RESUMO
Pump--probe fluorescence microscopy has been demonstrated to be a powerful tool for obtaining three-dimensional, time-resolved information in bioimaging applications. However, the use of this technique can be complicated by the fact that the different wavelengths used to achieve pump--probe microscopy can result in wavelength-dependent spherical aberration, thus limiting the usefulness of the technique. We address this issue by investigating the effects of refractive-index-mismatch-induced spherical aberration on pump--probe image formation. We model the effects by considering pump--probe imaging performed with an objective with a numerical aperture of 0.75 focusing through an oil-water interface. Our results show that spherical aberration has the greatest effect in degrading an axial point-spread function. In addition to signal loss, the redistribution of signal strength along the axial direction results in broadening of the FWHM of the plane response function. The inclusion of confocal detection tends to improve image resolution but at a significant loss of signal strength.
