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Biol Bull. 2016 Aug;231(1):26-39.

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM).

Author information

1
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892-7710; abhishk@gmail.com.
2
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892-7710;
3
Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065; and.
4
Program in Cellular Neuroscience, Neurodegeneration, and Repair, Department of Cell Biology, Yale University, New Haven, Connecticut 06511.

Abstract

Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, four-dimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning- methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online.

PMID:
27638693
PMCID:
PMC5481201
DOI:
10.1086/689589
[Indexed for MEDLINE]
Free PMC Article

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