Abstract
Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the micrometer scale) to small organisms (in the millimeter scale). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that make them ideal for a large range of applications. These include the study of cellular dynamics, in particular cellular motion which is essential to processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that provide better control over the substrate environment than traditional cell culture experiments. For example, to mimic in vivo conditions, experiment biochemical dynamics, and trap, move or count cells. Microdevices provide a higher degree of empirical complexity but, so far, most have been designed to be imaged through wide-field or confocal microscopes. Nonetheless, the properties of LSFM render it ideal for 3D characterization of active cells. When working with microdevices, confocal microscopy is more widespread than LSFM even though it suffers from higher phototoxicity and slower acquisition speeds. It is sometimes possible to illuminate with a light-sheet microdevices designed for confocal microscopes. However, these bioMEMS must be redesigned to exploit the full potential of LSFM and image more frequently on a wider scale phenomena such as motion, traction, differentiation, and diffusion of molecules. The use of microdevices for LSFM has extended beyond cell tracking studies into experiments regarding cytometry, spheroid cultures and lab-on-a-chip automation. Due to light-sheet microscopy being in its early stages, a setup of these characteristics demands some degree of optical expertise; and designing three-dimensional microdevices requires facilities, ingenuity, and experience in microfabrication. In this paper, we explore different approaches where light-sheet microscopy can achieve single-cell and subcellular resolution within microdevices, and provide a few pointers on how these experiments may be improved.
Highlights
Research in the area of cell motility (Ridley, 2003; Mayor and Etienne-Manneville, 2016) is essential to understand many core life processes like development and neurology, with especially strong biomedical significance in the fields of immunology, tissue repair, and tumor metastasis
Light-sheet microscopy is based on generating a sheet of light within the specimen and ensuring it coincides with the focal plane of a high numerical aperture objective placed at 90◦
Note that even though planes of light can be created directly from Gaussian beams using cylindrical lenses, beams of other shapes can be digitally scanned to form virtual light-sheets (Keller et al, 2008; Chen et al, 2014) by a technique known as digitally-scanned lightsheet microscopy (DSLM)
Summary
Research in the area of cell motility (Ridley, 2003; Mayor and Etienne-Manneville, 2016) is essential to understand many core life processes like development and neurology, with especially strong biomedical significance in the fields of immunology, tissue repair, and tumor metastasis. The authors suggest the use of electrically tunable lenses (ETLs) (see Fahrbach et al, 2013, for example) as an alternative scanning method to mechanical translation to achieve the required imaging speeds Both diagonally-scanned SPIM and an earlier publication by this group on axially swept light-sheet microscopy (Dean et al, 2015) show LSFM as a tool capable of imaging 3D single cells in their ECM matrix. Some earlier-described LSFM setups for standard sample mounting, such as inverted SPIM configurations, could be considered as a possible solution to high-scattering side interfaces Another microfluidic device of higher complexity has been designed for manipulating C. elegans specimens and delivering stimuli to monitor neural activity under widefield and brightfield illumination (Chronis et al, 2007).
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