From Cell Mechanics to Light Sheet Microscopy: Imaging Forces, Molecules, Cells and Embryos at High Spatiotemporal Resolution
Research Scientist, Betzig Lab
HHMI Research Campus
February 23, 2017
12:30 p.m. - 1:20 p.m.
Foege N130A, Wallace H. Coulter Seminar Room
By definition, living specimens are animate. Therefore, a full understanding of dynamic biological systems will only be obtained by observing them with enough 4D spatio-temporal resolution and for a sufficient duration, to capture the phenomena of interest. I will first present efforts toward understanding how mechanical forces drive cell protrusion, tissue formation and matrix remodeling within 3D environments. By utilizing synthetic hydrogels and finite element models, I will describe a technique to measure the traction forces that drive cell protrusion in 3D. When acting collectively, these protrusion events also drive matrix remodeling and tissue formation. To measure this process, I will describe a microfabricated platform that permits high throughput simultaneous measurement of tissue mechanics, geometry and protein conformation. However, the remodeling events that drive cell migration and tissue formation span multiple length and time scales ranging from a few microns and seconds for individual cell protrusion events to several millimeters and days for collective tissue condensation. Unfortunately, conventional widefield or confocal microscopes are either too slow, lack the spatial resolution, or induce too much photodamage to capture these phenomena in detail.
In the second portion of the presentation, I will describe Lattice Light Sheet Microscopy, a new sub-cellular light-sheet microscopy method capable of imaging fast 3D dynamic processes in vivo at signal to noise levels approaching those obtained by total internal reflection fluorescence (TIRF) illumination. By utilizing 2D optical lattices, we generate a thin (~800 nm full width half maximum) plane of light coincident with the focus of a high numerical aperture detection objective. Using this technique, we demonstrate substantial advantages in speed, sensitivity and reduced phototoxicity compared to conventional point scanning and spinning disc confocal microscopes as well as light-sheet microscopes utilizing single Gaussian or Bessel beams. We leverage these advantages to image samples ranging over three orders of magnitude in length scale from single molecules to whole embryos. Specific examples include: single molecule tracking of fluorescently tagged transcription factors in densely labeled embryonic stem cell spheroids, 3D imaging of microtubule growth phases and organelle dynamics throughout the course of cell division, 3D cell migration through collagen matrices, and protein localization throughout the course of dorsal closure in Drosophila embryos. Further, by combining lattice light sheet microscopy with point accumulation for imaging of nanoscale topography (PAINT) microscopy and novel fluorescent probes, we demonstrate 3D super-resolution localization microscopy over large fields of view and in thick 3D samples such as dividing cells and small embryos.
Wesley Legant, PhD, got his B.S. in Biomedical Engineering at Washington University in St. Louis, MO in 2005. He received his Ph.D. in Bioengineering from the University of Pennsylvania in 2012. Dr. Legant is currently a Research Scientist in the Betzig Lab at the HHMI Research Campus.