Journal
NATURE BIOTECHNOLOGY
Volume 31, Issue 11, Pages 1032-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2713
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Funding
- National Institutes of Health (NIH) [GM097576, HD075602]
- US National Institutes of Health (NIH) [R01 NS076558, U01HD075602]
- Intramural Research Programs of the NIH National Institute of Biomedical Imaging and Bioengineering
- National Institute of Heart, Lung, and Blood
- Center for Information Technology
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Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.
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