Abstract
In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids.
Highlights
Live imaging is a key tool in understanding the organization and function of cells, tissues and organisms, since it allows the visualization of dynamic processes within their native environment
We found that the resolution benefit of refractive index tuning increases with the distance of the object plane to the coverslip (Figure 4a, Supplementary file 1), as expected from the increasing impact of spherical aberrations with increasing distance to the objective
Our results establish Iodixanol supplementation as a simple, versatile and effective method for refractive index tuning in live imaging applications
Summary
Live imaging is a key tool in understanding the organization and function of cells, tissues and organisms, since it allows the visualization of dynamic processes within their native environment. Refractive index mismatches between tissue and surrounding medium result in spherical aberrations that misalign the optical paths and distort and attenuate the microscopic image. This effect increases with complexity and thickness of the specimen, making imaging in deep tissue layers difficult and technically demanding (Richardson and Lichtman, 2015). Recently developed optical clearing techniques can render tissues effectively transparent by equilibrating refractive index heterogeneity within biological samples (Chung et al, 2013; Hama et al, 2015) These protocols remain limited to fixed specimens due to their reliance on harsh mounting conditions and/or toxic chemicals (Richardson and Lichtman, 2015)
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