An ultrasensitive, high-speed camera has helped US researchers see the 3D spatial relationship between cellular structures - mitochondria and microtubules - with nanometre-scale resolution.
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, a low-light image of individual molecules is obtained, which 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.
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.
The team's 3D Storm images showed the hollow shape of the mitochondrial outer membrane.
They also observed two types of mitochondrial morphologies - globular and dispersed in some cells, tubular and interconnected in others.
The team could distinguish the individual points of contact between mitochondria and microtubules, even where they were densely packed.
