We develop novel optical imaging tools in an effort to open new windows into molecular, cellular, and neurobiology.
Bioimaging technologies developed in our lab: superresolution photoactivated localization microscopy (PALM); pulse splitters for mediation of nonlinear photodamage; Bessel beam plane illumination for rapid three-dimensional live cell imaging; and adaptive optics for deep tissue imaging.
Due to its comparatively benign effect on living systems, optical microscopy has been the workhorse for studies of structure and function at the cellular level and below for hundreds of years. Many questions at the forefront of molecular, cellular, and neurobiology remain beyond its current capabilities. In our group, we collaborate with scientists and engineers across disciplines to extend these capabilities in ways that we hope can be readily adopted by biologists at Janelia and elsewhere. The challenges of optical bioimaging, and hence the scope of our efforts, can be broken into four areas:
Focal adhesion proteins paxillin (green) and vinculin (red) assemble as interdigitated clusters in several adhesions at periphery of a human foreskin fibroblast cell, as seen at three different magnifications, using dual color PALM in conjunction with the photoactivatable fluorescent proteins Dronpa (green) and tdEos (red).
Credit: From H. Shroff, et al., Proc. Natl. Acad. Sci. 104, 20308 (2007).
Clustering of voltage-gated potassium channels at the plasma membrane of a cell, seen in a PALM image at two different magnifications (18.4 x 18.4 m and 4.6 x 4.6 m fields of view).
Credit: Sample courtesy of Mike Tadross, Johns Hopkins University.
The bacterial chemotaxis protein Tar forms banded structures in an E. Coli bacterium, as seen by PALM over a 2.5 x 2.5 mm field of view. Blue molecules, localized in the evanescent field produced by total internal reflection illumination, are closest to the substrate, while red molecules throughout the bulk of the bacterium were localized subsequently using epi-illumination.
Credit: From D. Greenfield, et al., PLoS Biol. 7, e1000137 (2009).
Combined PALM (red) and transmission electron microscopy (gray) images of a 50 nm thick resin-embedded section of the Drosophila optic lobe, with membrane expression of myr::tdEos in a subset of neurons (25 x 25 m field of view). Comparisons at right demonstrate comparable resolution by the two techniques (3 x 3 m field of view).
Credit: Sample courtesy of Barrett Pfeiffer, image by Haining Zhong.
Her2 receptors at the plasma membrane of an HEK cell exogenously labeled with a designed ankyrin repeat protein (DARPin) of picomoloar specificity bound to mEos.
Credit: Sample courtesy of Jonathan Marvin, image by Hari Shroff.
Photograph of the PALM instrument in our lab showing, from left to right, the EMCCD camera, the TIRF microscope, the sample stage and shutter controllers, and the laser combining sled.
Post-synaptic densities from a subset of synapses in a transfected mouse brain expressing PSD95::mEos2, as seen in a 70 nm thick resin-embedded section over a 14 x 14 m field of view (left), with two PSDs shown at higher magnification at right.
Credit: Sample and image by Haining Zhong.
PALM video at 25 sec/frame of tdEos::paxillin in a Chinese hamster ovary cell, showing the retrograde motion of adhesions at the periphery, and the formation and evolution of new adhesions at right.
Credit: From H. Shroff, et al., Nat. Mathods 5, 417 (2008).
Video demonstrating the acquisition of a PALM image: (left) single, isolated molecules of Kaede pop on and off by stochastic photoactivation and bleaching; (middle) diffraction limited image formed by the sum of all the frames at left; (right) PALM image built by plotting the centers of the diffraction limited spots at left.
Credit: From E. Betzig, et al., Science 313, 1642 (2006).
Top: dendrites loaded with calcium indicator OGB exhibit brightening and blebbing (inset) indicative of phototoxicity under repeated scanning by two photon microscopy.
Bottom: dendrites exhibit little physiological change when a 64x pulse splitter is used.
Credit: From Ji, et al., Nat. Methods 5, 197 (2008).
Photograph of a 128x pulse splitter, showing paths of light beams through the instrument, prior to their recombination at the output.
Repeated two photon imaging of nuclear histones in a Drosophila embryo show substantially less bleaching and phototoxicity when an 8x pulse splitter is used (right) than when one is not (left). Data courtesy of Thai Truong, Willy Suppatto, and Scott Fraser, Caltech.
Axial images of a 500 nm fluorescent bead under a 250 m thick fixed brain slice: (Left) bead image before adaptive optical correction, showing weak signal due to aberration; (middle) bead image before adaptive optical correction but after 4x intensity rescaling to show ghost images due to aberrations; (right) recovery of signal and a diffraction limited image of the bead after adaptive optical correction.
Credit: From N. Ji, et al., Nat. Methods 7, 141 (2009).
Corrective wavefront required to recover diffraction limited performance through a specific sample of fixed brain tissue, 250 m thick, showing substantial complexity indicative of highly inhomogeneous tissue.
Credit: From N. Ji, et al., Nat. Methods 7, 141 (2009).
Photograph of the two photon adaptive optical microscope in our lab.
Credit: Robert Merhaut Photography 2009
Lateral (top) and axial (bottom) view of a field of GFP-labeled dendrites 170 m deep in the cortex of a living mouse, as seen before (left) and after (right) adaptive optical correction.
Credit: Image courtesy of Na Ji.
Videos of GFP-labeled neurons over a 100 x 100 x 75 m volume in the cortex of a living mouse, as seen before (left) and after (right) adaptive optical correction.
Credit: Courtesy of Na Ji.