Super-Resolution 3D Microscopy Uncovers Cellular Interactions with Nanometer-Scale Resolution

19 Nov 2009
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An ultrasensitive, high speed camera has helped US researchers see the 3D spatial relationship between cellular structures – mitochondria and microtubules – with nanometre-scale resolution, for the first time.

Three-dimensional stochastic optical reconstruction microscopy (3D STORM) can resolve fine structural detail with a lateral (x,y) resolution of 20-30 nm and axial (z) resolution of 50-60nm, using photo-activatable fluorescent labels and a camera sensitive enough to detect single molecules.

The STORM approach uses sequential imaging of single fluorophore molecules as they toggle between bright and dark states. By exciting only a stochastic subset of single labels with an activating pulse of laser, one obtains a low light image of individual molecules that can be discerned as single diffraction-limited spots. This allows the position of each fluorescent molecule to be determined with nanometer precision. Such repeated cycles of pulses allow the position of all molecules to be determined, and subsequently the construction of a super-resolution image from these precisely determined fluorophore positions.

This is the first study to use 3D STORM to visualize the spatial relationships between nano-scale structures in cells. Understanding how these structures interact paves the way for future research into cellular processes.

The team from Harvard University used an ultra-sensitive iXonEM+ Electron-Multiplying CCD scientific camera from Andor Technology to capture whole monkey kidney cell images from an Olympus inverted microscope. Andor’s iXonEM+ EMCCD camera is capable of detecting single photons released by the isolated fluorophore molecules.

“Much of biology is governed by interactions between molecules and molecular assemblies, but many intracellular and molecular structures in cells are unresolvable with conventional light microscopy due to the diffraction limit,” says the senior author of the paper, Xiaowei Zhuang, an investigator at Howard Hughes Medical Institute and a professor at Harvard University. “Our research has demonstrated that 3D STORM has a spatial resolution at least 10 times greater than this classical limit.”

The team’s 3D STORM images showed the hollow shape of the mitochondrial outer membrane, which is typically hard to resolve using conventional wide-field or confocal fluorescence microscopy. They also observed two types of mitochondrial morphologies – globular and dispersed in some cells, tubular and interconnected in others. The different mitochondrial structures are thought to reflect cells at varying growth stages.

The team could distinguish the individual points of contact between mitochondria and microtubules, even where they were densely packed. In living cells, mitochondria are constantly transported and reorganised by motor proteins attached to microtubules – clear imaging of these structures will assist future research into how they interact.
“This new research shows 3D STORM can be used to aid understanding of molecular processes in cells,” said Xiaowei Zhuang. “The approach relies on single molecule detection and short exposure times – we needed a highly sensitive and fast camera to make this possible. “

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