JP2000056228A - System for detection by wavelengths to be used for laser scanning microscope, and image recording method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a system for controllable detection by wavelengths by combining a short wavelength transmission filter and at least one long wavelength transmission filter. SOLUTION: The light of a laser L is guided through a shutter device V, optical coupling system EO, mirror S2 and beam splitter ST2 toward a scanner SC. Reflected light and/or fluorescent light from a target passes through a dichroic beam splitter ST2, forward moves to an optical collimation system KO and pinhole PH and arrives at a detector but in front of the detector, a filter unit FE is arranged. It is preferable that the filter unit FE is composed of an exchangeable short wavelength transmission filter SP and long wavelength transmission filter LP. Thus, passing band width and the position of its selective area can be controlled as desired. Further, a notch filter NF can be additionally provided for stopping only the excited wavelength of high optical density as well. Thus, the sensitivity of detecting the fluorescent light is further improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a variable type of combination of a short wavelength transmission filter and a long wavelength transmission filter for enabling free programming of a bandpass filter used in a fluorescence microscope, particularly a laser scanning microscope. Or, it relates to a discontinuous serial arrangement. These filters are advantageously adaptable to newly marketed fluorescent coloring dyes.

[0002]

FIG. 1 schematically shows a microscope unit M and a scanning head S connected thereto. The microscope is provided with a light source LQ1 with an irradiation optical system that irradiates a target object on the sample table T through the beam separator ST1 as in the related art. The turning mirror S3 is a light source L
It is used to switch the transmitted light irradiation performed from the collector Q2 through the condenser KO. Observation is performed from the eyepiece OK through the lens barrel lens TL and the mirror S1. The scanning light path passes through this mirror or beam splitter S1 and further to the scanning lens SL and the scanning device SC.

The light of the laser L passes through a shutter device V, a coupling optical system EO, a mirror S2, and a beam splitter ST2, and is guided toward the scanning device SC. The reflected light and / or fluorescent light from the target object is output to the dichroic beam splitter ST2.
Through the collimation optical system KO and the pinhole PH to the detector, before which is arranged a filter unit FE according to the invention, which is shown enlarged and shown.

The filter unit FE preferably comprises a replaceable short wavelength transmission filter SP and a long wavelength transmission filter LP. Thereby, the pass band width and the position of the important region can be adjusted as desired. In addition to this, a notch filter NF that suppresses only the excitation wavelength having a high optical density can be provided. Thereby, the detection sensitivity for fluorescence is further increased.

The above-mentioned filter unit can of course be used for a plurality of detection lines. In this case, a rough preliminary selection of the spectral range can be made in a known manner, for example by means of a dichroic beam separator or a revolver-switching separator as in DE 197 275 A1, for each detection line, but with a slight difference. The adjustment takes place through the filter unit according to the invention.

FIGS. 2a to 2d are examples of a filter system according to the invention. FIG. 2a shows sliding filters FS1, FS2 arranged one behind the other, each having a circular filter F1, F2. In this system, two different filters are combined sharing the optical axis A.

In FIG. 2b, filters F1 and F2 are arranged on rotary disks FR1 and FR2, respectively, and are combined so as to share an optical axis A. The most advantageous is
This is a combination of at least one of the continuously variable edge filters VF1 and VF2. FIG. 2c shows a circular rotary type and FIG. 2d shows a sliding type. Such a variable edge pass filter can be obtained, for example, from COHERENT. FIG. 2d shows the course of the transmission curve corresponding to the individual filter areas. VF2 is a transmission filter for short wavelength SP, and VF1 is a transmission filter for long wavelength LP.

FIG. 3 shows two adjustment examples. As is clear from this, a short-wavelength transmission filter and a long-wavelength transmission filter are combined to adjust the mutual movement, whereby a bandpass filter for transmitting a detection wavelength having an arbitrary bandwidth can be configured. This is a wavelength tunable monochromator that continuously scans the entire area in small wavelength bandwidth units, enabling the recording of a complete fluorescence spectrum of the exogenous or endogenous fluorophore to be inspected in the sample, which is extremely useful. is there.

By adjusting the scanning device, the sample table, and the detector in this manner, it becomes possible to perform detection by wavelength and by time in the X, Y, and Z directions. Thereby, a five-dimensional (X, Y, Z, T, λ) sample image is formed. That is, a spectrum can be recorded for each pixel and each time of a scanning cycle.

[0010] In addition, the filter unit according to the present invention can be effectively used for spectrum selection of an irradiation surface.
For example, as a substitute for a shutter device, it can be used for narrowing a wide laser light source or for selecting a wavelength in an HBO or HAL lamp.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a microscope unit M and a scanning head S connected thereto.

FIG. 2 (a) a slide type filter, (b) a rotary disk filter, (c) a continuously variable circular rotary edge filter,
(D) Continuously variable slide type

FIG. 3 shows an example of adjusting a short wavelength transmission filter and a long wavelength transmission filter.

[Description of Signs] M Microscope unit S Scan head ST Beam separator LQ light source S2 Turning mirror SC Scanning device V Shutter EO Imaging optical system KO Collimating optical system PH Pinhole FE Filter unit SP Short wavelength transmission filter LP Long wavelength Transmission filter NF Notch filter FS filter FR Rotary filter VF Continuously variable filter

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Michael Stock (Original notation) Michael Stock Germany D-99510 Apolda Elf Luterstrasse 32 (Original notation) Er after Str. 32, D-99510 Apolda, Germany (72) Inventor Ulrich Simon (Original notation) Ulrich Simon Germany D-07751 Rosenstein Burgstrasse 35 (Original notation) Burgstr. 35, D-07751 Rotenstein, Germany (72) Inventor Torsten Antrac (Original notation) Torsten Anttrack Germany D-07745 Jena Anna Siamsen-Strasse 20 (Original description) Anna-Siemsen-Str. 20, D- 07745 Jena, German any

Claims (8)

[Claims] 1. A fluorescence microscope, in particular a laser scanning microscope, as an adjustable bandpass filter comprising at least one combination of at least one short wavelength transmission filter and at least one long wavelength transmission filter arranged in the detection light path. For controllable wavelength-specific detection used in optics 2. The system according to claim 1, wherein at least one filter is replaceable with another filter having another wavelength characteristic and / or its wavelength characteristic is adjustable. 3. The system according to claim 1, further comprising filter changing means for a short wavelength transmission filter and / or a long wavelength transmission filter. 4. The system according to claim 3, wherein the filter changing means is a sliding device or a rotating disk device. 5. The method according to claim 1, comprising at least one rotatable and / or sliding continuously movable short wavelength transmission filter and / or long wavelength transmission filter.
System based on one of the four 6. The system according to claim 1, wherein the filter and / or the filter changing means are replaceable. 7. The system according to claim 1, wherein the filter and / or the filter changing means are motor-driven. 8. A system according to claim 1, wherein the inspection results are recorded by time and by wavelength in a detection unit and are stored in a storage unit by using a laser scanning microscope. Way for