DE102006047911A1 - Arrangement for splitting detection light - Google Patents

Abstract

Arrangement for the division of detection light in a laser scanning microscope, wherein a spectral separation of different spectral components in transmitted and hidden parts takes place, wherein the division is effected by at least one tilted at an angle not equal to 90 ° and 0 ° to the optical axis coated filter, wherein the angle to the optical axis deviates less than 20 °, advantageously less than 10 °, from the optical axis.

Description

In A laser scanning system uses lasers of different power classes used. Furthermore, a laser scanning system is characterized by a large Number of variable modules marked as detector or to serve for lighting.

One confocal scanning microscope contains a light source module, which preferably consists of several laser beam sources exists that generate illumination light of different wavelengths. A scanning device, in which the illumination light as an illumination beam has a main color divider, an x-y scanner and a scan lens to scan the illumination beam through a beam deflection To lead a sample which is located on a microscope stage of a microscope unit. A measuring light beam coming from the sample generated thereby is transmitted via a Main color divider and an imaging optic on at least one confocal Detection aperture (detection pinhole) directed a detection channel.

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 DE 197 02 753 A1 directed.

to specific stimulation of the different dyes in a preparation used in a LSM laser with different wavelengths. The choice of Excitation wavelength depends on the absorption properties of the examined Dyes. The excitation radiation is generated in the light source module. Various lasers (argon, argon krypton, TiSa laser). Furthermore, the selection is carried out in the light source module the wavelengths and the setting of the intensity the needed Excitation wavelength, e.g. through the use of an acousto-optic crystal. 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 several 100 microseconds.

at a confocal detection (descanned detection) of the fluorescent light, The light comes from the focal plane (Specimen) and from the above and underlying levels is emitted via the scanner to a dichroic beam splitter (MD). This separates the fluorescent light from the excitation light. Subsequently, will 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 can the optical depth resolution of the microscope. Behind the panel is located another dichroic block filter (EF) again the excitation 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 done directly after the Objectively done (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 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 with the prior art either due to different absorption properties or emission (spectra) are 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 from Carl Zeiss Micolmaging GmbH realizes a very fast line scanner with an image generation rate of 120 frames per second
( http://www.zeiss.de/c12567be00459794/Contents-Frame/fd9fa0090eee01a641256a550036267b ).

The Connection of the light source modules with the scan module takes place in the Usually over Optical fibers.

The coupling of several independent lasers into a fiber for transmission to the scan head was, for example, in Pawley: "Handbook of Confocal Microscopy", Plenum Press, 1994, page 151 as in DE 196 33 185 A1 described.

In 2 the area after the main color divider is shown in the direction of the detection. In the 1 Here, NFT1 and NFT2, which are referred to as DBS, denote the detection radiation in a wavelength-dependent manner (preselection) in the direction of detectors D1-D3, which are preferably preceded by engine-changeable emission filters EF1-EF3 (main selection of the wavelength).

In 3 It is shown that NFT1 and 2 as well as EF 1-3 can be designed as motor-driven filter wheels in order to optimize the flexibility in the setting of detected wavelength ranges.

In the 1 For the sake of clarity, PH1-4 shown in FIG 2 - 5 n not available, as well as a pinhole for all partial beam paths, this vorgesetzt executable.

• • Emissionsfilter im Strahlengang vor dem Detektor zur Auswahl der detektierten Wellenlängen, senkrecht zur optischen Achse angeordnet. Gleichzeitig unterdrücken die Emissionsfilter das Licht außerhalb des gewählten Spektralbereiches
• • Dichroitische Strahlteiler (mit deutlich schwächeren Unterdrückungseigenschaften ausserhalb ihres transmissiven Wirkungsbereiches) zur Vorselektion von Wellenlängenbereichen, ebenfalls, auch wechselbar, nicht senkrecht, typisch unter 45° zur optischen Achse angeordnet

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 beamsplitters (with significantly lower suppression properties outside their transmissive range of action) for the preselection of wavelength ranges, also, also changeable, not vertical, typically at 45 ° to the optical axis

To reach the in 2 According to the state of the art, flexibility is required for the detection of three spectral ranges 5 filter wheels and with maximum variable detection and 5 drives for changing the filter.

Besides There is a high space requirement for the required elements.

This high cost to be reduced by the invention.

According to the invention was recognized that advantageously coated emission filters practically have no absorption. These filters reflect all light with wavelengths outside of the selected spectral range. It therefore seems possible, this reflected Light for additional spectral channels To use the reflected light from the incident light without Further filters can be separated, the emission filters slightly tilted out of the vertical incidence. A small angle (<< 20 °) was there recognized as particularly advantageous because the spectral properties typical emission filter (high suppression, steep band edges) for vertical Incidence and small angle deviating from the vertical incidence especially good to realize.

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.

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

The invention will be described below with reference to the 4 . 5 and 6 described in more detail.

It demonstrate:

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

5 :

The NEF 1-3 each on filter wheels for replacement

6 : Additional switchable filter F1-3 in the beam path of 4 ,

According to the invention are the emission filters are designed so that they have a defined part transmit the light radiation and reflect the rest almost completely.

To For this purpose, they are at an angle not equal to 90 degrees in the beam path arranged, which has an angle of incidence on the filter of unequal zero degrees realized

• – Reflektion des noch gewünschten Restspektrums
• – Nicht gewünschtes Restspektrum kann absorbiert werden
• – Einfallswinkel abweichend von 0°, aber noch klein (typischerweise 10°, auf jeden Fall deutlich kleiner 45°)
• – kleiner Einfallswinkel garantiert nahezu gleiche Eigenschaften für s- und p-polarisiertes Licht sowie hocheffiziente Unterdrückung der nicht gewünschten Spektralbereiche in Transmission.

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
• - Incidence angle 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 regions in transmission.

• – Erhöhung der Flexibilität, mit dem Filter kann Spektralbereich weiter eingeschränkt werden
• – Zur einseitigen Einengung des Spektrums reicht ein zusätzlicher Kurz- bzw. Langpaß, das ergibt zusammen mit dem vorgeschalteten Filter ein Bandpaß und ist kostengünstiger als zwei schaltbare Bandpässe
• – Das reflektiertes Licht vom nachgeschalteten Filter kann ebenfalls genutzt werden.

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.

Of the Unused portion of the spectrum may also be directed into a beam trap S become. Detection of the light with another detector e.g. for control purposes is also possible.

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

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

6 shows an arrangement with arranged in the beam path "conventional" emission filter wheels NEF 1-3.

exemplary Here, arrangements with 3 channels have always been shown, but they can be just two or any number of channels.

Claims (8)

Arrangement for the division of 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 is effected by at least one tilted at an angle not equal to 90 and zero degrees to the optical axis coated first filter , Arrangement according to claim 1, wherein the angle to the optical Axis less than 20 degrees, preferably less than 10 degrees from the deviates from optical axis. Arrangement according to claim 1 or 2, wherein the filter for splitting has a typical for emission filter suppression of the hidden spectral components of> 10 ^-3 . Arrangement according to one of the preceding claims, wherein the first filter at least in the direction of the hidden Radiation is arranged downstream of at least a second filter. Arrangement according to one of the preceding claims, wherein a cascade Splitting the detection light takes place at least in one plane, through which the optical axis leads. Arrangement according to one of the preceding claims, wherein the first or second filter downstream emission filter for Narrowing of the transmitted spectrum are provided. Arrangement according to claim 6, wherein the subordinate Emission filter is long or short pass and with the upstream Filter forms a bandpass. Laser scanning microscope with at least one arrangement according to any one of the preceding claims.