Process and arrangement for confocal microscopy

• US 6,462,345 B1
• Filed: 04/21/1999
• Issued: 10/08/2002
• Est. Priority Date: 07/04/1998
• Status: Expired

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: CARL Zeiss JENA GmbH (Carl-Zeiss-Stiftung), European Molecular Biology Laboratory
• Inventors: Simon, Ulrich, Moehler, Gunter, Wilhelm, Stefan, Meisel, Ulrich, Tille, Sebastian, Stelzer, Ernst Hans Karl
• Primary Examiner(s): Epps, Georgia
• Assistant Examiner(s): HANIG, RICHARD E
• Application Number:
  US09/295,556
• Time in Patent Office: 1,266 Days
• Field of Search: 250/458.1, 250/459.1, 250/461.1, 250/461.2, 356/317, 356/318, 356/417
• US Class Current: 250/458.1
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G02B 21/0056 based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
• CPC Class Codes: G02B 21/0064 multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/0084 time-scale detection, e.g. strobed, ultra-fast, heterodyne detection

Abstract

A process for confocal microscopy is disclosed in which laser light is coupled into a microscope beam path, directed successively with respect to time onto different locations of a specimen, and an image of the scanned plane is generated from the light reflected and emitted by the irradiated locations. A change in the spectral composition and in the intensity of light is carried out during the deflection of the laser beam from location to location, while the deflection continues in an uninterrupted manner. In this way, at least two locations of the specimen located next to one another are acted upon by light with different spectral characteristics and by laser radiation of different intensity. By periodically interrupting the coupling in of the laser light during the deflection of the microscope beam path, it is made possible that only selected portions of the image field are acted upon by the laser radiation. A laser scanning microscope for carrying out this process is also disclosed.

Claims

1. A process for confocal microscopy comprising the steps of:

coupling the laser light of different spectral ranges into a microscope beam path deflected in at least two coordinates and directing it successively with respect to time onto locations of a specimen;

permitting the specimen to be acted upon, location by location and line by line, by the laser light in at least one plane and generating an image of the scanned plane from light returned from the irradiated locations;

changing at least one of the spectral composition and the intensity of the laser light coupled into the microscope beam path while the deflection is continued without interruption, so that at least two adjacent locations of the specimen are acted upon by light of at least one of different spectral characteristics and different intensity.

2. The process for confocal microscopy according to claim 1, wherein at least one of the spectral composition and the intensity of the laser light is changed during the deflection by occasional additional coupling in of one of individual spectral components and of a plurality of spectral components and by occasional interruption of the coupling in of individual spectral components and of a plurality of spectral components.

3. The process for confocal microscopy according to claim 1, wherein the coupling in of the laser light is occasionally interrupted during the deflection.

4. The process for confocal microscopy according to claim 1, wherein at least one of the spectral composition and intensity of the laser light is changed during deflection on locations which are located adjacent to one another in a line, so that at least locations of this line are acted upon by laser radiation with at least one different spectral characteristics and different intensity.

5. The process for confocal microscopy according to claim 2, wherein the locations located adjacent to one another in a line are acted upon repeatedly by the coupled in laser light and, in this way, always the same locations are exposed to laser light with different spectral composition and/or with different intensity.

6. The process for confocal microscopy according to claim 1, wherein spectral components with wavelengths λ

_A1=633 nm, λ

_A2=568 nm, λ

_A3=543 nm, λ

A_A4=514 nm, λ

_A5=488 nm and/or λ

_A6=458 nm in the VIS range and with wavelengths λ

_A7=351 nm and/or λ

_A8=364 nm in the UV range are occasionally coupled in additionally or their coupling in is occasionally interrupted.

7. The process for confocal microscopy according to claim 1, wherein the light returned by every individual irradiated location of the specimen is evaluated with respect to its spectral characteristics and its intensity, wherein the evaluation is carried out synchronously in time with the irradiation of the same location and while taking into account at least one of the spectral composition and intensity of the laser light by which this location was irradiated.

8. The process for confocal microscopy according to claim 7, wherein the laser light returned by every individual irradiated location is detected with a plurality of detection channels, wherein the individual detection channels are arranged for receiving different spectral components.

9. The process for confocal microscopy according to claim 1, wherein at least one of the spectral composition and the intensity of the laser light which is coupled into the microscope beam path corresponds to the excitation radiation of a fluorescence dye contained in the specimen or applied to the specimen and the individual detection channels are configured for the reception of the emission radiation proceeding from the fluorescence dye.

10. The process for confocal microscopy according to claim 1, wherein a mathematical linking of data characterizing at least one of the spectral composition and the intensity of the laser light directed on a location, of data of the evaluation findings for the light returned by the same location and of the deflection positions corresponding to this location is carried out for the purpose of determining adjustment signals for changing at least one of the spectral composition and the intensity of the laser light directed on this location.

11. A laser scanning microscope for carrying out a process for confocal microscopy comprising the steps of:

coupling the laser light of different spectral ranges into a microscope beam path deflected in at least two coordinates and directing it successively with respect to time onto locations of a specimen;

permitting the specimen to be acted upon, location by location and line by line, by the laser light in at least one plane and generating an image of the scanned plane from light returned from the irradiated locations;

changing at least one of the spectral composition and the intensity of the laser light coupled into the microscope beam path while the deflection is continued without interruption, so that at least two adjacent locations of the specimen are acted upon by light of at least one of different spectral characteristics and different intensity, said microscope comprising;

a laser module for generating laser light with different selectable spectral components;

single-mode fibers for coupling the laser light into the microscope beam path;

a scanning device which deflects in at least two dimensions;

a microscope objective which focuses the laser light on a specimen;

a plurality of detectors for the reception of different spectral components of the light returned by the specimen;

an evaluation circuit which is connected subsequent to outputs of the detectors;

a plurality of individually controllable single-wavelength and multiple-wavelength lasers;

at least one of a filter which can be influenced acousto-optically and an acousto-optic modulator being provided in the laser module;

photomultipliers being provided as detectors;

color splitters which are arranged on drivable exchanging devices and which can be substituted for one another being provided for branching the reflection radiation and emission radiation proceeding from the specimen into individual detection channels; and

control inputs of the laser module, scanning device and exchanging devices being connected with the outputs of the evaluation circuit.

12. The laser scanning microscope according to claim 11, wherein a beam component of the laser light coupled into the microscope beam path is directed on an optoelectronic receiver whose output is connected with the driving unit.

13. The laser scanning microscope according to claim 11, wherein a mathematical linking of the output signals of the optoelectronic receiver with at least one of the output signals of the PMT and the deflection signals for the scanning device being provided in the evaluation circuit.