Process and arrangement for confocal microscopy

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 are carried out during the deflection of the laser beam from location to location, while the deflection continues in an uninterrupted manner. In this way , so that at least two adjacent 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. A laser scanning microscope for carrying out this process is also disclosed.

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, λA 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. 
     
     
       14. A process for confocal microscopy comprising the steps of:
   coupling laser light of different spectral ranges into a microscope beam path deflected in at least two coordinates and directing the laser light successively with respect to time onto locations of a specimen;        irradiating the specimen, 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 both the spectral characteristics and the intensity of the laser light coupled into the microscope beam path while the deflection continues without interruption, so that at least two adjacent locations of the specimen are acted upon by light of both different spectral characteristics and different intensities.      
     
     
       15. The process for confocal microscopy according to  claim 14 , wherein the spectral characteristics and the intensity of the laser light are changed during the deflection by at least one of ( a )  occasional additional coupling in of at least one of  (   1   )  individual spectral components and  (   2   )  a plurality of spectral components and  ( b )  occasional interruption of the coupling in of at least one of  (   1   )  individual spectral components and  (   2   )  a plurality of spectral components.    
     
     
       16. The process for confocal microscopy according to  claim 14 , wherein the coupling in of the laser light is occasionally interrupted during the deflection.  
     
     
       17. The process for confocal microscopy according to  claim 14 , wherein the spectral characteristics and intensity of the laser light are 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 different spectral characteristics and different intensities.  
     
     
       18. The process for confocal microscopy according to  claim 15 , 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, the same locations are always exposed to laser light with different spectral characteristics and different intensities.  
     
     
       19. The process for confocal microscopy according to  claim 14 , wherein spectral components with wavelengths λ A1   = 633  nm, λ   A2   = 568  nm, λ   A3   = 543  nm,    λA   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 additionally occasionally coupled in.  
     
     
       20. The process for confocal microscopy according to  claim 14 , wherein the coupling in of spectral components with wavelengths λ A1   = 633  nm, λ   A2   = 568  nm, λ   A3   = 543  nm, λA   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 is occasionally interrupted.    
     
     
       21. The process for confocal microscopy according to  claim 14 , wherein the light returned by every individual irradiated location of the specimen is evaluated with respect to its spectral characteristics and its intensity, and 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 characteristics and intensity of the laser light by which this location was irradiated.  
     
     
       22. The process for confocal microscopy according to  claim 21 , wherein the laser light returned by every individual irradiated location is detected with a plurality of detection channels, and wherein the individual detection channels are arranged for receiving different spectral components.  
     
     
       23. The process for confocal microscopy according to  claim 14 , wherein at least one of the spectral characteristics 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.  
     
     
       24. The process for confocal microscopy according to  claim 14 , wherein a mathematical linking of data characterizing at least one of the spectral characteristics 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 characteristics and the intensity of the laser light directed on this location. 
     
     
       25. A laser scanning microscope comprising:
   means for generating laser light with different spectral components;        fibers coupling the laser light into a microscope beam path;        means for deflecting the laser light in at least two dimensions;        means for focusing the laser light on a specimen;        a plurality of detector means for receiving at least one of  ( a )  different spectral components of the light returned by the specimen and  ( b )  different spectral components of the light emitted by the specimen, each detector means being associated with a detection channel for one of a reflection band and an emission band;        means for quickly changing at least one of the spectral characteristics and the intensity of the laser light during the deflection of the laser light from location to location in an uninterrupted manner, whereby at least two locations located next to one another on the specimen are acted upon by at least one of light with different spectral characteristics and laser light of different intensity;        means for branching the reflection radiation and emission radiation proceeding from the specimen into individual detection channels; and        means for synchronously driving the means for quickly changing at least one of the spectral characteristics and the intensity of the laser light and the means for deflecting the laser light.      
     
     
       26. The laser scanning microscope according to  claim 25 , wherein the means for quickly changing includes at least one of ( a )  means for periodically interrupting coupling in of at least one of  (   1   )  individual spectral components and  (   2   )  a plurality of spectral components and  (   3   )  the radiation of the laser light in its entirety and  ( b )  means for periodically coupling into the microscope beam path at least one of individual spectral components and a plurality of spectral components.    
     
     
       27. The laser scanning microscope according to  claim 25 , wherein the means for quickly changing at least one of the spectral characteristics and the intensity of the laser light comprises at least one of a filter which can be influenced acousto- optically and an acousto - optic modulator.    
     
     
       28. The laser scanning microscope according to  claim 25 , wherein the different spectral components are selectable.  
     
     
       29. The laser scanning microscope according to  claim 25 , wherein the means for branching comprise color splitters.  
     
     
       30. The laser scanning microscope according to  claim 25 , further comprising means for evaluating the light returned by every individual irradiated location of the specimen with respect to its spectral characteristics and its intensity synchronously in time with the irradiation of the same location and while taking into account at least one of the spectral characteristics and intensity of the laser light by which this location was irradiated.  
     
     
       31. The laser scanning microscope according to  claim 30 , further comprising means for mathematically linking data characterizing at least one of the spectral characteristics and the intensity of the laser light directed on a location with data from the evaluation means and with the deflection positions corresponding to this location, for determining adjustment signals for changing at least one of the spectral characteristics and the intensity of the laser light directed on this location.  
     
     
       32. A laser scanning microscope comprising:
   means for generating laser light with different spectral components;        fibers coupling the laser light into a microscope beam path;        means for deflecting the laser light in at least two dimensions;        means for focusing the laser light on a location of a specimen;        a plurality of detector means for receiving  ( a )  different spectral components of the light returned by the specimen and  ( b )  different spectral components of the light emitted by the specimen, each detector means being associated with a detection channel for one of a reflection band and an emission band;        means for quickly changing both the spectral characteristics and the intensity of the laser light while the laser light is deflected from location to location in an uninterrupted manner, so that at least two locations located next to one another on the specimen are acted upon by light with different spectral characteristics and laser light of different intensity;        means for branching the reflection radiation and emission radiation proceeding from the specimen into individual detection channels; and        means for synchronously driving the means for quickly changing both the spectral characteristics and the intensity of the laser light and the means for deflecting the laser light.      
     
     
       33. The laser scanning microscope according to  claim 32 , wherein the means for quickly changing includes at least one of ( a )  means for periodically interrupting coupling in of at least one of  (   1   )  individual spectral components and  (   2   )  a plurality of spectral components and  (   3   )  the radiation of the laser light in its entirety and  ( b )  means for periodically coupling into the microscope beam path at least one of individual spectral components and a plurality of spectral components.    
     
     
       34. The laser scanning microscope according to  claim 32 , wherein the means for quickly changing both the spectral characteristics and the intensity of the laser light comprises at least one of a filter which can be influenced acousto- optically and an acousto - optic modulator.    
     
     
       35. The laser scanning microscope according to  claim 32 , wherein the different spectral components are selectable.  
     
     
       36. The laser scanning microscope according to  claim 32 , wherein the means for branching comprise color splitters.  
     
     
       37. The laser scanning microscope according to  claim 32 , further comprising means for evaluating the light returned by every individual irradiated location of the specimen with respect to its spectral characteristics and its intensity synchronously in time with the irradiation of the same location and while taking into account at least one of the spectral characteristics and intensity of the laser light by which this location was irradiated.  
     
     
       38. The laser scanning microscope according to  claim 37 , further comprising means for mathematically linking data characterizing the spectral characteristics and the intensity of the laser light directed on a location with data from the evaluation means and with the deflection positions corresponding to this location, for determining adjustment signals for changing both the spectral characteristics and the intensity of the laser light directed on this location.  
     
     
       39. A laser scanning microscope for carrying out a process for confocal microscopy comprising:
   means for deflecting a microscope beam in at least two coordinates and directing the beam successively with respect to time onto locations of a specimen;        means for coupling laser light of different spectral ranges into the microscope beam;        means for irradiating the specimen with the laser light, location by location and line by line, in at least one plane;        means for generating an image of the scanned plane from light returned from the irradiated locations; and        means for changing at least one of the spectral characteristics 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.      
     
     
       40. The laser scanning microscope according to  claim 39 , wherein the means for changing includes at least one of:
   means for occasionally additionally coupling in of one of individual spectral components and of a plurality of spectral components and        means for occasionally interrupting the coupling in of individual spectral components and of a plurality of spectral components.      
     
     
       41. A laser scanning microscope for carrying out a process for confocal microscopy comprising:
   means for deflecting a microscope beam path in at least two coordinates and directing it successively with respect to time onto locations of a specimen;        means for coupling laser light of different spectral ranges into the microscope beam;        means for permitting the laser light to act upon the specimen, location by location and line by line, in at least one plane;        means for generating an image of the scanned plane from light returned from the irradiated locations; and        means for changing both the spectral characteristics 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 both different spectral characteristics and different intensity.      
     
     
       42. The laser scanning microscope according to  claim 41 , wherein the means for changing comprises at least one of:
   means for occasionally additionally coupling one of individual spectral components and of a plurality of spectral components and        means for occasionally interrupting the coupling in of individual spectral components and of a plurality of spectral components.