Microscope and method for operating a microscope

Abstract

The invention relates to a method for operating a microscope in which excitation light is focused on, or beamed to, different points of a specimen, in which an intensity of the excitation light is point-specifically varied and in which an intensity of the light reflected by said specimen in at least one spectral range is measured point-specifically and quantitatively. The method according to the invention is characterized in that the intensity and/or a spectral composition of the excitation light beamed to a specific point of said specimen is automatically adjusted by a regulating device on the basis of information previously gained from measured data of said specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity of the light reflected in the spectral range by this point during a pixel dwell time is within a predefined value interval. The invention also relates to a microscope.

1 - 36 . (canceled) 
   
   
       37 . A method for operating a microscope,
 in which excitation light is beamed to different points of a specimen,   in which an intensity of the excitation light is point-specifically varied and in which an intensity of the light reflected by the specimen in at least one spectral range is measured point-specifically and quantitatively,   wherein at least one of the intensity or a spectral composition of the excitation light beamed to a specific point of the specimen is automatically adjusted by a regulating device on the basis of information previously gained from measured data of the specimen concerning at least one of an estimated or an actual intensity of the light reflected in the spectral range by the specific point such that an integral of the intensity of the light reflected in the spectral range by the specific point during a pixel dwell time is within a predefined value interval.   
   
   
       38 . The method as defined in  claim 37 ,
 wherein the regulating device adjusts the intensity of the excitation light for the specific point such that the integral of the intensity of the light reflected by the specific point during a pixel dwell time is within a predefined value interval only when a signal criterion for this point is satisfied.   
   
   
       39 . The method as defined in  claim 37 ,
 wherein the excitation light includes a plurality of wavelengths for exciting a plurality of different dyes and   the intensity of the light reflected by the specimen is measured in a plurality of different spectral regions.   
   
   
       40 . The method as defined in  claim 37 ,
 wherein the regulating device adjusts the intensity of the excitation light beamed to the specific point on the basis of an intensity of the reflected light detected for the point in a previous image.   
   
   
       41 . The method as defined in  claim 37 ,
 wherein the value interval for the integral of the intensity of the light reflected by the specific point during the pixel dwell time is set such that a detector used is able to operate with maximum sensitivity and with a maximum signal to noise ratio.   
   
   
       42 . The method as defined in  claim 37 ,
 wherein the regulation is effected such that the intensity of the excitation light for the specific point is during a pixel dwell time constant to a maximum extent following a stabilization phase of the regulating process.   
   
   
       43 . The method as defined in  claim 37 ,
 wherein the regulating device adjusts the intensity of the excitation light for the specific point such that the integral of the intensity of the light reflected by the specific point during a pixel dwell time is within a predefined value interval only when a signal criterion for the specific point is satisfied.   
   
   
       44 . The method as defined in  claim 37 ,
 wherein the regulation is effected such that the integral of the intensity of the light reflected during a pixel dwell time by at least one point satisfying a signal criterion, is constant.   
   
   
       45 . The method as defined in  claim 37 ,
 wherein the same pixel dwell time is used for all points.   
   
   
       46 . The method as defined in  claim 37 ,
 wherein the same pixel dwell time is used for all points for which a signal criterion is satisfied.   
   
   
       47 . The method as defined in  claim 37 ,
 wherein the signal criterion for the specific point is satisfied if at least one of the estimated intensity and the actual intensity of the light reflected by the specific point is above a specifiable background threshold.   
   
   
       48 . The method as defined in  claim 37 ,
 wherein the signal criterion for the specific point is satisfied when an externally supplied point-specific signal has a predefined value.   
   
   
       49 . The method as defined in  claim 37 ,
 wherein the information concerning the estimated intensity of the light reflected by the specific point is provided by an initial measurement of the intensity of the light reflected by the point.   
   
   
       50 . The method as defined in  claim 37 ,
 wherein the regulating device adjusts the intensity of the excitation light beamed to the specific point on the basis of an intensity of the reflected light measured for this point in a previous image.   
   
   
       51 . The method as defined in  claim 37 ,
 wherein the information concerning the estimated intensity of the light reflected by the specific point is provided by a previous measurement of the intensity of the light reflected by an adjacent point.   
   
   
       52 . The method as defined in  claim 37 ,
 wherein for roughly ascertaining the intensity of the light reflected by individual points, a test pattern or test scan is recorded.   
   
   
       53 . The method as defined in  claim 37 ,
 wherein the specimen is pulse-illuminated and   a pulse rate of the excitation light is adapted to a triplet relaxation time of the dyes with which the specimen has been prepared.   
   
   
       54 . The method as defined in  claim 37 ,
 wherein the excitation light includes a plurality of wavelengths for exciting a plurality of different dyes and   the intensity of the light reflected by the specimen is measured in a plurality of different spectral ranges.   
   
   
       55 . The method as defined in  claim 54 ,
 wherein at least one of the intensity or the spectral composition of the excitation light beamed to the specific point of the specimen is automatically adjusted by the regulating device on the basis of information concerning estimated or actual intensities of the light reflected by the specific point in the different spectral ranges, as previously gained from measured data of the specimen, such that integrals of the intensity of the light reflected by the specific point in the different spectral ranges during the pixel dwell time are within value intervals in each case individually specifiable for the different spectral ranges.   
   
   
       56 . The method as defined in  claim 54 ,
 wherein the intensities measured for the different spectral regions are separated into portions pertaining to the respective dyes,   the separation is carried out on the basis of known information concerning the emission spectra of the different dyes while taking into consideration the position and width of the spectral ranges and   for each of the different dyes a weighting factor is determined from in each case at least one intensity portion of the respective dye pertaining to a spectral range.   
   
   
       57 . The method as defined in  claim 55 ,
 wherein the value intervals for the different spectral ranges are fixed such that the weighting factors for each dye lie in a value interval or have a predefined value, which value interval or value is individually specifiable for each of the different dyes.   
   
   
       58 . A microscope comprising
 a light source for emission of excitation light for microscopic investigation of a specimen,   an intensity modulator for varying an intensity of the excitation light, microscope optics for guiding the excitation light to different points of the specimen and for guiding the light reflected by the different points of the specimen to a detector and   the detector for point-specific and quantitative detection of the intensity of the light reflected by the specimen in at least one spectral range,   wherein a regulating device is present which cooperates with the intensity modulator and the detector and automatically adjusts at least one of the intensity or a spectral composition of the excitation light beamed to a specific point of the specimen on the basis of information, previously gained from measured data of the specimen, concerning an estimated or actual intensity of the light reflected in the spectral range by the specific point such that an integral of the intensity of the light reflected in the spectral range, as detected by the detector for the specific point, during a pixel dwell time is within a predefined value interval.   
   
   
       59 . The microscope as defined in  claim 58 ,
 wherein the regulating device adjusts the intensity of the excitation light for the specific point such that the integral of the intensity of the light reflected by the specific point during a pixel dwell time is within a predefined value interval only when a signal criterion for the specific point is satisfied.   
   
   
       60 . The microscope as defined in  claim 58 ,
 wherein the light source emits excitation light having a plurality of wavelengths for excitation of a plurality of different dyes and   a plurality of detectors is present for quantitative and point-specific determination of the light reflected by the specimen in a plurality of spectral ranges.   
   
   
       61 . The microscope as defined in  claim 58 ,
 wherein the regulating device adjusts the intensity of the excitation light beamed to the specific point on the basis of an intensity of the reflected light as determined for the specific point in a previous image.   
   
   
       62 . The microscope as defined in  claim 58 ,
 wherein the microscope is a scanning microscope.   
   
   
       63 . The microscope as defined in  claim 58 ,
 wherein the microscope is a line-scanning microscope.   
   
   
       64 . The microscope as defined in  claim 58 ,
 wherein the microscope is a wide field microscope.   
   
   
       65 . The microscope as defined in  claim 58 ,
 wherein the microscope is a fluorescence microscope.   
   
   
       66 . The microscope as defined in  claim 58 ,
 wherein the intensity modulator has at least one of an AOTF, an AOM, a Pockels cell, a Faraday cell, and a Kerr cell.   
   
   
       67 . The microscope as defined in  claim 58 ,
 wherein the intensity modulator has a spatial light modulator.   
   
   
       68 . The microscope as defined in  claim 67 ,
 wherein the spatial light modulator is disposed away from a detection beam path.   
   
   
       69 . The microscope as defined in  claim 58 ,
 wherein the regulating device is formed by a real time computer.   
   
   
       70 . The microscope as defined in  claim 58 ,
 wherein the regulating device is formed by an analog control circuit.   
   
   
       71 . The microscope as defined in  claim 58 ,
 wherein the light source for exciting a plurality of different dyes emits excitation light having a plurality of wavelengths.   
   
   
       72 . The microscope as defined in  claim 58 ,
 wherein a plurality of detectors is present for quantitative and point-specific determination of the light reflected by the specimen in a plurality of spectral ranges.   
   
   
       73 . The method as defined in  claim 37 ,
 wherein a microscope as defined in  claim 58  is used.   
   
   
       74 . The method as defined in  claim 37 ,
 wherein the excitation light is focused on different points of a specimen.   
   
   
       75 . The method as defined in  claim 51 ,
 wherein the previous measurement is carried out in one and the same scan process.   
   
   
       76 . The microscope as defined in  claim 58 ,
 which is designed for the execution of the method as defined in  claim 37 .   
   
   
       77 . The microscope as defined in  claim 65 ,
 which is designed as a total internal reflection fluorescence microscope.