Method of Aligning a Laser-Scanning Fluorescence Microscope and Laser-Scanning Fluorescence Microscope Having an Automatic Aligning System

A method of setting a laser-scanning fluorescence microscope comprising an objective lens, a fluorescence light detector and a pinhole aperture arranged in front of the fluorescence light detector to a correct alignment in which a center of an image of the pinhole aperture and an intensity maximum of excitation light coincide in a focal area of the objective lens, the method comprising:

• scanning a structure in a sample, the structure being marked with a fluorescent dye, with the intensity maximum of the excitation light to generate a first picture and a second picture of the sample,
• the first picture being generated at a smaller aperture opening of the pinhole aperture and comprising a first image of the structure, and
• the second picture being generated at a larger aperture opening of the pinhole aperture and comprising a second image of the structure;
• calculating an offset of a first position of the first image of the structure in the first picture with regard to a second position of the second image of the structure in the second picture;
• and
• shifting the intensity maximum of the excitation light with regard to the image of the pinhole aperture in the direction of the offset calculated to set the laser-scanning fluorescence microscope to the correct alignment.

A method of setting a laser-scanning fluorescence microscope comprising an objective lens, a fluorescence light detector and a pinhole aperture arranged in front of the fluorescence light detector to a correct alignment in which a center of an image of the pinhole aperture coincides with an intensity maximum of excitation light and an intensity minimum of fluorescence inhibition light in a focal area of an objective lens, the method comprising:

• scanning a structure in a sample, the structure being marked with a fluorescent dye, with the intensity maximum of the excitation light to generate a first picture and a second picture of the sample,
• the first picture corresponding to a higher intensity of the fluorescence inhibition light and comprising a first image of the structure, and
• the second picture corresponding to a lower intensity of the fluorescence inhibition light and comprising a second image of the structure;
• calculating an offset of a first position of the first image of the structure in the first picture with regard to a second position of the second image of the structure in the second picture;
• and
• shifting the image of the pinhole aperture with regard to the intensity maximum of the excitation light and the intensity minimum of the fluorescence inhibition light in the direction of the offset calculated to set the laser-scanning fluorescence microscope to the correct alignment.

A laser-scanning fluorescence microscope comprising

• an excitation light source of excitation light;
• a fluorescence inhibition light source of fluorescence inhibition light;
• a fluorescence light detector for fluorescence light;
• a pinhole aperture arranged in front of the fluorescence light detector;
• an objective lens focusing the excitation light and the fluorescence inhibition light into a focal area in a sample space and imaging the pinhole aperture into the focal area;
• and
• an automatic aligning system configured for setting the laser-scanning fluorescence microscope to a correct alignment in which an intensity maximum of the excitation light, an intensity minimum of the fluorescence inhibition light and the image of the pinhole aperture coincide in the focal area of the objective,
• by scanning a structure in a sample, the structure being marked with a fluorescent dye, with the intensity maximum of the excitation light to generate at least two pictures of the sample, the at least two pictures corresponding to at least one of
• different intensities of the fluorescence inhibition light,
• different aperture openings of the pinhole aperture arranged in front of a fluorescence light detector of the laser-scanning fluorescence microscope,
• different intensity maxima of several components of the excitation light,
• different intensity minima of several components of the fluorescence inhibition light, and
• different images of several pinhole apertures arranged in front of several fluorescence light detectors,
• and each of the at least two pictures comprising an image of the structure;
• by calculating at least one offset between at least two positions of the images of the structure in the at least two pictures;
• and
• by shifting, in the focal area of the objective lens, at least one of the intensity maximum of the excitation light, the intensity minimum of the fluorescence inhibition light and the image of the pinhole aperture image in the direction of the offset calculated.