Wu, Yicong, Fischer, Robert, Colon-Ramos, Daniel, Bao, Zhirong, Santella, Anthony, McAuliffe, Matthew, Winter, Peter, Senseney, Justin, Wawrzusin, Peter, Christensen, Ryan, York, Andrew, Waterman, Clare, Shroff, Hari
[
International Worm Meeting,
2013]
Optimal 3D time-lapse imaging requires microscopy that provides high resolution in all spatial dimensions, high speed, and minimal photobleaching and photodamage. By creating algorithms that register and appropriately combine two perpendicular specimen views, we developed a dual-view inverted selective plane illumination microscope (diSPIM) with 330 nm isotropic resolution and 200 Hz acquisition, enabling rapid 3D imaging at 2 volumes/second. Unlike spinning-disk confocal or Bessel beam illumination methods that dose the sample outside the focal plane, leading to significant photobleaching, we maintain this spatiotemporal resolution over hundreds of volumes (hundreds of thousands of images) with negligible photobleaching. DiSPIM enables the study of biological systems that are intractable using other optical microscopes, including 4D microtubule tracking in live cells, improved nuclear imaging over 14 hours of nematode embryogenesis, and visualization of neural wiring during C. elegans brain development.