Light-sheet fluorescence microscopy enables high-speed, high-resolution, gentle imaging of live biological specimens (spanning length scales from single cells to large organs) over extended periods (from hours to days). Here we report a reflective light sheet imaging technique that further improves the spatiotemporal resolution and collection efficiency of our dual-view light sheet microscope (diSPIM). By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to previous diSPIM. We further enhance spatial resolution to less than 300 nm in all three dimensions by combining a high numerical aperture (NA) lens with the use of reflective coverslips. We also present a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. We demonstrate the broad applicability of the methods in a variety of samples, investigating the dynamics of mitochondria, membranes, Golgi, and microtubules in single cells, and neurodevelopment in nematode embryos. Finally, we describe computational methods for untwisting worm embryos and analyzing calcium activity in freely moving embryos. Our imaging and data processing tools pave the way for establishing a 4D dynamic neurodevelopmental atlas in nematode embryos.
Yicong Wu, Evan Ardiel, Ryan Christensen, and Hari Shroff, "Pushing the envelope in biological imaging with dual-view light sheet fluorescence microscopy (Conference Presentation)," Proc. SPIE 10505, High-Speed Biomedical Imaging and Spectroscopy III: Toward Big Data Instrumentation and Management, 105050D (Presented at SPIE BiOS: January 29, 2018; Published: 15 March 2018); https://doi.org/10.1117/12.2293731.5752191696001.
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