Two-color excitation fluorescence microscopy through highly scattering media.

We study the performance of two-color excitation (2CE) fluorescence microscopy [Opt. Lett. 24, 1505 (1999)] in turbid media of different densities and anisotropy. Excitation is achieved with two confocal excitation beams of wavelengths lambda(1) and lambda(2), which are separated by an angular displacement theta, where lambda(1) not equal lambda(2), 1/lambda(e) = 1/lambda(1) + 1/lambda(2), and lambda(e) is the single-photon excitation wavelength of the sample. 2CE fluorescence is generated only in regions of the sample where the two excitation beams overlap. The 2CE fluorescence intensity is proportional to the product of the two excitation intensities and could be detected with a large-area photodetector. The requirement of spatiotemporal simultaneity for the two excitation beams makes 2CE fluorescence imaging a promising tool for observing microscopic objects in a highly scattering medium. Optical scattering asymmetrically broadens the excitation point-spread function and toward the side of the focusing lens that leads to the contrast deterioration of the fluorescence image in single- or two-photon (lambda(1) = lambda(2)) excitation. Image degradation is caused by the decrease in the excitation energy density at the geometrical focus and by the increase in background fluorescence from the out-of-focus planes. In a beam configuration with theta not equal 0, 2CE fluorescence imaging is robust against the deleterious effects of scattering on the excitation-beam distribution. Scattering only decreases the available energy density at the geometrical focus and does not increase the background noise. For both isotropic and anisotropic scattering media the performance of 2CE imaging is studied with a Monte Carlo simulation for theta = 0, pi/2, and pi, and at different h/d(s) values where h is the scattering depth and d(s) is the mean-free path of the scattering medium.