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  • Ultrahigh-Resolution Microscope Images Whole Cells in 3D

Photonics.com
Sep 2016
WEST LAFAYETTE, Ind., Sept. 12, 2016 — A novel superresolution nanoscope allows 3D imaging of an entire mammalian cell and its cellular constituents at a resolution that is 20 to 50 times higher than conventional microscopy, with imaging depth improved approximately tenfold over state-of-the-art iPALM and 4Pi-SMSN implementations. Until now, resolving details at this level was only possible using electron microscopy, which requires samples to be treated.

The whole-cell 4Pi single-molecule switching nanoscope, called W-4PiSMSN, allows volumetric reconstruction with 10- to 20-nm isotropic resolution of approximately 10-μm-thick samples. The novel nanoscope permits ultrahigh-resolution 3D imaging of virtually any subcellular structure, allowing researchers to resolve details smaller than the wavelength of light.

 Using a new 3D super-resolution instrument, researchers captured this visualization of a 'primary cilium,' the antenna of the cell.

Using a new 3D superresolution instrument, researchers captured this visualization of a "primary cilium," the antenna of the cell. Courtesy of Huang et al./
Cell. 


The W-4PiSMSN incorporates deformable mirrors using two objectives, one above and one below the sample, and uses a set of algorithms to pinpoint molecular positions of proteins deep inside cells, resolving features deep below the surface of the sample.

Researchers have demonstrated the wide applicability of W-4PiSMSN by imaging complex molecular architectures ranging from bacteriophages (viruses about 50 nm in diameter) to nuclear pores, cilia and synaptonemal complexes in large 3D cellular volumes. The development of W-4PiSMSN extends the application range of 4Pi-based SMSN to the imaging of organelles that span large volumes, exemplified by the mitochondrial network, the nuclear envelope, and synaptonemal complexes, which researchers were able to capture in virtual entirety.

The technology was used to visualize a mouse spermatocyte, revealing with unprecedented clarity the 'twisting paired lateral elements' of synaptonemal complexes, which link chromosomes together.

The technology was used to visualize a mouse spermatocyte, revealing with unprecedented clarity the "twisting paired lateral elements" of synaptonemal complexes, which link chromosomes together. Courtesy of Huang et al./Cell.

The research team included researchers at Purdue University, Yale University, the University of Cambridge, the Jackson Laboratory, Howard Hughes Medical Institute and the University of Oxford.

The advance could reveal biological phenomena never before seen, bringing novel medical insights.

"One goal is to further push the envelope in the direction of live-cell and tissue imaging, two major roadblocks of modern superresolution techniques, and therefore allow visualization of cellular functions live in their physiological conditions at the nanoscale,” said Fang Huang, professor of biomedical engineering at Purdue.

"We are interested in using our developments to study the cytokinetic apparatus, a core machinery during cell division," he added.

W-4PiSMSN is a versatile and powerful tool that promises a new perspective on how proteins distribute across entire organelles throughout whole cells, an as yet unmet challenge in cell biology.

The research was published in Cell (doi: 10.1016/j.cell.2016.06.016).



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.
microscope
An instrument consisting essentially of a tube 160 mm long, with an objective lens at the distant end and an eyepiece at the near end. The objective forms a real aerial image of the object in the focal plane of the eyepiece where it is observed by the eye. The overall magnifying power is equal to the linear magnification of the objective multiplied by the magnifying power of the eyepiece. The eyepiece can be replaced by a film to photograph the primary image, or a positive or negative relay...
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