Carmichael’s Concise Review
developed algorithms that decreased the computational time from months to about two days. They also designed customized objectives that provide long working distances and high numerical apertures. They provide specifics on the design of these objectives as well as detailed blueprints for the IsoView microscope.
Using this elegant instrument, Chhetri et al. demon - strated whole-animal functional imaging of fruit fly ( Drosophila ) embryos at a spatial resolution of about 2 μ m and a temporal resolution of 2 Hz for several hours. They also obtained spatially isotropic whole-brain functional imaging in zebrafish ( Danio rerio ) larvae and spatially isotropic multicolor imaging of rapid cellular dynamics across fruit fly embryos.
Compared to conventional light-sheet microscopy, IsoView microscopy improves three-dimensional spatial resolution and decreases resolution anisotropy. For Drosophila embryos, scattering and aberrations are relatively strong, but resolution varies with depth. T e authors performed a systematic depth-dependent analysis of the resolution improvement achieved by IsoView in this scenario (in Supplementary Figure 7 in [1]). Conventional resolution is approximately 1.8 μ m laterally and 5.5 μ m axially at a depth of 20 to 30 μ m, whereas IsoView provides 1.1 to 1.8 μ m laterally and axially. At maximum imaging depth, conven- tional resolution is approximately 3.0 μ m laterally and 9.0 μ m
axially, where IsoView provides 1.3 to 2.5 μ m laterally and axially.
Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides sub-second temporal resolution for specimens 400-fold larger than could previously be imaged. The functional imaging capabilities of this elegant microscope allows for the first time the ability to capture neuronal activity simultaneously throughout the entire nervous system of an intact living organism with more than 10,000 neurons. By contrast, the largest organisms that could previously be fully covered were C. elegans , which have a relatively simple nervous system with only a few hundred neurons. This is a remarkable achievement that provides enormous power to observe dynamic cellular events in whole organisms!
References [1] RK Chhetri et al., Nat Methods , doi:101038/NMETH.3632 , 2015.
[2] T e author gratefully acknowledges Drs. Philipp Keller and Raghav Chhetri for reviewing this article, as well as Dr. Charles Lyman for helpful editing.
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