Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)

Abstract

Multidirectional selective plane illumination microscopy (mSPIM) reduces absorption and scattering artifacts and provides an evenly illuminated focal plane. mSPIM solves two common problems in light-sheet-based imaging techniques: The shadowing in the excitation path due to absorption in the specimen is eliminated by pivoting the light sheet; the spread of the light sheet by scattering in the sample is compensated by illuminating the sample consecutively from opposing directions. The resulting two images are computationally fused yielding a superior image. The effective light sheet is thinner, and the axial resolution is increased by square root 2 over single-directional SPIM. The multidirectional illumination proves essential in biological specimens such as millimeter-sized embryos. The performance of mSPIM is demonstrated by the imaging of live zebrafish embryos.