Method for the microscopic three-dimensional reproduction of a sample

A method for microscopic three-dimensional imaging of a sample in which image information from different depth planes of the sample is acquired and is stored in a spatially resolved manner so as to be correlated to its spatial coordinates X, Y, Z, and in which the three-dimensional image of the sample is reconstructed electronically from the stored image information, comprising the steps of:

• obtaining a three-dimensional image of a sample comprising a fluorescing substance or of a sample to which a fluorescing substance is added by selective plane illumination microscopy;
• illuminating the sample by an excitation beam which is shaped as a light sheet;
• obtaining an image information of the sample based on the emission radiation coming from the sample;
• positioning at least one reference object comprising a fluorescing substance or a reference object having a fluorescing substance next to or in the sample;
• acquiring the detection direction from which the image information;
• changing the detection direction relative to the sample and relative to the reference object;
• acquiring image information from the reference object from each detection direction given by a change;
• storing the image information from the reference object from each detection direction given by a change in a spatially resolved manner in spatial coordinates X, Y, Z;
• acquiring image information from different depth planes of the sample from each detection direction given by a change;
• storing the image information from different depth planes in a spatially resolved manner in spatial coordinates X, Y, Z;
• obtaining transformation operators for translation, rotation and deformation based on the spatial coordinates X, Y, Z of the stored image information from the reference object so that image information from the same source from the different detection directions can be overlapped;
• reconstructing the three-dimensional image of the sample from the spatial coordinates X, Y, Z of the image information obtained from the sample based on these transformation operators.