Laboratory of Super-Resolution Fluorescence Microscopy
School of Life Sciences
Principal Investigator: Yongdeng Zhang ç« æ°¸ç™», Ph.D.
Research Highlights
1. Multicolor 4Pi-SMS imaging with salvaged fluorescence
Although super-resolution microscopy has opened the door to imaging at the nanoscale, the ultimate goal, simultaneously resolving multiple targets with high resolution in three dimensions (3D), has not yet been achieved. This has limited the impact of super-resolution microscopy in biology. To address this challenge, we have built a super-resolution microscope that enables, for the first time, simultaneous multi-color imaging of whole mammalian cells at ~20 nm isotropic 3D resolution with minimal cross-talk and negligible chromatic aberrations. This invention has enabled the visualization of, for example, Golgi cisternae (about 50 nm in thickness) and the close contacts between the endoplasmic reticulum and the plasma membrane (about 15-25 nm apart). As the optical gold standard, our system provides a tool to the cell biologist that combines the strength of specific labeling in the context of interaction partners and cellular landmarks with a level of detail that has traditionally been the realm of electron microscopy.
2. Long time-lapse nanoscopy with spontaneously blinking membrane probes
Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.
3. Visualization of type III protein secretion machines in live bacteria
Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine’s assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.