Fig. 2.

Imaging procedure for 3D site-selective tissue cytometry. (A) Alignment: Using a centering slide containing a cross-hair target, the field of view of wide-field fluorescence microscopy (WFM) and two-photon microscopy (TPM) are aligned. (B) Sample mounting: Each pancreas sample is compressed between two glass coverslips to a thickness of 0.5 mm. (C) Wide field microscopy: A series of wide-field fluorescence microscopy images are taken and are montaged to map the foci in the tissue. (D) Recording sites of interest: Positions of the foci are recorded. (E) Two-photon microscopy of sites of interest: A series of two-photon microscopy image stacks are taken at each focus location by using an automated x-y stage. (F) 3D reconstructed image cubes of a representative focus containing seven cells are shown for the blue channel (Left), the yellow channel (Center), and an overlay of blue and yellow channels (Right). The edge length of this cube is 50 μm.