Patent ID: 9697605
Date: 2017-07-04
CPC Classifications: G02B,G06T

Claim:
1. A method for microscopic three-dimensional imaging of a sample by selective plane illumination microscopy in which image information from different depth planes of the sample in a depth direction is acquired by a microscope apparatus 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, the method comprising: positioning at least one reference object, comprising a fluorescing substance and having a reference structure, next to or in the sample, which also comprises a fluorescing substance; illuminating the sample with an excitation beam from a light source, which excitation beam is shaped as a light sheet; a first acquiring step of acquiring, via the microscope apparatus having a microscope objective, reference-object image information from the reference object from a detection direction; a first storing step of storing the reference-object image information acquired from the detection direction in spatial coordinates X, Y, Z; a second acquiring step of acquiring, via the microscope apparatus having the microscope objective, depth-plane sample image information from different depth planes of the sample from the detection direction; a second storing step of storing the depth-plane sample image information acquired from the detection direction in spatial coordinates X, Y, Z; changing the detection direction, relative to the sample and relative to the reference object, to a different detection direction; repeating the first acquiring and storing steps and the second acquiring and storing steps for the different detection direction; obtaining mathematical transformation operators for translation, rotation and deformation based on the spatial coordinates X, Y, Z of the stored reference-object image information so that image information from the same source from the different detection directions can be overlapped; and reconstructing a three-dimensional image of the sample from the spatial coordinates X, Y, Z of the stored depth-plane sample image information based on these transformation operators; wherein the first acquiring step is performed before, after, or simultaneously with the second acquiring step.