Light Sheet Fluorescence Microscopy (LSFM) for Two-Photon Excitation Imaging of Thick Samples.

Over the last decades, fluorescence microscopy techniques have been developed in order to provide a deeper, faster and higher resolution imaging of three-dimensional biological samples. Within this framework, Light Sheet Fluorescence Microscopy (LSFM) became an increasingly useful and popular imaging technique able to answer several biological questions in the field of developmental biology [1]. Thanks to the spatial confinement of the excitation process within a thin sheet in the focal plane, it provides an intrinsic optical sectioning and a reduced phototoxicity. On the other side, Two-Photon Excitation (2PE), thanks to the use of IR wavelengths, has become an invaluable tool to improve imaging capabilities in terms of imaging depth and spatial resolution [2,3]. In this work we tested and compared the advantages provided by Two Photon Excitation in combination with two different light sheet based architectures: Selective Plane Illumination Microscopy (SPIM) and Inverted Selective Plane Illumination Microscopy (iSPIM) [4]. The two different optical approaches are characterized in terms of illumination intensity distributions and in terms of point spread function measurements, both in the linear and non linear regime. Additionally, particular attention has been addressed to the relationship between the sample holder and the specific sample geometry, showing the suitability of the inverted configuration when the sample geometry does not allow embedding in agarose gel (for example brain slices and retina). Furthermore, TPE-SPIM has been tested towards live imaging of nervous system in small animals, such as Danio Rerio