Laser scanning microscope and its operating method

Abstract

Laser scanning microscope and its operating method in which at least two first and second light distributions activated independently of each other and that can move in at least one direction illuminate a sample with the help of a beam-combining element, and the light is detected by the sample as it comes in, characterized by the fact that the scanning fields created by the light distributions on the sample are made to overlap mutually such that a reference pattern is created on the sample with one of the light distributions, which is then captured and used to create the overlap with the help of the second light distribution (correction values are determined) and/or a reference pattern arranged in the sample plane or in an intermediate image plane is captured by both scanning fields and used to create the overlap (correction values are determined) and/or structural characteristics of the sample are captured by the two scanning fields as reference pattern and used to create the overlap in which correction values are determined.

1 . Method of operating a laser scanning microscope, in which at least two independently controlled first and second light distributions that can move in at least one direction through a beam-combining element illuminate a sample and the light is detected by the sample as it comes in, the method comprising the steps of:
 causing the two scanning fields created by the light distribution on the sample to overlap, such that   a reference pattern is created on the sample with one light distribution, which is then captured by means of the second light distribution and used to create the overlap and/or   a reference pattern arranged in the plane of the sample or an intermediate image plane is captured by both scanning fields and used to create an overlap and/or   structural characteristics of the sample are captured by both scanning fields as a reference pattern and used to create the overlap, so that correction values are determined.   
   
   
       2 . Method of operating a laser scanning microscope according to  claim 1 , in which the first light distribution is moved over the sample to capture a sample image, and the second light distribution is used to manipulate the sample. 
   
   
       3 . Method of operating a laser scanning microscope according to  claim 1 , in which the reference pattern is a point distribution. 
   
   
       4 . Method of operating a laser scanning microscope according to  claim 1 , further comprising the steps of creating reference points by a sample manipulation system and capturing the reference points with an imaging system. 
   
   
       5 . Method of operating a laser scanning microscope according to  claim 1 , in which the reference pattern consists of at least three points. 
   
   
       6 . Method of operating a laser scanning microscope according to  claim 1 , further comprising the steps of creating a light point on the sample. 
   
   
       7 . Method of operating a laser scanning microscope according to  claim 5 , further comprising the steps of employing light reflected by the sample points. 
   
   
       8 . Method of operating a laser scanning microscope according to  claim 1 , further comprising the steps of capturing frequency-converted light, wherein the frequency-converted light is created through a non-linear or linear interaction of the illuminating light with the sample. 
   
   
       9 . Method of operating a laser scanning microscope according to  claim 8 , in which at least one luminescence point is created on the sample. 
   
   
       10 . Method of operating a laser scanning microscope according to  claim 8 , with creation through inelastic Light scattering. 
   
   
       11 . Method of operating a laser scanning microscope according to  claim 1 , further comprising the steps of creating reference patterns from points with light-inducing sample modification. 
   
   
       12 . Method of operating a laser scanning microscope according to  claim 1 , in which grids are used as reference patterns. 
   
   
       13 . Method of operating a laser scanning microscope according to  claim 1 , in which a statistical structure distribution of the sample itself serves as the reference pattern. 
   
   
       14 . Method of operating a laser scanning microscope according to  claim 1 , in which a coordinate transformation is used to determine correction values. 
   
   
       15 . Method of operating a laser scanning microscope according to  claim 13 , with an affine transformation having at least three reference points. 
   
   
       16 . Method of operating a laser scanning microscope according to  claim 4 , in which a point-scanning or line-scanning system, or a scanning point distribution system or a Nipkow system is used as the imaging system. 
   
   
       17 . Method of operating a laser scanning microscope according to  claim 4 , in which the manipulating system is a point scanning device, and the scanning takes place preferably in two directions. 
   
   
       18 . (canceled) 
   
   
       19 . Laser scanning microscope according to  claim 27 , having an imaging system and a manipulating system. 
   
   
       20 . Laser scanning microscope according to  claim 19 , in which the imaging system is a point-scanning system, a line-scanning system, a scanning point distribution system or a Nipkow system. 
   
   
       21 . Laser scanning microscope according to  claim 19 , in which the manipulating system is mapped and the scanning takes place preferably in two directions. 
   
   
       22 . Laser scanning microscope according to  claim 27 , with at least one laser as the light source. 
   
   
       23 . Laser scanning microscope according to  claim 27 , in which a movement takes place over the sample in at least one scanning direction. 
   
   
       24 . (canceled) 
   
   
       25 . Laser scanning microscope according to  claim 27 , in which at least one of the beam deflecting devices is provided with Galvo scanners. 
   
   
       26 . Laser scanning microscope according to  claim 23 , in which one coordinate transformation takes place through the modification of gain and offset values of the associated triggering unit. 
   
   
       27 . A laser scanning microscope comprising:
 first and second light distributions;   a beam-combining element for combining the first and second light distributions that are controlled independently of each other and that can move in at least one direction;   scanning means interposed between the beam combining element and the sample for causing first and second scanning fields to cause the combined light distributions to illuminate the sample;   at least one detector to detect the light coming from the sample; and   overlapping means for superimposing the first and second scanning fields on the sample through the use of at least one reference pattern by overlapping the first and second scanning fields.   
   
   
       28 . Laser scanning microscope of  claim 27 , wherein the overlapping means comprises:
 means for creating a first reference pattern on the sample with one light distribution;   means for capturing the reference pattern with the second light distribution; and   means for determining and adjusting correction values and for creating the overlap of the first and second light distributions.   
   
   
       29 . Laser scanning microscope of  claim 27 , wherein the overlapping means comprises:
 means for capturing a reference pattern arranged in the sample plane or in an intermediate plane of the two scanning fields; and   means for determining and adjusting correction values and for creating the overlap of the first and second scanning fields.   
   
   
       30 . Laser scanning microscope of  claim 29 , wherein the overlapping means comprises:
 means for capturing the structural characteristics of the means for determining and adjusting correction values, and   means for creating the overlap with the first and second scanning fields.