Light Sheet Microscopy Optimized for Depth Penetration to Study Embryogenesis

Abstract

Fluorescence light sheet microscopy (FLSM) has gained widespread recognition in recent years, due to its distinct advantages for the 3-dimensional (3D) imaging of living biological samples. FLSM uses a planar sheet of light to illuminate a sample, generating fluorescence over an optical section of the sample that is collected by a wide-field microscope camera oriented orthogonal to the light sheet. The orthogonal geometry between the illumination and detection pathways enables massive parallelization in both illumination and detection; futhermore, it permits optical and physical access to samples (3D cell cultures or whole embryos) in ways that are impossible in the collinear geometry of standard microscopes. Because of these features, FLSM significantly outperforms standard laser-scanning confocal microscopy in imaging speed, phototoxicity, and signal to noise in many imaging applications. An important aspect of any 3D imaging technique is its imaging depth limit (how deep into a sample useful information can be collected). In this respect, standard FLSM fares only slightly better than confocal microscopy. To overcome this hurdle, we have optimized FLSM for imaging of live thick samples by minimizing the degradation of the light sheet due to scattering, while preserving acceptable axial resolution. Using this approach we have imaged whole, live fruit fly embryos and zebrafish embryos. We achieve higher depth penetration than standard FLSM, while maintaining sub-cellular resolution, at imaging speed of about ten times faster than standard confocal microscopy.