DE102007040238A1 - Method for laser scanning microscopy and beam distributor - Google Patents

Abstract

Method for laser scanning microscopy, characterized by the use of thin-film technology from telecommunications in beam splitting into different wavelengths in the detection beam path of a laser scanning microscope and encapsulated beam splitter for a laser scanning microscope, consisting of thin-film filters (TTF) for beam splitting in several wavelengths.

Description

State of the art

In a laser scanning system lasers of different power classes are used. Furthermore, a laser scanning system is characterized by a large number of variable modules that serve as a detector or for illumination. In 1 schematically a beam path of a laser scanning microscope is shown.

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 DE19702753A1 directed.

to specific excitation of the different dyes in a preparation be in an LSM laser with different wavelengths used. The choice of the excitation wavelength depends according to the absorption properties of the dyes to be investigated. The excitation radiation is generated in the light source module. To be used different lasers (argon, argon krypton, TiSa laser). Farther the selection of the wavelengths takes place in the light source module and adjusting the intensity of the needed Excitation wavelength, z. B. by the use of an acousto-optical Crystal. Subsequently, the laser radiation passes 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 via the scanner, the scanning optics and the Tubus lens focused in the preparation. The focus is scanning punctiform the sample in x-y direction from. The pixel dwell times when scanning over the sample are usually in the range of less than a microsecond to several 100 microseconds.

at a confocal detection (descanned detection) of the fluorescent light, the light comes from the focus plane (Specimen) and from the above and underlying levels is emitted via the scanners on a dichroic beam splitter (MD). This separates the fluorescent light from the Excitation light. Subsequently, the fluorescent light focused on a diaphragm (confocal aperture / pinhole), which is located exactly in a plane conjugate to the focal plane. Thereby Fluorescence light components are suppressed out of focus. By Varying the aperture size can increase 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 measured by means 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 is particularly high. This area is only marginally larger as the detected area using a confocal array. Of the Use of a confocal aperture can thus be omitted and the detection can be done directly after the lens (non-descanned detection).

In another arrangement for detecting one by Mehrphotonenabsorption excited dye fluorescence is still a descanned detection, however, this time the pupil of the objective 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 then be computer-aided a three-dimensional Image of the sample will be generated. The LSM is thus for the investigation of suitable for thick preparations. The excitation wavelengths are determined by the dye used with its specific absorption properties. Dichroic tuned to the emission properties of the dye Filters ensure that only the fluorescent light emitted by the respective dye is measured by the point detector.

In Biomedical applications are currently becoming several different Cell regions labeled with different dyes at the same time (Multi fluorescence). The individual dyes can with the prior art either due to different absorption properties or emission properties (spectra) are detected separately. To an additional splitting of the fluorescence light takes place of several dyes with the secondary beam splitters (DBS) and a separate detection of individual dye emissions in separate point detectors (PMT x).

The LSM LIVE of Carl Zeiss Micolmaging GmbH realizes a very fast line scanner with an image generation of 120 frames per second ( http://www.zeiss.de/c12567be00459794/Contents-Frame/fd9fa0090eee01a641256a550036267b ).

When spectrally measuring fluorescent samples with a laser scanning microscope, the different spectral components of the Fluorescence light can be separated with suitable optical elements, the excitation light should be blocked efficiently.

Of the The prior art is the use of dichroic mirrors, Prisms or grids and block bridges as discrete, individually adjustable and adjustable components. The assembly and adjustment of these components is elaborate and delicate. The environmental influences (Temperature, dust, shocks) to which these assemblies are exposed, adversely affect performance, manufacturing costs, Servicebarkeit and reliability. Due to the complex structure Required service calls are also expensive and expensive.

Invention:

The invention is in 2 shown schematically.

One encapsulated component VT from telecommunications, preferably in TTF thin-film technology is suitable, the light of a Multiple wavelength laser or white light laser in single Split wavelength components. The solution presents a compact, encapsulated, fully adjusted assembly representing the Contains beam splitting. The laser sources are over Fibers are coupled in and out, with the input and output fibers are advantageously fixed, so that no adjustment of a fiber to Strahlaufspalter must be done as in the prior art.

In 2 is an input fiber that contains several wavelengths (multimode fiber) coupled to the manifold VT and out of the manifold VT come out several output fibers with different wavelengths λ1 .... λn.

It is according to the invention to the surprising use of one (or more) compact encapsulated beam distributor in fiber optic technology (eg Fa.CubOhttp: //www.cubeoptics.com/impressum.php ) in laser scanning microscopy.

The Solution represents a compact, encapsulated assembly the division of the wavelengths to be detected via Filtration divider realized and via separate fibers outputs.

The Invention enables a compact construction of a jet distributor, z. B. with the help of the CubO fiber pulplexer. The number of fiber outputs may vary. There are 32 fibers and 64 fibers possible. This solution allows hiding via shutter of the excitation light. It eliminates the assembly and adjustment at the mirrors, dividers, bars and bars. The encapsulated assembly ensures a robust construction that is resistant to the environmental influences temperature, dust and vibrations is and thus works more reliable. Advantageous is also the significant weight savings. The self-contained Beam distributor is technologically laser safe.

• – Der Strahlverteiler kann als beliebig z. B. als 32 bis 64 Kanal Version ausgeführt sein, auch sind mehr Kanäle denkbar
• – Es sind Kombinationen mit Shuttern und Strahlbeeinflussern möglich
• – Bauformunabhängige Spektraldetektoren können verwendet werden
• – Mehre spektrale (oder andere) Detektoren können integriert werden

The original application of the component of the company Cubo in the telecommunications industry allows low production costs.

• - The beam distributor can be as arbitrary z. B. be designed as a 32 to 64 channel version, also more channels are conceivable
• - Combinations with shutters and beam influencers are possible
• - Type independent spectral detectors can be used
• - Multiple spectral (or other) detectors can be integrated

Advantageously, a supply via optical fibers to channels of a multi-channel detector, preferably a multi-channel PMT ( DE 10033180A1 ) respectively.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• DE 19702753 A1 [0002]
• - DE 10033180 A1 [0020]

Cited non-patent literature

• - http://www.zeiss.de/c12567be00459794/Contents-Frame/fd9fa0090eee01a641256a550036267b [0010]
• - Fa.CubOhttp: //www.cubeoptics.com/impressum.php [0016]

Claims (6)

Method for laser scanning microscopy, characterized through the use of thin film technology from the Telecommunications in beam splitting into different wavelengths in the detection beam path of a laser scanning microscope. Encapsulated beam distributor for a laser scanning microscope, consisting of thin-film filters (TTF) for beam splitting in several wavelengths. Method for laser scanning microscopy, characterized through the use of encapsulated fiber telecommunications multiplexers when beam splitting into different wavelengths in the detection beam path of a laser scanning microscope. Jet distributor according to one of the claims 1-3, wherein from the encapsulated component optical fiber guides lead out, can be coupled to the different detectors are. Jet distributor according to one of the claims 1-4, wherein at least one multimode fiber for splitting is used. Jet distributor according to one of the claims 1-5, wherein a feed over optical fibers to channels of a multi-channel detector, preferably a multi-channel PMT he follows.