solid line, conjugated.

The illumination source 5 has a laser 14 on, passing through a plate device 16 , which will be explained in more detail, four partial beams Sa, Sb, Sc, Sd generated, in 1 only the center rays are drawn. The divider 16 focuses these four sub-beams Sa-d, of which in the schematic representation of the 1 only two are visible (the other two are perpendicular to the plane above the other) on the beam splitter 13 ,

In detail, the divider device 16 two dividers 17 and 18 and a deflecting mirror 19 on, the one from the laser 14 emitted beam S four parallel partial beams Sa, Sb and Sc and Sd generate. In the presentation of the 1 are the partial beams Sa and Sb on top of each other, as well as the partial beams Sc and Sd. lenses 20 and 21 in the dividing device 16 cause the focusing such that the partial beams Sa-c at four points on the surface of the beam splitter 13 be focused.

Of course, the generation of the four partial beams Sa-c can also be achieved by means of a differently configured divider device 16 or basically done differently, for example in the four lasers 14 be used. A possible further embodiment for the dividing device 16 is in 2 shown in the divider 18 and the mirror 19 with the divider 17 are reversed.

3 shows the beam splitter 13 in plan view of the beam splitter surface 22 , On the beam splitter surface 22 are four reflective mirror surface elements 23a . 23b . 23c and 23d arranged at the vertices of an imaginary square 24 lie at the center of the puncture point 25 the optical axis OA forms. Only there is the beam splitter surface 25 reflective, in the remaining area it is transmissive at least for detection radiation.

The illumination source 5 focuses the four sub-beams Sa-d on the four mirror surface elements 23a d. In the sample 3 The focused in the pupil and reflected partial beams come to interference, causing the in 4 illustrated multi-spot pattern 28 arises in the sample. The multi-spot pattern 28 is a regular arrangement of light spots 26 that of a dark area 27 are surrounded. The intensity difference between the light spots 26 and the dark area 27 , ie the depth of the zeros, can be varied by changing the intensities of the respective opposite partial beams Sa, Sd and Sb, Sc. In this case, diagonal (with respect to the optical axis) opposite partial beams or in each case separated by a partial beam between them partial beams are preferably adjusted in the same direction. With three partial beams one is adjusted opposite two (or vice versa).

The spatial extent of the pattern 28 can by varying the magnification scale of the microscope (eg variation of the focal length in 10 and 8b ) by adjusting the image field in the sample containing the pattern 28 covered, changed. Alternatively, the focal length of the lenses 10 . 21 to be adjusted.

The effect of the beam splitter 13 is as follows:
Due to the geometric arrangement of the mirror surface elements 23a -D and focused on sub-beams Sa-d, the dot pattern 28 with regularly arranged light spots 26 on the test 3 , When used in fluorescence microscopy is thus at the light spots 26 Fluorescence excited. This arises spatially incoherent and thus fills homogeneously the rear focal plane of the microscope 6 (between the elements 6 and 7 drawn as a solid line). The same applies to diffuse reflection, which can also be used for image acquisition.

Since the rear focal plane with the pupil, in which the beam splitter 13 is arranged, conjugated, the incoherent radiation to be detected also fills the entire pupil in which the beam splitter 13 is arranged and thus illuminates the entire beam splitter surface 22 out. Mirroring reflected illumination radiation, however, is applied to the mirror surface elements 23a -D focused and thus to the illumination source 5 thrown back.

As a result, the beam splitter passes 13 only the radiation to be detected which is spatially incoherent, ie undirected in the sample 3 originated. The beam splitter 13 thus causes a separation of detection radiation and specularly reflected illumination radiation without a chromatic adjustment would have to be made. This had the advantage that not only a higher yield is achieved, also the beam splitter 13 be used for a variety of illumination wavelengths and detection wavelengths. The usually provided with color dividers exchange mechanisms or change gears are not necessary.

The detection (in 1 If the pupil beam path is shown by dashed lines), this is done, for example, with the detector designed as a matrix detector 4 which optionally may be preceded by a pinhole mask. The illuminated spots are displayed confocally. This mask can lie in an intermediate image plane of the observation beam path in front of the detector or alternatively in the relay optics (between 10 and 8b ). The latter also improves the beam profile on the lighting side.

Scanning the sample 3 done by the action of the scanner 9 , This is a much smaller shift of the illuminating spots on the sample 3 necessary than is necessary with a single-point scanner. The individual light spots 26 be from the scanner 9 moved simultaneously, as they did caused by interference in the sample. It is therefore sufficient to move by means of the scanner 9 that the dark area 27 between adjacent light spots 26 scans. The scanning displacement of the light spots thus preferably takes place only within a period length of the periodic regular pattern 28 ,

The in 1 The structure shown can also be inverted to the effect that the detection beam path from the beam splitter 13 and the illumination source 5 be reversed. The beam splitter 13 is then negative with respect to the mirroring to that in 3 shown construction, ie the mirror surface elements 23a -D are holes in a mirror surface.

Claims (7)

Laser scanning microscope with an illumination beam source ( 5 ) and a detection beam path ( 2 ), which stimulated in a sample ( 3 ) and / or backscattered radiation along an optical axis (OA) to a detector device ( 4 ) and in which a beam splitter ( 13 ) is provided, via which of the illumination beam source ( 5 ) emitted illumination radiation in an illumination beam path (B) to the sample ( 3 ), wherein the beam splitter ( 13 ) on the sample ( 3 ) specularly reflected illumination radiation not to the detector device ( 4 ) and arranged in a pupil of the illumination beam path (B) and partially mirrored, characterized in that the beam splitter ( 13 ) one in the detection beam path ( 2 ) lying beam splitter surface ( 22 ) at least at three points ( 23a -D) is mirrored for the illumination radiation, which on the beam splitter surface ( 22 ) on a circle around the puncture point ( 25 ) of the optical axis (OA), and the illumination beam source ( 5 ) generates a number of sub-beams (Sa-d) corresponding to the number of points and these on the three points ( 23a -D) of the beam splitter ( 13 ) so focused that in the sample ( 3 ) an interference pattern ( 28 ) in the form of periodic over the sample ( 13 ) distributed illumination spots ( 26 ) arises. Laser scanning microscope with an illumination beam source ( 5 ) and a detection beam path ( 2 ), which stimulated in a sample ( 3 ) and / or backscattered radiation along an optical axis (OA) to a detector device ( 4 ) and in which a beam splitter ( 13 ) is provided, via which of the illumination beam source ( 5 ) emitted illumination radiation in an illumination beam path (B) to the sample ( 3 ), wherein the beam splitter ( 13 ) on the sample ( 3 ) specularly reflected illumination radiation not to the detector device ( 4 ) and arranged in a pupil of the illumination beam path (B) and partially mirrored, characterized in that the beam splitter ( 13 ) one in the detection beam path ( 2 ) lying reflective beam splitter surface ( 22 ) at least at three points ( 23a -D) is not mirrored for the illumination radiation, which on the beam splitter surface ( 22 ) on a circle around the puncture point ( 25 ) of the optical axis (OA), and the illumination beam source ( 5 ) generates a number of sub-beams (Sa-d) corresponding to the number of points and these on the three points ( 23a -D) of the beam splitter ( 13 ) so focused that in the sample ( 3 ) an interference pattern ( 28 ) in the form of periodic over the sample ( 13 ) distributed illumination spots ( 26 ) arises. Microscope according to Claim 1 or 2, characterized in that the illumination beam source ( 5 ) a laser ( 14 ) emitting a laser beam (S) and a dish device ( 16 ), which divides the laser beam (S) into the partial beams (Sa-d) and by means of an optical device ( 20 . 21 ) on the points ( 23a -D) focused. Microscope according to one of the above claims, characterized by a scanning device ( 8th ), which in the illumination direction the beam splitter ( 13 ) and a scanning by moving the interference pattern ( 28 ) to the test ( 3 ), wherein the displacement within a period of the periodically distributed illumination spots ( 26 ) he follows. Microscope according to one of the above claims by means for varying the intensity in each case diagonally opposite one another focused partial beams (Sa, Sc, Sb, Sd). Microscope according to one of the preceding claims, characterized in that the detection beam path in a matrix detector ( 4 ) ends. Microscope according to one of the above claims, characterized by means for focal length variation ( 10 . 8a . 8b ) between beam splitters ( 13 ) and sample ( 3 ).