Method and apparatus for confocal microscopy

The invention relates to a method for the confocal Laser scanning microscopy according to the preamble of claim 1.

The invention relates to a laser scanning microscope for Performing this method.

While the conventional light microscopy only the optical detection of an image plane is possible, provides confocal microscopy as a special further development development of light microscopy the possibility microstructures map in the Z-axis of the room and measured. With the light microscope, it is for example not possible to at high magnification an impression of the spatial to obtain the rough surface structure of a sample, since only a small portion of the sample sharp Darge can provide, while in-depth details the surface by the high proportion of scattered light and the lack of axial resolution are displayed blurry.

In confocal laser scanning microscope, the contrast Scattered light largely eliminated and only the structures be ready that in the focal plane of the lens . are If the radiation on different planes focused, so you can scan from the latter in the direction the Z-axis staggered planes three-dimensional images ei ner sample are calculated.

Given a first pinhole is punctiform reduced in the object plane ready, which as an illumination source Laser serve. The point-like laser beam is by means grid pattern of deflecting place for location and line Line on the sample moves. Through the microscope objective therethrough which is reflected from the sample and / or emit oriented light onto a second pinhole focused, the is arranged conjugated to the first pinhole. The Anord Regulation of these two pinholes has the consequence that only in formations from the focal plane to one or more De reach detectors that downstream of the second pinhole are.

The scattered light that is in line above and below the focus stands, is eliminated by the second pinhole. With a two-dimensional deflection determined information are imaging planes from several superimposed stored and processed into images.

This principle is the confocal laser scanning microscopy for example, described in Schroth: "Confocal Laser Scaning microscopy, a new method of investigation in the Material testing ", magazine material testing Jg. 39 (1997), Issue 6, pages 264 et seq.

DE 694 02 958 T2 describes a laser scanning microscope a frequency modulation, which many times higher than the Pixel frequency. Also the detector is modulated sinusoidally. Each fluorophore receives a different phase shift to the crosstalk at two wavelengths at the same time eliminate.

Within a sample image, the modulation is constant.

DE 39 41 726 A1 describes a high-frequency modulation of the (Single) excitation wavelength, to lie in the ns range To measure de fluorescence decay.

This is bound to pulsed laser, wherein each of the ge sent pulse is turned off at high frequency, which corresponds to a Intensity modulation with the pulse frequency corresponds.

DE 39 15 692 A1 relates to the measurement of fluorescence Lifetime.

The switchable irradiation is measured in minutes, where when after the shutdown, the decay times are measured.

EP 0440342 A2 relates to a general turn off the (Single) excitation wavelength after measurement to Ver avoidance of destruction.

EP 0916981 A1 describes a programmable white light source for fluorescence excitation of the sample with a certain wavelength.

The procedure used in contrast to the laser Scanning Microscopy wide field illumination, ie the sample is illuminated over its surface and not scanned point-like.

EP 0620468 A1 discloses among others the beam deflection by AOD.

The selection of the excitation laser wavelengths is (slowly) made with an optical filter / color splitter.

The deflectors used in the described solution permit the use of only one of excitation waves length per time, since the optical properties of the AODs are wavelength-dependent.

In Laser Focus World, May 1994, pp. 215-220, Biomedical Imaging "Confocal microscopes sample biological specimens" is a multi-wavelength excitation means (slower) me ical filter described.

DE 691 31 176 T2 describes the (image-wise) coupling under ferent wavelengths in a laser scanning micro microscope, each image with the respective same intensity or wavelength is applied.

From the "Messages of Science and Technology", Volume II, No. 1, pp. 9-19 June 1995, is known as a lighting source in laser scanning microscopes either single laser each having a wavelength, or "multi-line" - Mixed gas laser having a plurality of usable Wellenlän to use gen. This opens up the possibility, ne ben the classic contrasting methods brightfield, phase contrast and interference contrast confocal microscopy to be used for the fluorescence technique. It is assumed gone that different fluorochromes whose Anre gungs- and emission wavelengths in different spektra len bands are, the display structures same temporarily allow several Floreszenzfärbungen. Thus, various, depending on the spectral properties ferent dye molecules besides morphological information statements about physiological parameters obtained who the. If you use the confocal microscope for fluorometric Procedures to conclusions about changes allow the Concentration of ions and molecules derived. in this connection are also indicators of importance, in addition to In tensitätsabhängigkeit a shift of excitation or Emission spectrum show and thus quantification allow ion concentrations. Furthermore, in that context schla the photo-bleaching process gene, in which a defined inhomogeneity is generated to on the dynamics of subsequently adjusting Balance object information such as fluidity and Dif to obtain fusion.

From the aforesaid publication it is known, Ar- Kr laser for fluorescence excitation in the visible Spektralbe rich use with the lines 488 nm, 568 nm and 647 nm. These lines are combined in a laser beam and Optical fibers of the scanning device, respectively. to Anre transmission in the UV range is an Ar laser of wavelength 351 nm and 364 nm suggested. Here too, the Einkopp done ment in the scanning device via optical fibers.

Allow the procedures and arrangements described herein be used for receiving 3D data sets, the z. B. a reliable assignment of spatial cell or tissue structures within a microarchitecture or Loka lisation of several loci in the. Chromosomes in FISH allow experiments.

Under "FISH" is defined as the fluorescence in situ hybridized tion for the identification of DNA or RNA into cells.

However, there is the disadvantage that the respective Sample across the entire scan area with the laser module generated and coupled into the laser scanning device radiation is applied. Thus, the entire scan exposed area of ​​a relatively high radiation exposure, what particular living organisms in the investigation undesired effects and results are insufficient leads.

Furthermore, there is the disadvantage that, when excitation of the sample with different wavelengths, namely in the aforementioned Laser lines, no unambiguous detection and evaluation of emitted from a particular location of a sample and / or reflected radiation is possible, since the effect of the GE mutual "bleed-through" of the individual spectral lines occurs.

Based on that, the invention has the object, a process for laser-San microscopy of vorbeschriebe educate NEN type such that both lower Radiation exposure of the sample as well as a better picture Evaluation can be achieved.

According to the invention the object is achieved by the features of claim 1. It is either the coupling of one or more Spek tralanteile or the irradiation of light IMP EXP including temporarily interrupted or it is temporarily a individual or more spectral components in addition to the micro coupled skopstrahlengang while the deflection continued the microscope beam path continuously becomes.

Thereby it is achieved that at least two side by side lying locations of the sample with light of different Spek traleigenschaften and / or laser radiation with different inten- sities are acted upon. By temporarily Un interruption of the coupling of the laser light during the Deflection of the microscope beam path, it is possible only selected portions of the image field with the Laserstrah to apply treatment.

A protection of the sample is achieved insofar as a single Lich relevant to the image regions of interest a Sample subjected to laser radiation of higher intensity will.

In a preferred embodiment variant of the fiction, novel process it is provided that the spectral co composition and / or the intensity of the laser light during scanning a plurality of adjacent locations, the so form a scan line, is changed. Here, the Ab steering on the places that line across both multiple in the same direction or bidirectionally. He inventively is exemplary provided with every scan about the places that line of time, regardless of whether this occurs in the same direction or opposite, the change of the spectral composition or the In intensity always in relation to the same juxtaposed make the cities this line, whereby the quality of Image processing is increased while the energy input into the sample is limited. This is achieved at the same time, that single contiguous locations of the sample without the Effect of mutual "bleed-through" individual spectral areas can be viewed.

By "bleed-through" is understood as the superimposition of the Emission spectra of different dyes which, in the assignment different detection channels more difficult.

The different spectral composition of the Microscope beam path coupled laser radiation he it is sufficient, for example, by the multiple Linienla fibers, such as with wavelengths of 633 nm, 568 nm, 543 nm, 514 nm 488 nm and 458 nm radiation provided as needed or depending on the characteristics of the evaluated Sample with a single wavelength, with a choice several individual wavelengths or with all available A zelwellenlängen is coupled. In addition to this radiation in the VIS range can further wavelengths in the UV range, about 351 nm and 364 nm, for coupling provided who the.

In preferred embodiments of the invention, there is Coupling of the laser radiation in the microscope beam path Polarization Maintaining over single-mode fibers. The A position of the respectively provided for irradiating Laserli lines to a desired brightness is advantageously with an acousto-optic tunable filter (AOTF) ago taken, the even an acousto-optic. modulator (AOM) can be disposed downstream. The adjustment of the respective Laser wavelength to the ge respectively in the beam path presented microscope objective is performed on both the UV and also for the VIS region by variable beam collimation.

A further preferred embodiment of the invention Method is that the individual from each BE reflected aufschlagten location of the sample and / or light emitted Light with regard to its spectral properties, and its intensity is rated, the rating syn chron for applying the same place and taking into account the spectral composition and / or in occurs intensity of the laser light with which be this place was aufschlagt. This will provide an opportunity to scanned portion of the sample based on the individual evaluate places, resulting in a very high resolution and the highest possible accuracy in the image evaluation leads.

Within the scope of the invention is also that the each the individual acted place reflected and / or emitted laser light with multiple detection channels Detek Animal T is, the individual detection channels for Emp fang different spectral components are designed. There with very good conditions for studying given multifluorescence preparations, and it can ever the detection channel identical optical sections in the simultaneous generates receiving multiple fluorescence preparations will.

In this context, the invention provides that the spectral composition and / or the intensity of the in the microscope beam path coupled laser light of the excitation radiation of a contained in the sample or to the test applied fluorescent dye ent speaks and the individual detection channels for reception emanating from the fluorescent dye emission radiation are designed. Thus, it is possible to laser light for Anre to produce movement of different fluorescent dyes and from the detection conclusions regarding the distribution of these to draw fluorescent dyes on or in the sample.

Another very preferred embodiment of the invention is that permanently an evaluation of the spectral Composition and / or the intensity of the injected The laser light is carried out and a mathematical Ver linking of the evaluation results of a certain Site-directed laser radiation with the evaluation result sen reflected from this location and / or emitted Light is effected. In result of this operation, in for example the deflection position of the microscope beam path for two adjacent locations, determined by the Co ordinates x, y, z are determined for when evaluating above a predetermined threshold beyond lower differences in the spectral properties of the light proof are bar that reflects from these locations and / or emit Animal T is, from which the presence of an optical Boundary layer to be concluded between these two places. These deflection positions are stored according to the invention and the calculation of areas and / or volumes basis specified, the boundary of the optical layers within the Pro be enclosed.

With the deflection positions thus obtained and stored It is also possible to control signals for the spectral To composition and / or the intensity of the laser light for the admission of these places in a subsequent Ab duty cycle to identify and pretend thus a self automatic optimization in the image evaluation, taking into account the optical properties of the sample or the Fluo reszenzfarbstoffes is achieved.

In particular, the inventive method is advantageous used for the so-called photobleaching. It is currency rend scanning a selected area of ​​a sample to next with a relatively high irradiance charged and triggered by a bleaching process. With the immediately subsequent scanning cycles are a the releasing reactions optically detected and evaluated, resulting Information on the immediately after the bleaching process in the Sample substance dynamic processes, such Diffu sion and transport processes, can be recovered.

For this purpose, the scanning with very high time resolution must he follow what, according to the invention with sufficiently fast synchronism between the beam deflection occurring switchover rule different intensities and different spectral compositions of the individual locations of the Pro is achieved be light striking.

The fast switching between different Inten intensities and different spectral compositions the laser radiation is a acousto-optic tunable filter (AOTF) made of mutatis mutandis, however, much faster, and function of various in Strah accepts beam path against interchangeable filters and Moreover, even individual laser lines or any Combinations of lines with high temporal dynamics in the intensity can modulate.

Functioning and application of AOTF are, for example, described in detail in: String, Kenneth, R .: "Wave length Selection for illuminaton in Fluorescence Microsco py ", NIH, LKEM, Building 10 / 6N309, Bethexda, MD 20892, April 1993. Furthermore, concrete examples of AOTF in the US-patents US 5,444,528, US 5,377,003 and US 5,216,484 forth.

The time synchronization between the control of the AOTF to Modulation of the laser radiation and the scanning control of the Means for beam deflection is achieved in that of the said driving means to the scanning device output control signals each specific control signals be assigned for the AOTF. So effected the activation always syn of San-device and driving the AOTF chron, ie the output of a control pulse for the scan Means are time always also control pulses for the AOTF added.

This means on the other hand in that each deflection position and since with each location of the sample a characteristic intensity associated with and / or spectral composition of the light can be.

For this, the circuit arrangements for exercising the Ver proceedings with regard to very short maturities of Steuerim pulse from the output to the switching of Strahlmodula tion optimized by the AOTF. These are in the range of <10 ms. A variant of the method in which to control of the AOTF or the scanning device lead or Lead times for switching the intensity and spectral composition and / or vorauszube for distraction expected, thus precisely the intended place with the prior viewed irradiance and spectral Zusammenset tion is applied.

The invention further relates to a laser scanning Microscope for performing the aforementioned method steps, with a laser module for generating laser light with different selectable spectral components, with Sin gle-mode fibers for coupling the laser light into the Mi kroskopstrahlengang, with a minimum two-dimensionally deflecting scanning device, with a microscope objective, which focuses the laser light on a sample containing more eral detectors for receiving different Spektralan Part of the light reflected from the sample and / or light emitted Light, and having an evaluation circuit, the outputs of the the detectors downstream.

According to the invention in such a laser-scanning microscope the laser module several individually controllable single and / or Multiwavelength laser provided the laser module is a Beam combiner, an acousto-optically tunable filter (AOTF) and / or an acousto-optic modulator (AOM) according to connected, the single model fibers Kollimationsopti ken downstream whose distances to the respective fiber end are variable and controllable with Stelleinrichtun gene are coupled. The detectors are photomultiplier (PMT) are provided each of which a reflection zugeord or emission band and thus a detection channel is net. Branching to the outgoing of the sample Radiation in the individual detection channels are on Tei lerrädern arranged and ge by rotation of the divider gears against one another interchangeable filters and / or color divider before present, each splitter wheel also with a driving ble actuator is coupled. Furthermore, the Control inputs of the laser module, the AOTF, the AOM, the Scanning device and the controls for the Tei lerräder and collimation optics with the outputs of the Evaluating circuit.

In one embodiment variant of the laser scanning microscope is directed at the sample microscope beam path on are branched and one of the branches on an optoelectronic directed receiver whose output also with the on control unit is connected.

Furthermore, in a preferred embodiment variant provided that a mathematical in the evaluation circuit Of the outputs of the optoelectronic Emp catcher with the output signals of the PMT and / or the carried deflection signals for the scanning device, wherein the Output of the evaluation optimized actuation signals for the laser module, the AOTF, the AOM, the scanning device and are provided for the adjusting device is available.

The invention will now by way of Ausführungsbei Game will be explained in more detail. In the accompanying drawing show openings

<b>Fig.</b> 1 shows the basic structure of a laser scanning microscope

<b>Fig.</b> 2 shows the principle of deflection of the laser light over the individual places a sample

In <b>Fig.</b> 1 is a laser module <b>1</b> shown that the La fibers <b>2</b>. <b>3</b> and <b>4</b> for the generation of laser light in the visible out area of ​​the wavelengths 633 nm, 543 nm and 458 nm is equipped. The radiation emanating from these lasers radiation is over several beam combiner <b>5</b>, An AOTF <b>6</b> and a fiber <b>7</b> in a scanning device <b>8th</b> coupled that with one in the x and y coordinates beam deflecting unit <b>9</b> is equipped.

In a second laser module <b>10</b> a UV laser is provided, its light through an AOTF <b>11</b> and an optical fiber <b>12</b> in the scanning device <b>8th</b> is coupled.

In both beam paths are optical fibers <b>7</b> and <b>12</b> collimation <b>13</b> downstream, their distances to each respective fiber end can be changed and for this purpose with a controllable adjusting device (drawing not shown) are coupled.

From the beam deflecting device <b>9</b> the laser radiation through a scan lens <b>14</b> in the beam path the microscope shown simplified <b>15</b> coupled and here on a sample <b>16</b> addressed. It happened the Laser radiation a tube lens <b>17</b>, A beam splitter <b>18</b> and the microscope objective <b>19</b>,

The reflectors of the respective location of the sample applied oriented and / or emitted light passes through the micro skopobjektiv <b>19</b> Return to the beam deflecting device <b>9</b>. happens after a beam splitter <b>20</b> and is using the imaging optics <b>21</b> by branching into several Detekti onskanäle <b>22</b> on photomultiplier <b>23</b> directed, of which a respective one of the detection channels <b>22</b> assigned. For the purpose of branching in the individual detection channels <b>22</b> the light by a deflecting prism <b>24</b> on dichroiti cal beam splitter <b>25</b> addressed. Each detection channel<b>22</b> are both in direction as well as perpendicular to the Radiation direction adjustable and veränderba in their diameters re pinholes <b>26</b> and emission filters <b>27</b> provided.

The outputs of the photomultiplier <b>23</b> lead to the Si gnaleingängen an evaluation circuit <b>28</b>, Which in turn with a control device <b>29</b> connected is. The Ausgän ge the drive device <b>29</b> are connected to the signal inputs the laser modules <b>1</b> and <b>10</b> and to signal inputs of Actuating devices for influencing the position of opti rule elements or modules, such as the Po sition of collimation <b>13</b>, The pinholes <b>26</b> u. ä. connected (not shown in detail).

One example is in the scanning device <b>8th</b> be paddock te laser radiation through a beam splitter <b>30</b> branched, one of the branches on one optoelectronic receivers eng <b>31</b> is addressed, the more angeord on filter wheels designated and played by rotating the filter wheels exchangeable line filter <b>32</b> and also against each other replaceable neutral density filters <b>33</b> are upstream. The exit Recipient <b>31</b> also is at a signal input the evaluation <b>28</b>, The filter wheels, on which the line filter <b>32</b> and the neutral density filter <b>33</b> are arranged, coupled with actuating devices, whose control inputs with signal outputs of the control device <b>29</b> connected are (not graphically illustrated).

When operating the laser-scanning microscope is the optical axis <b>38</b> the microscope beam path by the scanning facility <b>8th</b>, as in <b>Fig.</b> 2 symbolically represented, in Direction of coordinate X from place to place and in direction the coordinate y from line to line grid pattern on a scanned object plane <b>34</b> performed in which the auszuwer tend detail <b>35</b> a sample is.

According to the state of the art so far has been with laser light currencies rend scanning constant spectral Zusammenset tion or intensity coupled into the microscope beam path, which has led to that particular case hochauflö Send structural studies of extremely low contrast Whether projects, such as single cells, organelles, Orga organisms or parasites, consistently high radiation Bela Stung was required to pictures with sufficient light to obtain field- or phase contrast.

In order to reduce the radiation exposure and the quality To increase the image analysis nevertheless, inventions provided according to that during scanning a Zei le and / or the object plane <b>34</b> the coupling of individual or more spectral components, optionally also of ge entire spectrum, is temporarily interrupted or old natively purpose, single or multiple spectral time , be additionally injected.

During the change of the spectral composition and the intensity of the laser light is the beam deflecting facility <b>9</b> interruption in activity. In this way it is achieved that, for example, the places <b>36</b> and <b>37</b> intra half a scan line or within the scanned Sample are differently applied. Thus, it is possible places <b>37</b>That within the evaluated details <b>35</b>, For example, a cell is, a ge suspend ringeren radiation.

Conversely, when scanning the locations <b>37</b> an increase the intensity and / or a change in the spectrum of the made of laser radiation, if this is desirable, such as in the application of the invention Process for the purpose of photobleaching, which is it, arrives, selected areas of the sample with a very high to light irradiance to immediately the then incipient dynamic processes to track can.

With the inventive method and with the fiction, according to an arrangement, it is further possible that of each Specifically, the applied places <b>36</b> and <b>37</b> reflected and / or emitted light in each Detektionskanä len <b>22</b> to receive, with the individual detection channels <b>22</b> each for receiving different spectral components modified emanating from the respective spot light are.

A special feature of the present process that the detection and evaluation of each of the acted place outgoing light in synchronization with the Beauf carried Nominal situation concerned. To that extent, for each of the places <b>36</b> and <b>37</b> the excitation of the sample wavelength and the emission wavelength can be evaluated, from which conclusions on the properties of the sample can be derived exactly at the viewed scene.

With the inventive arrangement, it is also pos Lich, on the basis of optoelectronic of the receiver <b>31</b> output signals permanently the composition and to control intensity of the laser light on the Sample is addressed and these signals to balance itself Smallest intensity fluctuations over the Ansteuereinrich tion <b>29</b> to use.

With a in the evaluation <b>28</b> integrated racks circuit is in each case the excitation radiation and emis onsstrahlung evaluated, the bezo on one and the same place gene. In this way is precisely to establish whether at the Deflection of the laser radiation from one location to another, for example immediately adjacent locations <b>36</b> and <b>37</b>, A change in the emission wavelength or the Inten intensity of the emitted radiation can be observed that in their extent over a predetermined threshold value addition goes. If that is the case, the presence of an optical interface in the neighboring towns <b>36</b> and <b>37</b> getting closed.

Since these cities <b>36</b>. <b>37</b> as for any other sampled Location of the sample and the data of the deflection positions in the driving means <b>29</b> and / or the evaluation circuit <b>28</b> are available, can be with the procedural invention ren against relevant deflection positions of course derarti Calculate ger optical boundary layers and inter alia with Interpretation of these deflection positions finally the surface or Calculate the volume of the optical boundary layers is included.

It should be noted for completeness that in the <b>Fig.</b> 2 object plane shown <b>34</b> only to a scan of the sample relates. is of course it possible to scan a plurality of planes of the sample by the Laser radiation to different coordinates in the z Direction, ie face to the illustrated vertical focus Siert is.