WO2009027024A1

WO2009027024A1 - Method for laser scanning microscopy and beam distributor

Info

Publication number: WO2009027024A1
Application number: PCT/EP2008/006748
Authority: WO
Inventors: Dieter Huhse; Jörg PACHOLIK; Thomas Paatzsch
Original assignee: Carl Zeiss Microimaging Gmbh
Priority date: 2007-08-25
Filing date: 2008-08-16
Publication date: 2009-03-05
Also published as: DE102007040238A1

Abstract

A method for laser scanning microscopy, characterized by the use of thin-film technology from the telecommunications field during the beam splitting into different wavelengths in the detection beam path of a laser scanning microscope and an encapsulated beam distributor for a laser scanning microscope, comprising thin-film filters for beam splitting into a plurality of wavelengths.

Description

Method for laser scanning microscopy and beam distributor

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 Fig. 1, a beam path of a laser scanning microscope is shown schematically.

An LSM is essentially divided into 4 modules as shown in FIG. 1: light source, scanning module, detection unit and microscope. These modules are described in more detail below. Reference is additionally made to DE19702753A1. 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). Furthermore, in the light source module, the selection of the wavelengths and the adjustment of the intensity of the required excitation wavelength, e.g. through the use of an acousto-optic crystal. Subsequently, the laser radiation passes through a fiber or a suitable mirror arrangement in the scan module.

The laser radiation generated in the light source is focused by means of the diffraction-limited diffraction lens via the scanner, the scanning optics and the tube lens into the specimen. The focus scans the sample punctiformly in the x-y direction. The pixel dwell times when scanning over the sample are usually in the range of less than one microsecond to several 100 microseconds.

In the case of a confocal detection (descanned detection) of the fluorescent light, the light which is emitted from the focal plane (specimen) and from the planes above and below passes through the scanners to a dichroic beam splitter (MD). This separates the fluorescent light from the excitation light. Subsequently, the fluorescent light is focused on a diaphragm (confocal aperture / pinhole), which is located exactly in a plane conjugate to the focal plane. As a result, fluorescent light portions outside the focus are suppressed. By varying the aperture size, the optical resolution of the microscope can be adjusted become. Behind the aperture is another dichroic block filter (EF) which again suppresses the excitation radiation. After passing through the block filter, the fluorescent light is measured by means of a point detector (PMT). When using multiphoton absorption, the excitation of dye fluorescence occurs in a small volume where the excitation intensity is particularly high. This area is only marginally larger than the detected area using a confocal array. The use of a confocal aperture can thus be dispensed with and the detection can take place directly after the objective (non-descanned detection).

In a further arrangement for detecting a dye fluorescence excited by multiphoton absorption, a descanned detection also takes place, but this time the pupil of the objective is imaged into the detection unit (nonconfocally descanned detection).

From a three-dimensionally illuminated image, only the plane (optical section) which is located in the focal plane of the objective is reproduced by both detection arrangements in conjunction with the corresponding one-photon absorption or multiphoton absorption. By recording a plurality of optical sections in the x-y plane at different depths z of the sample, a three-dimensional image of the sample can then be generated computer-aided. The LSM is therefore suitable for the examination of thick specimens. The excitation wavelengths are determined by the dye used with its specific absorption properties. Dichroic filters tuned to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye is measured by the point detector.

In biomedical applications, several different cell regions are currently labeled with different dyes simultaneously (multifluorescence). The individual dyes can be detected separately with the prior art either due to different absorption properties or emission properties (spectra). For this purpose, 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 Micolmaging GmbH realizes a very fast

Line scanner with 120 frames per second imaging

(http://www.zeiss.de/c12567be00459794/Contents-

Frame / fd9fa0090eee01 a641256a550036267b).

In the spectral measurement of fluorescent samples with a laser scanning microscope, the different spectral components of the fluorescent light must be separated with suitable optical elements, the excitation light should be blocked efficiently.

The prior art is the use of dichroic mirrors, prisms and block lands as discrete, individually adjustable and adjustable components. The assembly and adjustment of these components is complex and delicate. The environmental influences (temperature, dust, shocks) to which these assemblies are exposed adversely affect performance, manufacturing costs, service availability and reliability. Due to the complex structure required service calls are also complex and expensive.

Invention:

The invention is shown schematically in FIG.

An encapsulated component VT from telecommunications, preferably in TTF

Thin-film technology is suitable, the light of a multi-wavelength laser or

White light laser to split into individual wavelength components. The solution is a compact, encapsulated, fully adjusted assembly that contains the beam split. The laser sources are coupled in and out via fibers, wherein the input and output fibers are advantageously fixedly adjusted so that no adjustment of a fiber to the beam splitter must be made as in the prior art.

In Fig. 2, an input fiber containing a plurality of wavelengths (multimode fiber) is coupled to the distributor VT and a plurality of out of the distributor VT come out

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 the Fa. Cubθhttp: //www.cubeoptics.com/impressum.php) in laser scanning microscopy.

The solution is a compact, encapsulated assembly that realizes the division of the wavelengths to be detected via filter divider mirrors and outputs them via separate fibers.

The invention enables a compact construction of a jet distributor, e.g. with the help of the CubO fiber multiplexer. The number of fiber outputs can vary. There are 32 fibers and 64 fibers possible. This solution allows the shutter of the excitation light via shutter. It eliminates the assembly and adjustment work on the mirrors, dividers, gratings and bars. The encapsulated assembly provides a rugged construction that is resistant to environmental influences such as temperature, dust and shocks, making it significantly more reliable. Another advantage is the considerable weight savings. The self-contained beam distributor is technologically laser safe.

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

The beam distributor can be designed as any, e.g. run as 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) take place.

Claims

claims

1.

Method for laser scanning microscopy, characterized by

Use of telecommunications thin film technology in the

Beam splitting into different wavelengths in the detection beam path of a

Laser Scanning Microscope.

Second

Encapsulated beam distributor for a laser scanning microscope, consisting of

Thin-film filters (TTF) for beam splitting into several wavelengths.

Third

Method for laser scanning microscopy, characterized by

Use of encapsulated fiber multiplexers in telecommunications in the

Beam splitting into different wavelengths in the detection beam path of a

Laser Scanning Microscope.

4th

A jet distributor according to any one of claims 1-3, wherein the encapsulated component

Lead out light guides to which different detectors can be coupled.

5th

A beam distributor according to any one of claims 1-4, wherein at least one multi-mode fiber is used for splitting.

6th

A jet distributor according to any one of claims 1-5, wherein a

Feeding via optical fibers to channels of a multi-channel detector, preferably a multi-channel PMT takes place.