The behavior of two-photon fluorescence imaging through a scattering medium is analyzed by use of the Monte Carlo technique. The axial and transverse distributions of the excitation photons in the focused Gaussian beam are derived for both isotropic and anisotropic scatterers at different numerical apertures and at various ratios of the scattering depth with the mean free path. The two-photon fluorescence profiles of the sample are determined from the square of the normalized excitation intensity distributions. For the same lens aperture and scattering medium, two-photon fluorescence imaging offers a sharper and less aberrated axial response than that of single-photon confocal fluorescence imaging. The contrast in the corresponding transverse fluorescence profile is also significantly higher. Also presented are results comparing the effects of isotropic and anisotropic scattering media in confocal reflection imaging. The convergence properties of the Monte Carlo simulation are also discussed.

We report subdiffraction resolution in far-field fluorescence microscopy through laser-diode-stimulated emission depletion of molecular markers. The diode-generated focal intensities lead to a resolution improvement by ∼45% in both lateral directions. Excitation and stimulated emission are performed with electronically synchronized diode pulses of 50–70 ps and 300–400 ps duration, respectively. The subdiffraction resolution is utilized to resolve neighboring individual molecules.

We demonstrate theoretically, experimentally, and in an imaging application the possibility to generate a single predominant sharp diffraction maximum in the effective point-spread function of a fluorescence microscope that coherently uses two opposing lenses. This is achieved through binary pupil filters that preclude the origination of the unfavorable strong interference side maxima that are otherwise present in these systems. Mathematical postprocessing, which has so far been a prerequisite to gain artifact-free images, is now optional or obsolete.

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We explore the current limits of the axial resolution of optical sectioning microscopy using a single lens, by combining the resolving power of novel 1.45 numerical aperture oil immersion lenses with superresolving binary aperture filters. We quantify the axial resolution brought about by the increase in semiaperture angle to max á =72.8 degrees and demonstrate an absolute gain in axial resolution through binary pupil filters. Implemented in a confocalized two-photon excitation microscope, the combination of both effectively improves the axial resolution to 330 nm full-width-half-maximum, which is by 34% over that of a standard 1.4 NA system.