Satoshi Kawata

Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM(00) mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-mum-diameter polystyrene spheres.

Excitation with a focused, pulsed optical beam in scattering media: diffraction effects.

To gain a better understanding of the spatiotemporal problems that are encountered in two-photon excitation fluorescence imaging through highly scattering media, we investigate how diffraction affects the three-dimensional intensity distribution of a focused, pulsed optical beam propagating inside a scattering medium. In practice, the full potential of the two-photon excitation fluorescence imaging is unrealized at long scattering depths, owing to the unwanted temporal and spatial broadening of the femtosecond excitation light pulse that reduces the energy density at the geometric focus while it increases the excitation energy density in the out-of-focus regions. To analyze the excitation intensity distribution, we modify the Monte Carlo-based photon-transport model to a semi-quantum-mechanical representation that combines the wave properties of light with the particle behavior of the propagating photons. In our model the propagating photon is represented by a plane wave with its propagation direction in the scattering medium determined by the Monte Carlo technique. The intensity distribution in the focal region is given by the square of the linear superposition of the various plane waves that arrive at different incident angles and optical path lengths. In the absence of scattering, the propagation model yields the intensity distribution that is predicted by the Huygens-Fresnel principle. We quantify the decrease of the energy density delivered at the geometric focus as a function of the optical depth to the mean-free-path ratio that yields the average number of scattering events that a photon encounters as it propagates toward the focus. Both isotropic and anisotropic scattering media are considered. Three values for the numerical aperture (NA) of the focusing lens are considered: NA = 0.25, 0.5, 0.75.

Reply to comment on “Excitation with a focused, pulsed optical beam in scattering media: diffraction effects”

We address the issues that were raised by Tycho and Jørgensen [Appl. Opt. 41, 4709 (2002)] concerning our strategy [Appl. Opt. 39, 5244 (2000)] for incorporating the wave properties of light in the description of a propagating focused excitation beam in a highly scattering medium. We explain that the strategy is consistent with the Huygens-Fresnel principle and does not violate the energy conservation principle.