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Marriage of Microscopy Techniques Reveals Cells’ 3D Ultrastructure

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Scientists at the Howard Hughes Medical Institute (HHMI) have combined superresolution fluorescence microscopy and electron microscopy to shed new light on the structures and organizations within cells.

Superresolution fluorescence microscopy on its own is able to show cellular structures with a high degree of clarity and detail, though the technique does have its limits. Only a few of the more than 10,000 proteins in a cell can be revealed, which does not provide researchers with enough information to study the interconnections and dependencies of cellular structures.

Electron microscopy, on the other hand, comes with its own set of pros and cons. The technique is able to reveal all cellular structures in high resolution, but the crowded intricacies of a cell’s interior are almost indecipherable without fluorescence markers.

To gain a better understanding of cells’ inner workings, the researchers combined the two methods to create a new technique called cryo-SR/EM.

To get an image, the cells must first be frozen under high pressure to halt the cells’ activity and prevent the formation of damaging and obstructive ice crystals. The samples are then placed in a cryogenic chamber where they are imaged in 3D with superresolution fluorescence microscopy at temperatures less than 10° above absolute zero. The cells are then removed, encased in resin, and imaged with a powerful electron microscope. The microscope fires an ion beam at the cells’ surface, which mills away layer by layer, photographing each newly exposed layer. The imagery from both techniques is later stitched together with a computer program to create a 3D reconstruction.

Combining the two techniques has given scientists a clear picture of how specific cellular features relate to their surroundings, said Harald Hess, a senior group leader at the HHMI’s Janelia research campus.

“This is a very powerful method,” Hess said.

Janelia research scientist David Hoffman and senior scientist Gleb Shtengel spearheaded the project under the leadership of Hess and Janelia senior fellow Eric Betzig, an HHMI investigator at the University of California, Berkeley.

The research was published in Science (www.doi.org/10.1126/science.aaz5357).

BioPhotonics
Mar/Apr 2020
GLOSSARY
fluorescence
The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.
Research & TechnologyBiophotonicssuper-resolution microscopyfluorescenceelectron microscopy3D imagingbiologycellular structuresuperresolution microscopyMicroscopyimagingBioScan

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