Two-photon fluorescence excitation within a light sheet based microscopy architecture

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  • 04 April 2012

Author(s): F. Cella Zanacchi; Z. Lavagnino; M. Pesce; F. Difato; E. Ronzitti; A. Diaspro.

Light-sheet microscopy, such as ultramicroscopy, single plane  illumination microscopy (SPIM) [1] and digital scanned laser  microscopy (DSLM) [2], represents a useful tool for biological investigations of thick samples. Such techniques have been found  particularly useful in developmental biology applications since they provide the capability to perform fast imaging of living samples  reducing photobleaching effects. The high signal to noise ratio and the intrinsic optical sectioning capability provided by SPIM suggest  this technique as the best choice for imaging of thick scattering  samples. Nevertheless, imaging in depth of large samples suffers from a decreasing in the image quality due to scattering effects.  Two photon excitation microscopy [3] became a popular tool to  perform imaging in turbid media since it improves the penetration depth capability and it reduces the image quality degradation due  to scattering [4] and light matter interactions. Therefore, two  photon excitation within the light sheet illumination scheme has been exploited in order to reduce scattering effects due to light-sample interactions. In this work two photon excitation imaging in SPIM scheme has been performed in order to achieve an  improvement in the penetration depth while imaging living biological samples.