Like a kid hovering over an ant with a magnifying glass, you can easily fry a worm with a microscope. But if you could do it without zapping the subjects, long exposure imaging would be immensely helpful for studying a cell process like development in a living Caenorhabditis elegans embryo. In a pair of just published papers—one in Nature Biotechnology yesterday and another in Nature Methods on October 6—Hari Shroff, tenure-track investigator at the NIH, unveiled a pair of new microscopes that offer an alternative solution to the problem of light-blasted subjects.
Shroff’s lab is in the windowless basement of Building 13 at the NIH. His lab is a suite of medium-sized rooms, each with a table the perfect height and size for a game of pool. Shroff’s tables though have shiny metal tops drilled with regular rows of holes like the backer board in woodshop class. Lenses and mirrors are mounted on the tables, seemingly in a haphazard pattern except that everything is in perfect mathematical alignment. To keep it that way, the tabletops float on air to minimize movement or vibrations.
Shroff’s first new microscope, which he developed in collaboration with Yicong Wu, a staff scientist in his lab, can image a live sample for over 14 hours without killing it. Standard epifluorescence microscopes spray light throughout the sample, which can fry it while confocal microscopes focus intensely on one point but still have light propagating through the entire sample. Shroff’s device avoids the frying by quickly passing sheets of light over the subject (the sheet ensures that only the focal plane is illuminated).
In the new Nature Biotechnology paper, Shroff called his new high-resolution microscope diSPIM (dual-view inverted selective plane illumination microscopy). In the first prototype tested, Shroff found that while the image looked great from one direction, if the view is rotated 90 degrees, the picture becomes distorted. To solve this problem, he added a second objective at 90 degrees from the first to form a V. The dual-view microscope takes images sequentially in each plane, with rapidly scanning sheets of light. This creates an image with isotropic resolution and minimal bleaching, Shroff explained.
Shroff’s original goal was to build an instrument that would allow him to reconstruct the C. elegans nervous system during development. His new microscope can easily image worm neurons in three dimensions. Still the curves of the worm’s body make tracing neurons difficult so Shroff has started to develop a computer program that traces along the backbone and straightens the image.
Shroff’s second high-resolution microscope for live cells, described in Nature Methods on October 6, goes by the name of iSIM (instant structured illumination microscopy). Developed in collaboration with Andrew York, a research fellow in Shroff’s lab, the iSIM uses structured light that looks like an array of spots. It mathematically processes the multiple spots to recover an image with twice the resolution of an epifluorescence microscope.
“The mathematical details are pretty simple for a computer to do.” Shroff explained “You throw away the light in between the spots, then shrink each spot by a factor of two, then add all the resulting images to get a higher resolution image.” That concept isn’t new, says Shroff but he and York did devise a new way to mathematically process the images optically with hardware. “It’s blazingly fast compared to any other super resolution microscope.”
iSIM does not have the same power of resolution as other new super-resolution systems such as PALM (photoactivated localization microscopy) or STORM (stochastic optical reconstruction microscopy) but unlike PALM and STORM, Shroff’s iSIM can image live cells over a much longer time without damaging them. It allows a user to acquire raw super-resolution images, unlike PALM or STORM where tens or even hundreds of thousands of images are stitched together for a reconstruction. The iSIM is fast enough that a researcher can watch microscopic events at super-resolution, in real time. Shroff has used the scope to image the cytoskeleton in blood cells flowing through a living zebrafish, a feat that would be impossible for PALM or STORM systems.
During his graduate work at the University of California, Berkeley, Shroff built a TIRF (total internal reflection fluorescence) microscope for in vitro studies but recalls, “It was always a frustration that what I built was good for in vitro work… which is relatively easy… I wanted to build microscopes to look at stuff inside cells.”
Shroff decided to build microscopes for living cells at the Physiology Course held each year at the Marine Biological Laboratory in Woods Hole, MA. “Innovation in microscopy was a big thing at the Physiology course, and [there I learned] I could fit into that niche,” he says. The work the Physiology students were attempting provided Shroff with his design requirements, “How could my microscopes enable the kind of cell biology that these people were doing?”
To gain more experience in building microscopes, Shroff did a postdoc at the Howard Hughes Medical Institute’s Janelia Farm where he worked on PALM and STORM in Eric Betzig’s lab. PALM and STORM have become hugely popular in labs, says Shroff but he “didn’t want to be one of many [working in the field].” He started as a tenure-track investigator at the National Institutes of Health in 2009, and in his first project he took PALM to the next level by building a three-dimensional PALM system.
As a PI, Shroff finds his time in the lab limited. But he still enjoys working with his postdocs and actually building microscopes, “I try to do something every day with my hands, if my postdocs will let me,” Shroff says.
The next development for his two new microscopes is to rebuild them for easier use by bench biologists. Surveying his experimental tables of mirrors, objectives, light sources, and lasers, Shroff says he looks forward to the day that this new table top of technology will be in the hands of researchers, giving them a longer, clearer, non-lethal look at living cells.