Changes in the PSF under refractive index mismatch

The Point Spread Function (PSF) of a Fluorescence Microscope reflects the aberrations arising from having a Refractive Index (r.i.) mismatch between the Lens Immersion Medium and the Specimen Embedding Medium: the PSF becomes more strongly aberrated as you move deeper within the sample.

The Huygens Software corrects these aberrations by generating depth-dependent theoretical PSFs during deconvolution.

Below are two PSFs of a 2-photon microscope using an oil lens (r.i. = 1.51) and a water medium (r.i. = 1.34), at depth zero (on the sample border) and at depth 20 μm inside the sample.


PSF at z = 0.

PSF at z = 20 μm.

For a thorough study on refractive index mismatch problems see Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. Hell, S., G. Reiner, C. Cremer, E.H.K. Stelzer.
J. Micr., Vol 169, pp. 391-405 (1993)