WO2008043459A2

WO2008043459A2 - System for detection light division

Info

Publication number: WO2008043459A2
Application number: PCT/EP2007/008555
Authority: WO
Inventors: Ralf Wolleschensky; Wolfgang Bathe; Jörg STEINERT; Dieter Huhse
Original assignee: Carl Zeiss Microimaging Gmbh
Priority date: 2006-10-06
Filing date: 2007-10-02
Publication date: 2008-04-17
Also published as: DE102006047911A1; WO2008043459A3

Abstract

The invention relates to a system for dividing the detection light in a laser scanning microscope, the separation into spectra of different spectral components being carried out into transmitted and extracted components. The invention is characterized in that the division is carried out by at least one coated filter that is tilted in relation to the optical axis at an angle unequal 90 and zero degrees, the angle in relation to the optical axis deviating less than 20 degrees, preferably less than 10 degrees from the optical axis.

Description

Arrangement for splitting detection light

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. A confocal scanning microscope includes a light source module, which preferably consists of a plurality of laser beam sources that generate illumination light of different wavelengths. A scanning device, in which the illumination light is coupled as an illumination beam, has a main color splitter, an x-y scanner and a scanning objective to guide the illumination beam by beam deflection over a sample which is located on a microscope stage of a microscope unit. A measurement light beam generated thereby from the sample is directed via a main color splitter and imaging optics to at least one confocal detection aperture (detection pinhole) of a detection channel.

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

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 a confocal detection (descanned detection) of the fluorescent light, the light passes from the focal plane (Specimen) and from the above and below Layers is emitted via the scanner 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 depth resolution of the microscope can be adjusted. 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 parts (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 / fd9fa0090eee01a641256a550036267b).

The connection of the light source modules with the scan module is usually about

Optical fibers.

The coupling of several independent lasers into a fiber for transmission to the scan head has been described for example in Pawley: "Handbook of Confocal Microscopy", Plenum Press,

1994, page 151 and in DE19633185 A1.

In Fig. 2, the area after the main color splitter in the direction of detection is shown.

The secondary color splitters designated as DBS in FIG. 1 are here NFT1 and NFT2, which are the

Wavelength-dependent detection radiation (preselection) in the direction of detectorsDI-

D3, which still preferably motor-changeable emission filter EF1-EF3 (

Main selection of the wavelength) are arranged upstream.

In Fig. 3 it is shown that NFT1 and 2 and EF 1-3 can be designed as a motorized filter wheels to the flexibility in the setting of detected

Optimize wavelength ranges.

The pinholes PH1-4 shown in FIG. 1 are not present for reasons of clarity in FIG. 2-5 n, since a pinhole for all beam paths .these vorgesetzt executable would be.

Emission filters and dichroic beam splitters have hitherto been used differently in the prior art:

• Emission filter in the beam path in front of the detector for selecting the detected wavelengths, arranged perpendicular to the optical axis.

At the same time, the emission filters suppress the light outside the selected spectral range

• Dichroic beam splitters (with significantly lower suppression properties outside their transmissive range of action) for preselection of wavelength ranges, also, also changeable, non-perpendicular, typically less than 45 ° to the optical axis To achieve the flexibility shown in FIG. 2 according to the state of the art, 5 filter wheels are required to detect three spectral ranges, and 5 drives for changing the filters are required for a maximum of variable detection. In addition, there is a high space requirement for the required elements.

This high cost is to be reduced by the invention.

According to the invention, it was recognized that advantageously coated emission filters have virtually no absorption. These filters reflect all light with wavelengths outside the selected spectral range. It therefore seems possible to use this reflected light for other spectral channels. In order for the reflected light to be able to be separated from the incident light without further filters, the emission filters are easily tilted out of the vertical incidence. A small angle («20 °) was found to be particularly advantageous because the spectral properties of typical emission filters (high suppression, steep band edges) can be implemented particularly well for vertical incidence and small angles deviating from the normal incidence.

Furthermore, with the filters, a transmission of the non-reflected beam portion with high suppression of unwanted radiation (advantageously greater than 10 ^"3 ) in this

Area.

This arrangement advantageously results in the possibility of a simpler, more compact and cost-reduced "cascading" of the detection channels in one or more planes.

The invention is described below with reference to Figures 4, 5 and 6 in more detail.

Show it:

4:

Emission filters NEF 1-3 arranged according to the invention, detectors D1-D3, jet trap S

Figure 5:

The NEF 1-3 each on filter wheels for replacement

Fig. 6:

Additional switchable filter F1-3 in the beam path of FIG. 4.

According to the invention, the emission filters are designed such that they transmit a defined part of the light radiation and reflect the rest almost completely. For this purpose, they are arranged at an angle other than 90 degrees in the beam path, which realizes an angle of incidence on the non-zero-degree filter

So there is a design of the emission filter so that reflected spectrum can still be used:

- Reflection of the desired residual spectrum

Unnecessary residual spectrum can be absorbed

Angle of incidence deviating from 0 °, but still small (typically 10 °, in any case significantly less than 45 °) small angle of incidence guarantees almost the same properties for s- and p-polarized light as well as highly efficient suppression of unwanted

Spectral ranges in transmission.

In a further step, a downstream, preferably switchable, emission filter follows in front of the detector with the following advantages:

- Increased flexibility, with the filter spectral range can be further restricted

- For one-sided narrowing of the spectrum an additional short or long pass, which together with the upstream filter bandpass and is less expensive than two switchable bandpasses

The reflected light from the downstream filter can also be used.

The unused portion of the spectrum may also be directed into a beam trap S. Detection of the light with another detector e.g. for control purposes is also conceivable.

Fig. 4 shows a simple structure without downstream filters: The filters can also be switched interchangeable:

Fig. 5 shows fixed filters for main selection, additional switchable filters, preferably long and short passes, for narrowing the spectra of the fixed filters:

Fig. 6 shows an arrangement with arranged in the beam path "conventional" emission filter wheels NEF 1-3. By way of example, arrangements with 3 channels have always been shown here, but it is also possible for only two or any number of channels.

Claims

claims

1. Arrangement for dividing detection light in a laser scanning microscope, wherein a spectral separation of different spectral components in transmitted and hidden parts, characterized in that the division by at least one tilted at an angle not equal to 90 and zero degrees to the optical axis coated first Filter takes place.

2. Arrangement according to claim 1, wherein the angle to the optical axis deviates less than 20 degrees, advantageously less than 10 degrees from the optical axis.

3. Arrangement according to claim 1 or 2,

Wherein the filter for splitting a typical for emission filter suppression of the hidden spectral components of> 10 ^'3 has.

4. Arrangement according to one of the preceding claims, wherein at least one second filter is arranged downstream of the first filter at least in the direction of the blanked radiation.

5. Arrangement according to one of the preceding claims, wherein a cascaded splitting of the detection light takes place at least in a plane through which the optical axis leads.

6. Arrangement according to one of the preceding claims, wherein the first or second filter downstream emission filters are provided for narrowing the transmitted spectrum.

7. Arrangement according to claim 6, wherein the downstream emission filter is long or short pass and forms a bandpass filter with the upstream filter.

8. Laser scanning microscope with at least one arrangement according to one of the preceding claims.