Abstract
Imaging depth in turbid media by two-photon fluorescence microscopy depends on the ability of the optical system to detect weak fluorescence signals. We have shown that use of a wide area detector in transmission geometry allows increasing imaging depth in turbid media due to efficient photon collection. Compared to the conventional epi-detection scheme used in most commercial microscopes, the transmission detector was found to be 2-3 orders of magnitude more sensitive when used for in depth imaging in scattering samples simulating brain optical properties.
Highlights
Since its introduction in 1990 [1], two-photon fluorescence microscopy, has been widely applied to image biological tissues
Additional attenuation may be due to absorption, for most biological tissues and the depths studied, attenuation of excitation light by media absorption is negligible and scattering is the dominant process
In this paper we present a quantitative comparison of the two-photon excitation imaging of tissue phantoms and a mouse brain tissue sample using DIVER and epi-detection schemes at the same time and show that, depending on the imaging depth and turbidity of the sample, the DIVER detector could be up to 3 orders of magnitude more sensitive than conventional epi-detection
Summary
Since its introduction in 1990 [1], two-photon fluorescence microscopy, has been widely applied to image biological tissues. Objectives with a high numerical aperture allow for focusing light in a smaller spot, which effectively increases excitation intensity and, as a result, induced fluorescence For such objectives peripheral photons travel longer distances to the focal area and there is more scattering than for central photons so that the effective NA is decreased. In the DIVER (Deep Imaging Via Emission Recovery) detection scheme, Fig. 1(B), the excitation and detection optics are separated, that allows more efficient collection of fluorescence photons. The DIVER detection scheme collects the scattered fluorescence photons reaching the surface of the sample with minimum losses It is based on two concepts: 1) the use of a wide area detector; 2) maintaining the uniformity of the refractive index in the optical path from the sample surface to the detector.
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