3D Imaging by Multiphoton Selective Plane Illumination

Abstract

Light-sheet based techniques, such as single plane illumination microscopy (SPIM) [1] and digital scanned laser microscopy (DSLM) [2], have been found particularly useful in developmental biology applications since they provide the capability to perform fast imaging of living samples reducing photobleaching effects. In particular, single plane illumination provides high signal to noise ratio and optical sectioning capability due to the planar excitation, representing a useful tool for biological investigations of thick biological samples.On the other side, two photon excitation microscopy (2PE) has been demonstrated to be a suitable tool for improving penetration depth since it allows for reduction of the scattering effects and light-sample interactions. To improve image quality and penetration depth of light sheet illumination microscopy, two photon excitation can be coupled with planar illumination [3] thus reducing the scattering effects due to light-sample interactions.We characterized the two photon excitation process within the light sheet and we applied this technique to image large biological samples [4]. 3D imaging of mammary cell spheroids in depth has been performed using the resulting hybrid architecture.Keywords: Two photon excitation, Single plane illumination microscopy.(1) Huisken, J., et al. Science 305, 1007-1009 (2004).(2) Keller, P. J., Schmidt, A. D., Wittbrodt, J., and Stelzer, E.H.K. Science, 322 (2008).(3) Truong et al. Nature Methods 8,757-760, (2011)(4) Cella Zanacchi et al. Proc. of SPIE, vol.7903 “Multiphoton Microscopy in the Biomedical Sciences XI” (2011)