Refractive index mismatch induced spherical aberration (RIMISA) is a ubiquitous problem for in-depth optical biological imaging and it is an effect most researchers like to avoid. Associated with RIMISA is a broadening of the point-spread-function (PSF) which leads to a degradation of image resolution and the loss of specimen signal. It is these features that render RIMISA to be useful for one important application in biological application, the determination of sample refractive index. We will show how RIMISA under multiphoton and confocal microscopy can be used to determine the refractive indices of uniformly luminescent specimens and a comparison will be made between the multiphoton and confocal approaches.© (2005) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.