Refractive index mismatch induced intensity and phase variations in fluorescence confocal, multiphoton and 4Pi-microscopy

We examine the focal shift and the phase when focusing into a refractive index mismatched sample. We show that, while the phase at the location of the intensity maximum is not a linear function of the focusing depth, it can be linearly approximated in intervals that depend on the aperture, on the refractive index and the wavelength. The first axial interval is the maximum object thickness up to which confocal or multiphoton axial images only differ by a wavelength-independent factor from the true axial dimension. The first axial interval also defines the maximum thickness up to which 4Pi-microscopy can be employed. The imaging properties of 4Pi-microscopy of samples that are mounted in mismatched media are investigated. We show how to compensate mismatch induced phase shifts to obtain superresolved 4Pi-confocal images of mismatched objects. (C) 1998 Elsevier Science B.V. All rights reserved.