Application Notes

The Effects of Refractive Index Mismatch Between Immersion Liquid and Tissue Clearing Reagent on Image Resolution in Deep Areas

April 2022

Purkinje cells, which have a characteristic shape, are difficult to capture with a confocal system using visible light excitation, from the surface to the deep regions of the cerebellum, even if they are subjected to tissue clearing treatment, so observation with a multiphoton confocal system by IR light excitation is more suitable. Nevertheless, especially during detailed observations in deep areas, the difference between the refractive index of the immersion liquid of the objective lens and that of the tissue affects brightness and resolution. This article focuses on the effect this difference in refractive index has at different observation depths. In addition, we captured the state of the PSF along the optical axis for each depth using fluorescent beads, conducted nonlinear deconvolution processing using this result as a parameter, and verified its beneficial effect on image elongation along the optical axis.