GB2416261A - Laser scanning microscope with parallel illumination and simultaneous, locally resolved detection - Google Patents

Abstract

In a laser scanning microscope 1, the sample is illuminated 2 at several parallel points or regions, lying on a line, and the points or regions are detected 5 simultaneously via a locally resolving detector 28. Detection beam paths are provided with exchangeable, switchable beam splitters and/or filters. Scanning may be performed by resonance, multipoint or Nipkow scanners. The optical section thickness may be changed by adjustable confocal diaphragms, and beam splitters may allocate wavelengths to the detectors. Spectral detection and diaphragm adjustment may be synchronous. The apparatus is particularly suited to analysis of live cells in 3D tissue or environments, confining the laser illumination to a region of interest (ROI) and protecting cells outside the ROI.

Description

1 241 6261 Laser scanning microscope with linear scanning and usage The

present invention relates to laser scanning microscopes having linear scanning and is concerned in particular with a very rapid imaging microscope which has several freely configurable detection channels and good 3D resolution.

In order to achieve high image rates in confocal or 4D-microscopy, measures are necessary for the sample scanning to be performed in parallel. This previously meant that the resolution in 2D or 3D was limited by non-changeable confocal diaphragms (e.g. in the case of a lo Nipkow disc) or a limitation of the number of detectors used and their fixed spectral arrangement (generally, a CCD camera attached externally). This limited considerably the possible applications and samples and also the quality ofthe image results in 3D.

In accordance with the present invention there is provided a laser scanning microscope for the detection of at least one sample region by a relative movement between the illumination light and the sample, wherein the illumination light illuminates the sample in parallel at several points or regions and several points or regions are detected simultaneously with a detector arrangement and several illuminated sample points lie on a line and several points are detected simultaneously go with a locally resolving detector, and wherein detection beam paths are provided with exchangeable and/or switchable beam splitters and/or filters.

A rapid confocal or 4D microscope in accordance with the invention can possess the combination of high image rates as a result of parallel scanning the sample and an adjustable confocal diaphragm. In addition, internal detectors can be used, the arrangement of which is such that several selectable colour splitters allow a flexible configuration of the detection channels. As a consequence, new applications in imaging are possible which were hitherto not possible using rapid, confocal or 4D microscopes. In particular, when rapidly changing the so detection channels and the detected wave length it is possible in an advantageous manner to separate superimposed spectral signals such as fluorescence signals, such as is described in principle in DE 19915137A1 and US 6028306.

The invention can be used advantageously to a similar extent in the case of multiple point illuminations as in US6028306 and Nipkow arrangements as in US6028306, W08807695, and EP539691 A. The invention is further explained hereinunder, by way of example only, with reference to the accompanying drawings, in which: Fig. 1 is a schematic illustration of one embodiment of a laser scanning microscope in accordance with the present invention; Fig. 2 illustrates how it is possible to select a region of interest; Fig. 3 shows schematically a further possible construction of a laser scanning microscope in accordance with the invention using a Nipkow disc approach; Fig. 4 shows schematically a further possible construction of a laser scanning microscope in accordance with the invention using multipoint scanning; Fig. 5 shows an arrangement of two internal detectors having an exchangeable beam so splitter; and Fig. 6 shows an arrangement of an adjustable confocal slit diaphragm for improved 3D detection.

Figure I schematically illustrates a laser scanning microscope 1 which is made up substantially of five components: a radiation source module 2 which generates excitation radiation for laser scanning microscopy, a scanning module 3 which conditions the excitation radiation and suitably deflects it for scanning over a sample, a microscope module 4, shown only schematically for simplification, which directs the scanning radiation provided by the So scanning module in a microscopic beam path on to a sample, and a detector module 5 which obtains and detects optical radiation from the sample. As illustrated in Figure 1, the detector module 5 can be spectrally multi-channelled in design.

For a general description of a point-wise scanning laser scanning m icroscope, reference is made to DE 19702753AI which thus forms part of this description.

The radiation source module 2 generates illumination radiation which is suitable for laser scanning microscopy, i.e. in particular radiation which can trigger fluorescence. Depending upon the application, the radiation source module comprises several radiation sources for this purpose. In the illustrated embodiment, the radiation source module 2 is provided with two lasers 6 and 7 which have in each case a light valve 8 and an attenuator 9 connected downstream thereofand which couple their radiation via a coupling point 10 into a light- conducting fibre 11. The light valve 8 operates as a beam deflector which renders it possible to switch offthe beam without having to switch offthe operation ofthe lasers in the laser unit 6, 7 itself. The light valve 8 is formed e.g. as an AOTF which for the purpose of switching off to the beam deflects the laser beam, prior to it being coupled into the light-conducting fibre 11, in the direction of a light trap, not illustrated.

In the exemplary illustration of Figure 1, the laser unit 6 comprises three lasers B. C, D, whereas the laser unit 7 includes only one laser A. The illustration is thus exemplary of a combination of individual and multiple wavelength lasers which are coupled individually or even jointly to one or several fibres. The lasers can also be coupled simultaneously via several fibres, whose radiation is mixed at a later stage by colour combiners after passing through adaptive optics. It is thus possible to use the most varied wavelengths or wavelength ranges for the excitation radiation.

The radiation coupled into the light-conducting fibre 11 is combined by means of displaceable collimator optics 12 and 13 via beam-combining mirrors 14, l 5 and is changed in terms of the beam profile in a beam forming unit.

z5 The collimators 12, 13 ensure that the radiation supplied from the radiation source module 2 to the scanning module 3 is collimated into an infinity beam path. In each case, this is performed in an advantageous manner with an individual lens which by displacement along the optical axis under the control of a central actuating unit, not illustrated, has a focussing function, in that the distance between the collimator 12, 13 and the respective end of the light o conducting fibre can be altered.

The beam forming unit which will be discussed in more detail hereinunder uses the rotationally symmetrical, Gaussian profiled laser beam, as provided downstream ofthe beam- combining mirrors 14, 15, to generate a linear beam which is no longer rotationally symmetrical but rather has a suitable cross-section to generate a rectangularly illuminated

field.

This illumination beam which is also defined as linear serves as excitation radiation and is directed to a scanner 18 via a main colour splitter 17 and zoom optics to be described below.

The main colour splitter will also be discussed later, suffice to mention at this juncture that it performs the function of separating sample radiation, which returns from the microscope module 4, from the excitation radiation.

to The scanner 18 deflects the linear beam uniaxially or biaxially, after which it is bundled by a scanning objective 19 and a tube lens 20 and an objective of the microscope module 4 into a focus 22 which is located in a preparation or in a sample. Optical imaging is performed so that the sample is illuminated in a focal line by means of excitation radiation.

s Fluorescence radiation which is excited in such a manner in the linear focus passes via the objective and the tube lens of the microscope module 4 and the scanning objective 19 back to the scanner 18, so that a resting beam is then provided in the reverse direction downstream of the scanner 18. Therefore, it is said that the scanner 18 descans the fluorescence radiation.

so The main colour splitter 17 allows the passage of the fluorescence radiation which is at different wavelength ranges than the excitation radiation, so that it can be diverted via a diverting mirror 24 in the detector module 5 and then analysed. In the embodiment of Figure 1, the detector module S comprises several spectral channels, i.e. the fluorescence radiation coming from the diverting mirror 24 is divided in a secondary colour splitter 25 into two spectral channels.

Each spectral channel has a slit diaphragm 26 which effects confocal or partial confocal imaging in relation to the sample 23 and whose size determines the depth of sharpness, by which the fluorescence radiation can be detected. The geometry of the slit diaphragm 26 thus so determines the sectioning plane within the (thick) preparation, from which fluorescence radiation is detected.

Disposed downstream of the slit diaphragm 26 is also a block filter 27 which blocks out any undesired excitation radiation which has passed into the detector module S. lithe linearly fanned radiation which is separated in this manner and emanates from a specific depth portion is then analyscd by a suitable detector 28. The second spectral detection channel is also constructed in a similar manner to the colour channel depicted and also comprises a slit diaphragm 26a, a block filter 27a and a detector 28a.

The use of a confocal slit aperture in the detector module 5 is merely exemplary. Of course, it is also possible to produce a single point scanner. The slit diaphragms 26, 26a are then replaced by apertured diaphragms and the beam forming unit can be dispensed with.

Furthermore, for this type of construction, all of the optics are rotationally symmetrical in 0 design. Then, instead of single point scanning and detection, it is naturally also possible in principle to use any multiple point arrangements, such as point clouds or Nipkow disc concepts, as will also be explained later with reference to Figure 3 and 4. However, it is then essential that the detector 28 is then locally resolving, as several sample points are detected in parallel during a pass of the scanner.

Figure I shows that the Gaussian beam bundles located downstream of the moveable, i.e. displaceable, collimators 12 and 13 are combined via a mirror staircase in the form of beam combining mirrors 14, 16 and in the illustrated design with a confocal slit diaphragm are then converted into a beam bundle having a rectangular beam cross-section. In the embodiment of so Figure 1, the beam forming unit utilises a cylindrical telescope 37, downstream of which is disposed an asphere unit 38 followed by cylindrical optics 39.

After conversion, a beam is provided which in a profile plane substantially illuminates a rectangular field, wherein the intensity distribution along the field longitudinal axis is not Gaussian but rather box-shaped.

The illumination arrangement having the asphere unit 38 can be used for uniformly filling a pupil between a tube lens and an objective. In this way, the optical resolution of the objective can be fully exploited. This variation is thus also expedient in a single point or multiple point so scanning microscope system, e.g. in a line-scanning system (in the case of the latter in addition to the axis in which focussing occurs on to or into the sample).

lithe e.g. linearly conditioned excitation radiation is directed on to the main colour splitter 17.

In a preferred embodhnent, this is designed as a spectrally neutral splitter mirror in accordance with DE 10257237 A1, the disclosure content of which is fully incorporated herein. The term,,colour splitter" thus also includes splitter systems which act in a non- spectral manner. Instead of the spectrally independent colour splitter described, it is also possible to use a homogenous neutral splitter (e.g. 50/50, 70/30, 80/20 or the like) or a dichroic splitter. In order to be able to make a selection depending upon the application, the main colour splitter is preferably provided with mechanical means which permits simple replacement, e.g. by a corresponding splitter wheel which contains individual, interchangeable splitters.

0 A dichroic main colour splitter is then particularly advantageous, if coherent, i.e. directed radiation is to be detected, such as reflection, Stokes or anti-Stokes Raman spectroscopy, coherent Raman processes of a higher order, generally parametric non-linear optical processes, such as second harmonic generation, third harmonic generation, sum frequency generation, two and multiple photon absorption or fluorescence. Several of these methods of non-linear optical spectroscopy require the use oftwo or several laser beams which are collinearly superimposed. In this case, the illustrated beam combination of the radiation from several lasers proves to be particularly advantageous. It is fundamentally possible to use the dichroic beam splitters widely used in fluorescence microscopy. Also, for Raman microscopy it is advantageous to use holographic notch splitters or f lters upstream of the detectors in order to suppress Rayleigh scattering.

In the embodiment of Figure 1, the excitation radiation or illumination radiation is supplied to the scanner 18 via motor-controllable zoom optics 41. Therefore, the zoom factor can be adapted and the scanned field of vision can be varied continuously within a specific adjustment range. Particularly advantageous are zoom optics, in which during adaptation of the focus position and the imaging scale, the pupil position is retained in the continuous tuning procedure. The three motor- driven degrees of freedom of the zoom optics 41 as illustrated in Figure I and symbolised by arrows correspond precisely to the number of degrees of freedom which are provided for the purpose of adapting the three parameters, so imaging scale, focus position and pupil position. Particularly preferred are zoom optics 41, whose output-side pupil is provided with a fixed diaphragm 42. In a practical, convenient embodiment, the diaphragm 42 can also be specified by the delimitation ofthe reflective surface ofthe scanner 18. 'I'he output-side diaphragm 42 having the zoom optics 41 ensures that a fixed pupil diameter is always imaged on to the scanning objective 19 regardless of the adjustment to the zoom magnification. Therefore, for any adjustment of the zoom optics 41 the objective pupil remains fully illuminated. The use of an independent diaphragm 42 advantageously prevents the occurrence of undesired scatter radiation in the region of the scanner 1 8.

The cylindrical telescope 37 cooperates with the zoom optics 41, can also be actuated by a motor and is disposed upstream of the asphere unit 38. In the embodiment of Figure 1, this is selected for reasons of a compact structure, however, this does not have to be the case.

to If a zoom factor of less than 1.0 is desired, the cylindrical telescope 37 is automatically pivoted into the optical beam path. It prevents the aperture diaphragm 42 from being incompletely illuminated, if the zoom objective 41 is reduced in size. The pivotable cylindrical telescope 37 thus guarantees that even with zoom factors less than 1, i.e. irrespective of the adjustment of the zoom optics 41, there is always an illumination line of constant length at the location of the objective pupil. Therefore, in comparison to a simple field of vision zoom, it is possible to avoid laser output losses in the illumination beam.

As it is impossible to avoid a sudden increase in image brightness in the illumination line as the cylindrical telescope 37 is being pivoted in, it is provided in the control unit, not zo illustrated, that the advance rate of the scanner 18 or an amplification factor of the detectors in the detector module S is adapted accordingly when the cylindrical telescope 37 is activated, in order to keep the image brightness constant.

In addition to the motor-driven zoom optics 41 and the motor-activatable cylindrical telescope :s 37, remote-controllable adjusting elements are also provided in the detector module of the laser scanning microscope of Figure 1. For example, in order to compensate for longitudinal colour errors, circular optics 44 and cylindrical optics 39 are provided upstream of the slit diaphragm and cylindrical optics 39 are provided immediately upstream of the detector 28 and are each displaceable in the axial direction by means of a motor.

In addition, for compensation purposes, a correction unit 30 is provided which will be described briefly hereinunder.

Together with circular optics 44 disposed upstream and the first cylindrical optics 39 also disposed upstream and the second cylindrical optics disposed downstream, the slit diaphragm 26 forms a pinhole objective of the detector arrangement 5, wherein the pinhole is produced in this case by the slit diaphragm 26. In order to avoid any undesired detection of excitation s radiation reflected in the system, the second cylindrical lens 39 also has a block filter 27 disposed upstream of it which has suitable spectral properties at its disposal in order to allow passage only of desirable fluorescence radiation to the detector 28, 28a.

Changing the colour splitter 25 or the block filter 27 inevitably causes some tilting or to wedging errors during inwards pivoting. The colour splitter can result in an error between the sample region and the slit diaphragm 26, the block filter 27 can cause an error between the slit diaphragm 26 and the detector 28. In order to eliminate the need to readjust the position of the slit diaphragm 26 or the detector 28, a plane-parallel plate 40 is disposed between the circular optics 44 and the slit diaphragm 26, i.e. in the imaging beam path between the sample and the detector 28 and can be moved to various tilting positions under the control of a controller. For this purpose, the plane-parallel plate 40 is adjustably mounted in a suitable holding device.

Figure 2 illustrates how with the aid ofthe zoom optics 41 within the maximum available scanning field SF it is possible to select a region (region of interest) ROI. If the control of the scanner 18 is left such that the amplitude does not change, as is essential e.g. in the case of the no resonance scanner, a magnification of greater than 1.0 set on the zoom optics serves to narrow the selected region of interest ROI centred about the optical axis of the scanning field SF.

Resonance scanners are described in Pawley, Handbook of Biological Confocal Microscopy, Plenum Press 1994, page 461 ff.

If the scanner is controlled in such a manner that it scans a field asymmetrically with respect :5 to the optical axis, i.e. with respect to the non-operative position of the scanner mirrors, then it is possible in combination with a zoom effect to achieve an offset displacement OF of the selected region ROI. The already mentioned effect of the scanner 18 to descan and the renewed passage through the zoom optics 41 then cancel out the selection of the region of interest ROI in the detection beam path in the direction towards the detector. It is thus so possible to make any selection within the scanning image SF for the region of the interest ROI. In addition, it is possible to acquire images for various selections of the region of interest ROI and then to combine these images to produce a high- resolution image.

If there is a desire not only to displace the selected region of interest ROI by an offset OF with respect to the optical axis but in addition also to rotate it, there is an expedient embodiment which in a pupil of the beam path between the main colour splitter 17 and the sample 23 provides an Abbe-Konig prism which is known to produce a rotation of the image field. This is then also cancelled out in the direction towards the detector. It is now possible to measure images with various offset displacements OF and various angles of rotation and subsequently to calculate them to produce a high-resolution image, e.g. in accordance with an algorithm as described in the publication, Gustafsson, M, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination", in "Three-dimensional and to multidimensional microscopy: Image acquisition processing Vll", Proceedings of SPIE, Vol. 3919 (2000), p 141-150.

Figure 3 shows a further possible construction of a laser scanning microscope I, in which a Nipkow disc approach is utilised. The light source module 2 which in Figure 3 is illustrated in a greatly simplified manner illuminates a Nipkow disc 64 via a mini lens array 65 through the main colour splitter 17, as described e.g. in US 6,028,306, WO 88 07695 or DE 2360197 Al.

The pinholes ofthe Nipkow disc which are illuminated via the mini lens array 65 are imaged into the sample located in the microscope module 4. In turn, the zoom optics 41 are provided in order also to be able to vary the sample-side image size in this case.

As an alternative to the construction in Figure 1, the illumination is performed in the case of the Nipkow scanner during passage through the main colour splitter 17 and the radiation to be detected is reflected out. Furthermore, as an alternative to Figure 1, the detector 28 is now designed to have local resolution, so that the multipoint illumination achieved with the Nipkow disc 64 is also scanned accordingly in parallel. Furthermore, disposed between the Nipkow disc 64 and the zoom optics 41 are suitable fixed optics 63 which have a positive refractive force and which convert the radiation exiting divergently through the pinholes of the Nipkow disc 64 into suitable bundle diameters. For the Nipkow structure of Figure 3, the main colour splitter 17 is a classic dichroic beam splitter, i.e. not the aforementioned beam so splitter comprising the slit-like or point-like reflective region.

al he zoom optics 41 correspond to the design explained above, wherein of course the scanner 18 is rendered superfluous because of the Nipkow disc 64. It can still be provided if there is a wish to select a region of interest ROI as explained with reference to Figure 2. The same applies to the Abbe-Konig prism.

Figure 4 schematically illustrates an alternative approach with multipoint scanning, wherein several light sources are irradiated obliquely into the scanner pupil. It is also possible in this case to produce a zoom function as illustrated in Figure 2 by utilising the zoom optics 41 for imaging purposes between the main colour splitter 17 and the scanner 18. By simultaneously irradiating light bundles at various angles in a plane which is conjugated with respect to the pupil, light points are generated in a plane conjugated with respect to the object plane and lo these light points are guided by the scanner 18 simultaneously via a partial region of the entire object field. The image information is produced by the evaluation of all of the partial images on a locally resolving matrix detector 28.

A further possible embodiment involves multipoint scanning, as described in US 6,028,306, the disclosure of which is fully incorporated in this respect herein. In this case, a locally resolving detector 28 is also provided. The sample is then illuminated by a multipoint light source which is produced by a beam expander with a micro lens array disposed downstream which illuminates a multi-aperture plate in such a manner that a multipoint light source is thus produced.

Figure 5 shows by way of example the arrangement of two internal detectors having an exchangeable beam splitter for the detection of several colours on a rapid (parallelising) line scanner.

With reference to a schematically illustrated line scanner (see detail in Figure 1), it is shown that by means of exchangeable dichroic beam splitters, it is possible e.g. by way of a splitter wheel T to alternate very quickly between various spectral detection wavelengths and to perform parallel detection via several line detectors which in this case are designated by way of example by the reference numerals I and 2.

so Figure 6 shows an arrangement of an adjustable confocal slit diaphragm for improved 3D detection by optical sectioning on a rapid (parallelising) line scanner.

In this case, an adjustable slit diaphragm is illustrated, in order to be able to vary the section thickness in the Z-direction as an alternative to or in addition to changing the spectral detection mode.

In the case of beam multiplication with axial or lateral division, it is also possible to use a neutral splitter instead of a dichroic beam splitter. The light paths which are axially or laterally offset with respect to each other can be selectively allocated to the detectors e.g. by the corresponding axial or lateral positioning of the slit diaphragms.

The invention described represents a significant extension of the possible applications of rapid confocal laser scanning microscopes. The significance of such a further development can be appreciated by reference to the standard literature on Cell Biology and the rapid cellular and sub-cellular procedures) described therein and the examination methods used with a plurality of dyestuffs2.

to See e.g.: JIB. Alberts et al. (2002): Molecular Biology of the Cell; Garland Science.

2G. Karp (2002): Cell and Molecular Biology: Concepts and Experiments; Wiley Text Books.

2R. Yuste et al. (2000): Imaging neurons - a laboratory Manual; Cold Spring Harbor Laboratory Press, New York.

2R.P. Haugland (2003): Handbook of fluorescent Probes and research Products, I 0th Edition; Molecular Probes Inc. and Molecular Probes Europe BV.

The invention is of particularly great significance for the following processes and procedures: The development of organisms The invention described is suitable inter alla for the examination of development processes which are characterized primarily by dynamic processes in the range of tenths of a second to several hours. Exemplary applications at the level of groups of cells and entire organisms are so described e.g. here: In 2003, Abdul-Karim, M.A. et al. described in Microvasc. Res., 66:113- 125 a long time analysis of blood vessel changes in a live animal, wherein fluorescence images were recorded at intervals over several days. The 3D data records were evaluated with adaptive algorithms, in order to illustrate the movement trajectories in a schematic manner.

In 2003, Soll, D.R. et al. described in Scientific World Journ. 3:827841 a software-based movement analysis of microscopic data of nuclei and pseudo pods of live cells in all 3 spatial dimensions.

In 2002, Grossmann, R. et al. described in Clia, 37:229-240 a 3D analysis of the movements of micro glial cells of the rat, wherein the data was recorded for up to 10 hours.

to At the same time, after traumatic damage rapid reactions of the glial also occur, so as to produce a high data rate and corresponding data volume.

This relates in particular to the following main points: Analysis of live cells in a 3D environment, whose neighbouring cells react sensitively to laser illumination and which must be protected from the illumination of the 3D-ROI; Analysis of live cells in a 3D environment with markings which are to be bleached in a targeted manner by laser illumination in 3D, e.g. FRET- experiments; Analysis of live cells in a 3D environment with markings which are to be bleached in a targeted manner by laser illumination and at the same time are also to be observed outside the so ROI, e.g. FRAP- and FLIP- experiments in 3D; Targeted analysis of live cells in a 3D environment with markings and medicines which comprise manipulation-induced changes by laser illumination, e.g. activation oftransmitters in 3D; Targeted analysis of live cells in a 3D environment with markings which comprise manipulation-induced colour changes by laser illumination, e.g. paGFP, Kacde; Targeted analysis of live cells in a 3D environment with very weak markings which require e.g. an optimum balance between confocality and detection sensitivity.

Live cells in a 3D tissue formation with varying multiple markings, e.g. CFP, GFP, YFP, DsRed, HcRed and the like.

so Live cells in a 3D tissue formation with markings which comprise colour changes which are dependent upon function, e.g. Ca remarkers. 1 ive cells in a 3D tissue formation with markings which comprise

development-induced colour changes, e.g. transgenic anknals with GFP.

Live cells in a 3D tissue formation with markings which comprise manipulation-induced colour changes by laser illumination, e.g. paGFP, Kaede.

Live cells in a 3D tissue formation with very weak markings which require a restriction in confocality in favour of detection sensitivity.

The last point referred to combined with the preceding points.

Transportation procedures in cells The invention described is ideally suited for the examination of inner cellular transportation to procedures, since in this case fairly small motile structures, e.g. proteins must be illustrated at high speed (generally in the range of hundredths of a second). In order to record the dynamic of complex transportation procedures, applications such as FRAP with ROI-bleaching are also frequently utilised. Examples of such studies are described below: In 2000, Umenishi, F. et al. described in Biophys. J., 78:1024-1035 the analysis ofthe spatial mobility of aquaporin in GFP-transfected culture cells. For this purpose, points in the cell membranes were locally bleached in a targeted manner and the diffusion of the fluorescence in the surrounding area was analysed.

so In 2002, Gimpl, G. et al. described in Prog. Brain Res., 139:43-55 experiments with ROI bleaching and fluorescence imaging for the analysis of the mobility and distribution of GFP marked oxytocin-receptors in fibroblasts. Considerable demands are placed upon the spatial positioning and resolution and the direct time sequence of bleaching and imaging.

In 2001, Zhang et al. described in Neuron, 31:261 -275 live cell imaging of GFP-transfected nerve cells, wherein the movement of granuli was analysed by combined bleaching and fluorescence imaging. The dynamic of the nerve cells places considerable demands upon imaging rate.

Interactions of molecules The invention described is particularly suitable for illustrating molecular and other sub cellular interactions. In this case, very small structures are illustrated at high speed (in the range of hundredths of a second). In order to resolve the spatial position of the molecules which is required for the interaction, indirect techniques, such as e.g. FRET with ROI- bleaching can also be utilised. Example applications are described below: In 2004, Petersen, M.A. and Dailey, M.E. described in Glia, 46:195-206 the dual-channel recording of live rat hippocampus cultures, wherein the two channels are recorded for the markers lectin and sytox spatially in 3D and over a relatively long period of time.

In 2003, Yamamoto, N. et al. described in Clin. Exp. Metastasis, 20:633638 two-colour to imaging of human fibrosarcoma cells, wherein green and red fluorescent protein (GFP and REP) were observed simultaneously in real time.

In 2003, Bertera, S. et al. described in Biotechniques, 35:718-722 multicolour imaging of transgenic mice marked with timer reporter protein which upon synthesis changes its colour from green to red. The image is recorded in 3D as a rapid series in the tissue ofthe live animal.

Signal transmission between cells The invention described is exceptionally well suited for the examination of generally extremely rapid signal transmission procedures. These generally neurophysiological procedures place very high demands upon time resolution, as the activities imparted by the ions occur in the range of hundredths of a second to less than thousandths of a second.

Example applications of examinations in the muscular or nervous system are described below: In 2000, Brum G et al. described in J Physiol. 528: 419-433 the localization of rapid Ca+ activities in muscle cells of the frog after stimulation with caffeine as a transmitter. The localization and micrometer-precise resolution was only achieved by the use of a rapid confocal microscope.

In 2003, Schmidt H et al. described in J Physiol. 551-13-32 the analysis of Ca+ ions in nerve cell extensions of transgenic mice. The examination of rapid Ca±transients in mice with modified proteins which bond Ca+ could only be carried out using high-resolution, l 15 confocal microscopy, as the localization of the Ca+ activity within the nerve cell and its precise time kinetics also play an important role.

Claims (9)

1. Claims I. Laser scanning microscope for the detection of at least one

   sample region by a relative movement between the illumination light and the sample, wherein the illumination light illuminates the sample in parallel at several points or regions and several points or regions are detected simultaneously with a detector arrangement and several illuminated sample points lie on a line and several points are detected simultaneously with a locally resolving detector, and wherein detection beam paths are provided with exchangeable and/or switchable beam to splitters and/or filters.
2. 2. Laser scanning microscope as claimed in claim 1, wherein adjustable confocal diaphragms are provided to change the optical section thickness.
3. 3. Laser scanning microscope as claimed in any ofthe preceding claims, wherein several internal detectors are provided with switchable and/or exchangeable beam splitters for allocating the wavelength for these detectors.
4. 4. Laser scanning microscope as claimed in any of the preceding claims, no wherein an actuating unit is provided for the synchronous actuation of spectral detection and diaphragm adjustment.
5. 5. Laser scanning microscope as claimed in any of the preceding claims, wherein: several illumination regions are illuminated in parallel by several points or lines, to which detectors are allocated, by means of the rapid change of the spectral detection, fluorescence spectra are recorded and different wavelengths are spectrally separated, and a separating method is applied.

   so
6. 6. Laser scanning microscope as claimed in any of claims l to 5, having a resonance scanner, Nipkow scanner or multipoint scanner.
7. 7. Use of arrangements and/or methods as claimed in at least one of the preceding claims for the examination of development processes, in particular dynamic processes in the range of tenths of a second to several hours, in particular at the level of groups of cells and entire organisms, in particular according to at least one of the following points: Analysis of live cells in a 3D environment, whose neighbouring cells react sensitively to laser illumination and which must be protected from the illumination of the 3D-ROI; Analysis of live cells in a 3D environment with markings which are to be bleached in a targeted manner by laser illumination in 3D, e.g. FRET- experiments; lo Analysis of live cells in a 3D environment with markings which are to be bleached in a targeted manner by laser illumination and at the same time are also to be observed outside the ROI, e.g. FRAP- and FLIP-experiments in 3D; Targeted analysis of live cells in a 3D environment with markings and medicines which comprise manipulation-induced changes by laser illumination, e.g. activation of transmitters 4s in 3D; Targeted analysis of live cells in a 3D environment with markings which comprise manipulation-induced colour changes by laser illumination, e.g. paGFP, Kacde; Targeted analysis of live cells in a 3D environment with very weak markings which require e.g. an optimum balance between confocality and detection sensitivity.

   so Live cells in a 3D tissue formation with varying multiple markings, e.g. CFP, GFP, YFP, DsRed, HcRed and the like.

   Live cells in a 3D tissue formation with markings which comprise colour changes which are dependent upon function, e.g. Ca±markers.

   Live cells in a 3D tissue formation with markings which comprise development-induced colour changes, e.g. transgenic animals with GFP.

   Live cells in a 3D tissue formation with markings which comprise manipulation-induced colour changes by laser illumination, e.g. paGFP, Kaede.

   I,ive cells in a 3D tissue formation with very weak markings which require a restriction in confocality in favour of detection sensitivity.

   so The last point referred to combined with the preceding points.
8. 8. Use of arrangements andlor methods as claimed in at least one of the preceding claims for the examination of inner cellular transportation procedures, in particular for illustration - 18 purposes small motile structures, e.g. proteins, at high speed (generally in the range of hundredths of a second) in particular for applications such as FRAP with ROI-bleaching.
9. 9. Use of arrangements and/or methods as claimed in at least one of the preceding claims for the illustration of molecular and other sub-cellular interactions, in particular the illustration of very small structures at high speed preferably using indirect techniques such as e.g. FRET with ROI-bleaching for the resolution of sub-molecular structures.

   I O. Use of arrangements and/or methods as claimed in at least one of the preceding claims in to rapid signal transmission procedures, in particular neurophysiological procedures with high time resolution, since the activities imparted by ions occur in the range of hundredths of a second to less than thousandths of a second, in particular in examinations in the muscular or nervous system.

   I I. A laser scanning microscope substantially as herebefore described, with reference to and as illustrated in the accompanying drawings.