DE102006034915A1 - Laser Scanning Microscope - Google Patents

Abstract

Laser scanning microscope with a lighting module for coupling and combining a plurality of light sources, characterized in that the coupling and unification via an acousto-optic modulator (AOTF) takes place.

Description

at Some applications of laser scanning microscopy require that to work with very high scanning speeds to fast-running operations to capture, as in physiology in the observation extremely fast processes.

An LSM is essentially as in 1 presented in 4 modules: light source, scan module, detection unit and microscope. These modules are described in more detail below. It is additionally on DE 197 02 753 A1 directed. For the specific excitation of the different dyes in a preparation, lasers with different wavelengths are used in one LSM. The choice of the excitation wavelength depends on the absorption properties of the dyes to be investigated. The excitation radiation is generated in the light source module. Various lasers are used here (argon, argon krypton, TiSa laser). The combination of different wavelengths in the light source module is carried out as in 1 shown in the light source module on the right via beam splitters or mirrors. Furthermore, in the light source module, the selection of the wavelengths and the adjustment of the intensity of the required excitation wavelength, for example by the use of an acousto-optical crystal (AOTF).

Then arrives the laser radiation over a fiber or a suitable mirror arrangement in the scan module.

The Laser radiation generated in the light source is using the lens diffraction limited over Focus the scanner, the scan optics and the tube lens into the specimen. The focus rasterizes punctiform sample in the x-y direction. The pixel dwell times when scanning over the Samples are usually in the range of less than a microsecond up to a few seconds.

at a confocal detection (descanned detection) of the fluorescent light, The light comes from the focal plane (Specimen) and out of the about that- and underlying levels is emitted via the scanners to a dichroic Beam splitter (MDB). This separates the fluorescent light from the excitation light. Subsequently, will the fluorescent light on an aperture (confocal aperture / pinhole) focused, which are exactly in a conjugate to the focal plane Level is located.

Thereby Fluorescence light components are suppressed out of focus. By Varying the aperture size can the optical resolution of the microscope. Behind the panel is located another dichroic block filter (EF) which once again excites the radiation suppressed. After passing through the block filter, the fluorescent light is of a point detector (PMT).

at Using a multiphoton absorption, the excitation of the Dye fluorescence in a small volume at which the excitation intensity especially is high. This range is only slightly larger than the detected range when using a confocal arrangement. The use of a confocal aperture can thus be omitted and the detection can be directly after the lens done (non-descanned detection).

In another arrangement for detecting one by Mehrphotonenabsorption excited dye fluorescence continues to be a descanned Detection, but this time the pupil of the lens is in the detection unit imaged (non-confocal descanned detection).

From a three-dimensionally illuminated image is by both detection arrangements in conjunction with the corresponding one-photon or multiphoton absorption only the plane (optical section) reproduced in the Focusing plane of the lens is located. By recording several optical sections in the x-y plane at different depths z Sample can subsequently computer-aided a three-dimensional image of the sample will be generated.

The LSM is therefore suitable for the examination of thick specimens. The excitation wavelengths become by the dye used with its specific absorption properties certainly. Matched to the emission properties of the dye Dichroic filters ensure that only that of the respective Dye emitted fluorescent light from the point detector measured becomes.

In biomedical applications, several different cell regions are currently labeled with different dyes simultaneously (multifluorescence). The individual dyes can be compared with the prior art either due to different absorption properties or emission properties (spectra) be detected separately. For this purpose, there is an additional splitting of the fluorescent light of several dyes with the secondary beam splitters (DBS) and a separate detection of the individual dye emissions in separate point detectors (PMT x). The LSM LIVE of Carl Zeiss Microlmaging GmbH realizes a very fast line scanner with an image generation of 120 frames per second ( http://www.zeiss.de/c12567be00459794/Contents-Frame/fd9fa0090eee01a641256a550036267b ).

different discrete laser wavelengths be over united a tunable AOTF in a beam path.

The can Laser L1 emitting multiple wavelengths and single wavelength lasers L2, L3.

laser with multiple wavelengths are advantageously irradiated under the zeroth diffraction order in the AOTF and other lasers, for example, under the +/- first diffraction order.

It is also possible to irradiate several lasers at different angles, for this purpose the frequency of the acoustic wave of the AOTF is adjusted accordingly. The following relationship exists between the diffraction angle between diffracted and undiffracted radiation (theta), the wavelength (lambda) and the frequency of the acoustic waves (F): Theta = lambda * F / v, where v is the velocity of the acoustic wave in the crystal.

Assuming typical values v = 4000 m / s and F = 100 MHz, the following angles result for the following wavelengths: Lambda [nm] Theta [mrad] 405 0101 488 0122 543 0136 633 0158

The Angle splitting (theta) at constant frequency (F) is thus tiny. The splitting can be additionally increased, by the acousto-optic crystal with different frequencies (F) is operated. These frequencies can also be parallel in the Crystal are fed.

Previous laser modules (see above) already had AOTF for fast wavelength-dependent beam switching or attenuation ( DE 198 29 981 A1 ), new and advantageous here is the use for variable and flexible combination of different light sources.

A another possibility is the use of a prism P (or mirror element or other deflecting element), that for the multiline laser L1 permeable and by which a plurality of light beams L2, L3 are deflected, that they are for the relatively small angles of incidence of the AOTF (e.g., when used a constant frequency (F)) are suitable.

This prism P or mirror element can advantageously be arranged rotatably for adjusting the angle of incidence or displaceable for setting the impact location. As in the prior art, a coupling point to a microscope, here for example two ports for two scan modules of an LSM is provided. The coupling into light-guiding fibers F takes place in the prior art, for example via collimator optics KO
Advantageously, at the output of the AOTF to forward the radiation in the direction of the microscope, a fiber be firmly coupled (pre-adjustment of the crystal to the fiber).

Claims (5)

Laser scanning microscope with a lighting module for coupling and combining several light sources, characterized in that the coupling via an acousto-optic modulator (AOTF) takes place. Laser scanning microscope according to claim 1, wherein different wavelengths on unterschiedli diffraction orders corresponding different angles are radiated into the AOTF. Laser scanning microscope according to claim 1 or 2, one being several wavelengths containing light source over the zeroth diffraction order of the AOTF is radiated. Laser scanning microscope according to one of claims 1-3, wherein the light sources are lasers. Laser scanning microscope according to one of claims 1-4, wherein the irradiation of at least one wavelength via an adjustable mirror for angle adjustment.