An ultrasensitive, high-speed camera has allowed researchers at Harvard University to see the 3D spatial relationship between cellular structures (mitochondria and microtubules) with nanometre-scale resolution.
'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,' said 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 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.
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.
'This new research shows 3D STORM can be used to aid understanding of molecular processes in cells,' said Zhuang. 'The approach relies on single molecule detection and short exposure times – we needed a highly sensitive and fast camera to make this possible.'
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.
