DE102006011277A1 - Laser scanning microscope for detecting fluorescent radiation, has detection module with detection unit that detects linear sections in such a manner that linear probe radiation bundle is produced for each section - Google Patents

Abstract

The microscope has a lighting module (BM) for illuminating two linear sections of a probe (26) with laser radiation of different wavelength. A sample radiation is produced based on interaction of the laser radiation with the probe. A scanning module (27) causes a relative motion between the laser radiation for illuminating the sections and the probe. A detection module (DM) has a detection unit (D1) and a CMOS sensor (38), where the detection unit detects the sections in such a manner that a linear probe radiation bundle is produced for each section. An independent claim is also included for a laser scanning-microscopic method.

Description

The The invention relates to a laser scanning microscope and to a laser scanning microscopy method, in particular suitable for the detection of fluorescence radiation are.

For the fluorescence microscopy Surveying various detection principles are known. So can For example, a laser beam focused punctiform on the sample and locally filtering the fluorescent light emitted by the sample through a pinhole aperture and registered with a downstream, non-spatially resolving detector become. The disadvantage here, however, that on the one hand many moving Parts (two scanners to deflect the focused laser beam over the entire sample), which limits the service life. Furthermore, the dye excitation is in focus due to the very high peak intensities slightly saturated, so that this an upper limit of the maximum speed for detecting a spatially resolved fluorescence image the sample sets. Furthermore, the difficulty arises that one strong fading of the dyes occurs.

The Sample may be with a line focused laser radiation are illuminated, this illumination line then over the sample is scanned to obtain an image of the entire sample. The Detection takes place, for example, with a CCD or CMOS line detector. Light from other planes is preferably confocally suppressed in this method. If However, one has several dyes in the sample with different Laser wavelengths need to be stimulated so must the Measure the sample either several times with one of the excitation wavelengths or stimulate fluorescence simultaneously with all excitation laser lines and spectrally detects the light excited by all lines. at the first alternative leads this to a higher one Measuring time. In the second alternative one can not distinguish which ones Fluorescence radiation from which laser radiation was generated.

It is possible, too, the sample flat to illuminate and to measure with a spatially resolving detector. Spectral information can but only be won serially. To a confocal suppression too reach, must further elaborate measures be taken so that this Total procedure only for Samples with a few dyes is suitable.

outgoing It is the object of the invention to provide a laser scanning microscope and to provide a laser scanning microscopy method, the one possible has a simple structure and allows high measuring speeds.

According to the invention Task solved by a laser scanning microscope with a lighting module that has a first line-shaped and one of them spaced apart second line-shaped sections to one examining sample with laser radiation of different wavelengths, being in each section due to an interaction of the laser radiation With the sample sample radiation is generated, with a scan module, the a relative movement between the laser radiation for illumination the sections and the sample transversely to the linear sections causes and with a detection module, which is a first detection unit as well as a spatially resolving Detector comprises, wherein the first detection unit spatially separated the line-shaped sections detected confocally, that for everyone Section a line-shaped Samples ray beam is generated, which detects each spatially resolved by means of the detector becomes.

There the laser scanning microscope (preferably at the same time) two line-shaped sections illuminated and these (preferably simultaneously) seize confocal detected locally can, it is possible the sample radiation both after emission wavelengths and after excitation wavelengths (preferably simultaneously) Detect separately. This is particularly advantageous when Fluorescent radiation of several dyes in the sample are introduced, to be detected. So it is possible in particular, two to separate eight different dyes in the sample and their proportions too determine. To have to only the appropriate number of line-shaped sections, from each other are spaced, illuminated with laser radiation of different wavelengths and accordingly detected confocally.

Around a local resolved To obtain image of the detected sample radiation, can by means of the scan module only the sample relative to the laser radiation to be moved. This can be done, for example, with a conventional one x-y stage of a microscope can be realized. So it just has to a moving part can be provided to the desired image to obtain. This simplifies the construction, lowers the manufacturing costs and leads too long service life.

The Sample radiation of the linear Sections can be detected simultaneously by means of the detector, So that the Detection speed increased can be.

The detection module may have a second detection unit, which spectrally splits the sample bundles of rays coming from the first detection unit transversely to the linear expansion, the detector each detecting at least one spek in the detection of the sample beams tral split wavelength detected triggered locally. This can achieve a very high spectral resolution during detection.

The Lighting module can more than two spaced-apart, line-shaped sections with Laser radiation of different wavelengths moistened. Especially can For example, illuminated three to ten sections simultaneously and also detected.

The first detection unit may be for locally segregated confocal Detecting a slit, which for each section a line-shaped gap contains. With such a slit aperture you can in a simple manner the desired realize confocal detection.

The Lighting module can illuminate each section of the respective Laser radiation linear Focus on or in the sample. Preferably lies transversely to the direction of extension a diffraction-limited focusing in order to a possible to achieve good confocal detection.

The Lighting module may in particular still be designed so that the depth of focus changeable in the sample is. This makes it possible different optical sections in different sample depths perform.

The linear Sections preferably run parallel to each other. This facilitates the structure of the illumination and the detection module.

The Detection module can as sample radiation in particular by detect the laser radiation excited fluorescence radiation.

The Scanning module may include at least one scanner mirror (e.g. Ablenkspiegel), the laser radiation for the sections simultaneously over the sample steers. By means of the scanner mirror or the relative movement partially or completely causes. When the laser radiation is focused linear, takes place the deflection preferably transversely (in particular perpendicular) to the linear expansion the linear focused laser radiation.

The Detection module may contain a filter that is out of the sample radiation (ie before the confocal detection) and / or from the sample beams (ie after confocal detection) filters out the wavelengths of the laser radiation. With that you can e.g. in the detection of fluorescence radiation as sample radiation the common undesirable Excitation wavelengths be filtered out.

Of the spatially resolving Detector is in particular a flat, spatially Resolved Detector. It can be designed, for example, as a CMOS or CCD detector be.

Further the detector can be used as a linear, spatially resolving detector be formed, in which case the second detection unit preferably contains a deflection unit upstream of the detector, the the wished Part of the spectrally split sample beam directs to the detector. The deflection unit may, for example, a rotatably mounted deflection mirror be.

to spectral splitting of the sample beam, the second detection unit For example, a deflection prism, a grid or any other contain optical or dispersive element, the desired spectral Splitting up.

The Illumination module can be a laser source for generating the laser radiation containing the different wavelengths.

Further For example, the lighting module may include an imaging unit that the sections are linear focused laser beam illuminated. The imaging unit is designed in particular that the Focus depth in the sample is adjustable and adjustable.

Further For example, the illumination module for generating the laser beams can Transmitting laser radiation through a slit diaphragm, which is for everyone illuminating portion has a transmissive gap, wherein the individual columns are spaced from each other.

The Microscope may further comprise a control unit, the Control of the microscope is used. The control unit is in particular designed so that the realized functions of the laser scanning microscope can be.

Further the microscope may also comprise an evaluation module to which the data fed to the detector are. The evaluation module then generates the desired data from this data (preferably spatially resolved) Images of the sample radiation.

The scanning module may cause the sample to be guided in a fluid stream to provide at least a contribution to the relative movement. A further contribution to the relative movement can be made, for example, by means of a movable sample table or a deflection mirror in the scan module. Of course, the relative movement can be achieved completely by means of the liquid flow who the.

The Laser scanning microscope can thus be used as an imaging cytometer or imaging flow cytometer.

If the laser radiation linear is focused, the scan module can be formed so that the liquid flow in an object plane of the microscope perpendicular to the linear extension the linear focused laser radiation flows.

The Task is further solved by a laser scanning microscopy method in which a first line-shaped and a second line-shaped portion of a sample to be examined, spaced therefrom Sample (preferably simultaneously) with laser radiation different wavelength is lit, being in each section due to an interaction the laser radiation is generated with the sample sample radiation, and in which the linear Sections (preferably simultaneously) are confocal detected locally segregated, that for everyone Section a line-shaped Samples ray beam is generated, which detects spatially resolved in each case in a linear expansion becomes.

With this method it is possible spectrally selective and selective excitation wavelengths to the sample radiation detect.

at the method can the sample beams before the detection in each case transversely to the linear expansion spectral can be split and can each have at least one spectral augmented Wavelength detected locally resolved become.

Especially in the detection of fluorescence radiation, this leads to the advantage that several dyes spectrally resolved and spectrally selectively detected by excitation wavelength simultaneously can.

at The method may be used to illuminate each section Laser radiation linear be focused on or in the sample. This leads to a better confocal Suppression.

Especially are illuminated parallel to each other extending linear sections. This facilitates the detection of the sample radiation.

Especially becomes as sample radiation by the laser radiation excited fluorescence radiation detected.

The locally resolved detection can be linear or even flat carried out become. If they are linear carried out In particular, a line-shaped, spatially resolving detector can be used be provided, which is preceded by a deflection, the the wished Part of the spectrally split sample beam directs to the detector.

For locally separated Confocal detection can be used in particular a slit diaphragm be that for each section has a linear gap contains. this leads to to an excellent confocal suppression of the sample radiation.

Further Can also be used to illuminate the sections of a slit that will be for everyone to be illuminated portion has a transmissive gap, wherein the individual gaps are spatially spaced from each other.

at The method can be a relative movement between the laser radiation for illuminating the sections and the sample transversely to the extension direction the linear Sections are carried out.

The Invention will now be described by way of example with reference to the drawings explained in more detail. It demonstrate:

1 a schematic view of a first embodiment of a laser scanning microscope according to the invention;

2 a plan view of the slit 17 from 1 ;

3 a top view of the illuminated sample 26 from 1 ;

4 a plan view of the slit 30 from 1 ;

5 a top view of the detector 38 from 1 ;

6 a schematic representation of the intensity distribution of the first line 39 of the detector 38 from 5 ;

7 A second embodiment of the laser scanning microscope according to the invention,

8th a schematic view of a second embodiment of the laser source 1 , and

9 A third embodiment of the laser scanning microscope according to the invention.

At the in 1 In the embodiment shown, the laser scanning microscope comprises a laser source 1 , the four lasers 2 . 3 . 4 . 5 as well as for every laser 2 - 5 an optical fiber 6 . 7 . 8th . 9 contains. The lasers 2 - 5 generate laser radiation with different wavelengths, here laser radiation in the UV range as well as blue, green and red laser radiation, which into the optical fibers 6 - 9 and the lasers 1 - 4 opposite ends as a laser beam 10 . 11 . 12 . 13 with substantially circular beam cross-section and Gaussian intensity distribution is delivered.

The optical fibers 6 - 9 is a lens 15 , a diffractive optical element 16 as well as a slit diaphragm 17 downstream. The Lens 15 that forms from the fibers 6 - 9 emerging laser beam on the aperture 17 from. The diffractive-optical element 16 assumes the function of a Powell lens or Powell mirror, so that the individual laser beam 10 - 13 in the plane of the aperture 17 each have a line-shaped cross-section which extends here in each case perpendicular to the plane of the drawing. At the same time the diffractive optical element causes 16 in that the laser beams 10 - 13 in the plane of the aperture 17 have an approximately homogeneous intensity profile.

The aperture 17 points, as in the plan view of 2 is shown schematically, four transmissive areas 18 . 19 . 20 and 21 on which the line-shaped laser beams 10 - 13 meet and thus through the aperture 17 can pass through. The areas 18 - 21 are preferably each formed as a gap or slot and the remaining areas of the diaphragm are absorbing and / or reflective for the laser radiation. At the place of the aperture 17 There are thus locally spaced, linear laser beams with different wavelength, namely a UV illumination line (gap 18 ), a blue lighting line (gap 19 ), a green lighting line (gap 20 ) and a red illumination line (gap 21 ).

To a convergent spherical wave (here the from the fibers 6 - 9 emerging laser beam 10 - 13 ) on a gap (here column 18 - 21 the aperture 17 ) with a diffractive-optical element, a special one-dimensional diffractive profile is conventionally used. However, since the effect of the diffractive optical element depends on the wavelength of the radiation, the diffractive optical element used here has 16 in the second dimension a continuously changing profile adapted to the wavelength, so that it is for the laser beam 10 - 13 with the different wavelengths side by side on the diffractive-optical element 16 meet, is optimally suited. For example, in the second dimension, a linear scaling of the profile of the diffractive optical element can be provided.

The through the aperture 17 transmitted laser beam 10 - 13 be using one of the aperture 17 downstream first tube lens 22 mapped to infinity, go through an excitation filter 23 (For example, a plasma filter), which is used to filter out unwanted secondary modes of the laser 1 - 4 serves, and then over a divider cube 24 and a lens 25 on the sample to be examined 26 Focused on a sample table 27 lies. To simplify the illustration is after the aperture 17 only the direction of propagation of the laser beam 10 - 13 and the sample radiation described below. Thus, the sample becomes 26 with four line-focused laser beams 10 - 13 illuminated, wherein the linearly focused laser beam 10 - 13 are aligned parallel to each other and spaced from each other. It will thus be on or in the sample 26 , depending on the focus by means of the lens 25 , linear and spaced sections A1, A2, A3, A4 of the sample 26 illuminated, as shown in the schematic plan view of the sample in 3 is shown.

The sample table 27 is as indicated by the double arrow P in 1 and 3 is indicated, in a direction transverse to the direction of extension of the linearly focused on or in the sample laser beam 10 - 13 movable, so that the sample 26 relative to the illumination pattern M passing through the four on or in the sample 26 linearly focused laser beam 10 - 13 is formed, can be moved. The laser beams 10 - 13 thus become with a lighting module BM, here the lasers 1 - 4 , the optical fibers 6 - 9 , the Lens 15 , the diffractive-optical element 16 , the aperture 17 , the first tube lens 22 , the filter 22 , the divider cube 24 as well as the lens 25 contains, on or in the sample 26 focused and serve in the embodiment described here to excite fluorescent light (sample light) in the sample.

The fluorescent light thus generated is by means of the lens 25 imaged infinity and passes through the divider cube 24 and a divider cube 24 downstream detection filter 28 (eg a notch filter), which should eliminate the excitation wavelengths. The sample light then passes through a second tube lens 29 put it on a second slit diaphragm 30 which has the same design as the slit diaphragm 17 and schematically in plan view 4 is shown.

Thus, this is true of the UV laser beam 10 excited fluorescent light on the first gap 31 , The from the blue laser beam 11 excited fluorescent light hits the second gap 32 that from the green laser beam 12 excited fluorescent light hits the third gap 33 and that of the red laser beam 13 excited fluorescent light hits the fourth gap 34 , In this way, the fluorescent light of the individual be Sections A1-A4 were locally confocal detected in such a way that for each section A1-A4, a line-shaped sample beam is generated. All line-shaped sample beams are parallel to each other and in turn spaced from each other and along the line direction contain the corresponding spectral information from the assigned location in the corresponding section A1-A4 of the sample. The objective 25 , the divider cubes 24 , the filter 28 , the second tube lens 29 as well as the aperture 30 form a first detection unit D1.

The through the aperture 30 transmitted sample beams are by means of a first detector lens 35 mapped to infinity, then by means of a downstream prism 36 spectrally split transversely to the linear extension direction and with a prism 36 downstream second detector lens 37 on a two-dimensional, spatially resolving CMOS sensor 38 (Detector) shown. The detector lenses 35 . 37 and the prism 36 form a second detection unit D2, which together with the first detection unit D1 and the sensor 38 form the detection module DM of the microscope

The CMOS sensor 38 is in a schematic plan view in 5 shown, each pixel is shown as a square. In the example shown here, the sensor includes 38 to simplify the illustration, eight lines (x direction) and twelve columns (y direction). The sensor 38 is aligned so that the line-shaped extension of the sample beam coincides with the y-direction, so that along the x-direction, the spectral splitting takes place.

In 6 is the intensity distribution I of the first line 39 of the sensor 38 shown schematically. Assuming that the detection filter 28 can not completely suppress the excitation radiation, the largest intensities are each at the wavelength of the excitation radiation and are here marked with r, g, b and UV for the red, green, blue wavelength or the UV wavelength. Each excitation radiation is followed by the spectral splitting (here to the right) the spectrum of the respective fluorescence radiation 40 . 41 . 42 . 43 at. It is thus possible to sample 26 stimulate simultaneously with all desired excitation wavelengths (here four) and to resolve the fluorescence spectrally as well as after excitation wavelength. Because the sample 26 illuminated line and confocal line is detected, respectively, the corresponding section A1-A4 of the sample 26 spatially resolved along the direction of extension of sections A1-A4. Through the movement of the sample table 27 along the double arrow P is then a strip-like scanning of the sample 26 allows so that an image of the fluorescence radiation of the sample can be assembled.

The CMOS sensor 38 can advantageously be used to read only the columns (x-coordinate) corresponding to the fluorescent radiation to be detected. For a quick evaluation is possible.

Further can Advantageously, individual columns are interconnected and read out together, which is also referred to as hardware binning. This allows the spectral resolution the detection changed and be adjusted.

Alternatively it is possible to use the prism 38 to replace another prism, which has a different dispersion behavior, so that at the same deflection angle by means of the prism different spectral resolutions are present.

In the described embodiment of the laser scanning microscope advantageously only a minimum of moving parts is needed, here only the sample table 27 , Since the microscope with a single scan over the sample area to be examined the entire spectral information of the sample 26 As a function of the excitation and detection wavelength, the microscope is also very well suited for the realization of a fast, image-giving flow cytometer.

If you want to create three-dimensional sample images, the sample becomes 26 several times with different focal depths of the linearly focused laser beam 10 - 13 scanned.

In 7 is a modification of the embodiment of 1 shown, wherein like components or components are designated by the same reference numerals and reference is made to the description of the above statements. Unlike the in 1 embodiment shown, the embodiment of 7 no planar, spatially resolving sensor, but a linear spatial resolution sensor 44 (ie a one-dimensional resolution sensor and not a two-dimensional resolution sensor as in 1 ), which extends perpendicular to the plane of the drawing. Between the second detector lens 37 and the sensor 44 is a about a perpendicular to the plane extending axis of rotation rotatable deflecting mirror 45 arranged from the spectral splitting of the line-shaped sample beams the line with the desired wavelength to be detected on the sensor 44 directs. Thus, in this embodiment, the individual lines can be detected only in chronological succession.

In 8th is a modification of the laser source 1 and the downstream optics to the slit diaphragm 17 shown schematically. Again, the same elements as in the embodiment of 1 with the same reference numerals and to the description of which reference is made to the above statements. The laser beams 10 . 11 . 12 and 13 be with the help of the lenses 46 . 47 . 48 and 49 collimated and by means of a deflection mirror 50 as well as three dichroic mirrors 51 . 52 and 53 to a common laser beam 55 superimposed. The superimposed laser beam is by means of a grating 55 as a function of the wavelengths split into four partial beams, which then by means of the lens 15 and the diffractive optical element 16 on the slit diaphragm 17 as four laser beam with line-shaped cross section and almost homogeneous intensity distribution meet. The further course of the beam is similar to that in 1 respectively. 7 shown embodiment.

Instead of the prism 36 For spectral splitting of the sample beams also a grating or any other dispersive optical element can be used to achieve the desired spectral splitting.

The filters 23 and 28 are each slightly inclined to the optical axis to minimize the influence of unwanted reflections on the filters. The filters 23 and 28 can also be omitted.

In a modification of the embodiments of 1 and 7 becomes the prism 36 replaced by a prism with corrected dispersion, which thus performs only the desired deflection of the sample beam and no spectral splitting. It can also be a deflecting mirror instead of the prism 36 be provided. The prism 36 can also be completely eliminated. In this case, only the prism 36 subsequent optical elements are arranged so that the sample beams are still on the detector 38 . 44 to meet.

In this case, it is preferable that at least the filter 28 is provided to filter out the wavelengths of the exciting laser radiation. Furthermore, it is also possible just in front of the detector 38 . 44 to provide a corresponding filter. For example, a so-called comb filter can be used.

In 9 a further embodiment of the laser scanning microscope according to the invention is shown. This further embodiment differs from the first embodiment of FIG 1 essentially by the way in which the laser beams focus in a line-shaped manner 10 to 13 about the sample 26 to be moved. The same elements as in the 1 shown embodiment, therefore, in the illustration in 9 the same reference numerals and to the description thereof reference is made to the above statements. The difference between the two embodiments is shown by the lens 25 , now the laser beams 10 to 13 in an intermediate image plane 56 maps. The intermediate image plane 56 are a tube lens 57 , a scanner mirror 58 as well as a scan lens 59 arranged in this order, which together form a scanning unit 60 form. The sample 28 lies on a sample table 61 ,

The scanner mirror 58 is rotatable, as indicated by the arrow S1, so that the laser beam 10 to 13 transverse to their line orientation across the sample 26 can be performed, as indicated by the double arrow S2. The relative movement between the laser beam 10 to 13 and the sample 26 takes place here due to the rotation of the scanner mirror 60 , Therefore, the sample table must 61 not be movable. Of course, it is also possible to provide a movable sample table and possibly the movement of the sample table 61 together with the scan unit 60 for deflecting the laser beam 10 to 13 to use.

Claims (16)

Laser scanning microscope with an illumination module (BM), which has a first line-shaped and a second line-shaped section (A1, A2, A3, A4) of a sample to be examined (FIG. 26 Illuminated with laser radiation of different wavelengths, wherein in each section (A1-A4) due to an interaction of the laser radiation with the sample ( 26 ) Is generated with a scan module ( 27 ; 60 ), which is a relative movement between the laser radiation for illuminating the sections (A1-A4) and the sample ( 26 ) transversely to the line-shaped sections (A1-A4), and with a detection module (DM), a first detection unit (D1) and a spatially resolving detector ( 38 . 44 ), wherein the first detection unit (D1) detects the line-shaped sections (A1-A4) in a locally separated confocal manner in such a way that a line-shaped sample beam is generated for each section (A1-A4), in each case by means of the detector ( 38 . 44 ) is detected locally dissolved. Microscope according to Claim 1, in which the detection module (DM) has a second detection unit (D2) which spectrally splits the sample beams coming from the first detection unit (D1) transversely to the line-shaped extension. 38 . 44 ) is detected locally resolved at the detection of the sample beam at least one spectrally split wavelength. Microscope according to one of the above claims, in which the first detection unit (D1) for spatially separate confocal detection comprises a slit diaphragm ( 30 ), which for each section (A1-A4) has a line-shaped gap ( 31 . 32 . 33 . 34 ) contains. Microscope according to one of the preceding claims, in which the illumination module (BM) for illuminating each section (A1-A4) directs the respective laser radiation onto or into the sample ( 26 ) focused. Microscope according to one of the above claims, at the linear one Sections (A1-A4) are parallel to each other. Microscope according to one of the preceding claims, in which the scanning module ( 60 ) a scanner mirror ( 58 ), the laser radiation for the individual sections simultaneously over the sample ( 26 ) steers. Microscope according to one of the above claims, at the detection module (DM) as sample radiation by the laser radiation excited fluorescence radiation detected. Microscope according to Claim 7, in which the detection module (DM) comprises a filter ( 28 ), which filters out the wavelengths of the laser radiation from the sample radiation and / or the sample beam. Microscope according to one of the preceding claims, in which the detector ( 38 . 44 ) detects the sample beams simultaneously. Microscope according to one of Claims 1 to 8, in which the detector is in the form of a linear detector ( 44 ) and the second detection unit (D2) forms a detector ( 44 ) upstream deflection unit ( 45 ) containing the desired part of the spectrally split sample beam onto the detector ( 44 ) steers. Microscope according to one of the preceding claims, in which the illumination module (BM) comprises a laser source ( 1 ) for generating the laser radiation having the different wavelengths. Microscope according to one of the preceding claims, in which the illumination module for generating the laser beam bundles the laser radiation through a slit diaphragm ( 17 ) transmits a transmissive gap for each section to be illuminated ( 18 . 19 . 20 . 21 ) having. Microscope according to one of the preceding claims, in which the illumination module (BM) comprises an imaging unit ( 22 . 25 ) which illuminates the sections (A1-A4) with linearly focused laser beams. Microscope according to one of the above claims, in which the scanning module causes the sample ( 26 ) is guided in a liquid flow to provide a contribution to the relative movement. Laser scanning microscopy method, in which a first line-shaped and a second line-shaped portion of a sample to be examined, spaced therefrom Sample illuminated with laser radiation of different wavelengths being, in each section due to an interaction of the laser radiation With the sample sample radiation is generated, the line-shaped sections so locally seized confocally be that for each section a linear Sample beam generated is, in each case in linear Extension locally disbanded is detected. The method of claim 15, wherein the sample beams are in front of the detection in each case transversely to the linear expansion spectral be split and each at least one spectrally split Wavelength is detected locally resolved.