Correcting optical aberrations in fluorescence microscopy

X-ray crystallography is still the only way to obtain the full structure of a protein, but crystallysing membrane proteins is notoriously fiddly.

Membrane proteins have a habit of resisting crystallisation attempts, so for many years the best and easiest technique for probing biological molecules in the one to 10 nanometre range has been fluorescence resonance energy transfer (FRET).

FRET measures the transfer of energy from one fluorescent probe called the donor to another probe, the acceptor, and thus the distance between them.

It is often used to investigate protein-protein interactions, but its efficiency depends on the orientation between the two probes and knowing how the probes are oriented relative to each other can be difficult.

A new experimental technique developed by Dr Pascal Vallotton, head of the biotech imaging group at CSIRO Mathematical and Information Sciences, may prove a cheaper and easier way to measure distances with nanometer accuracy in both two and three dimensions, using conventional fluorescence instrumentation.

Called Differential Aberration Correction (DAC) microscopy, the technique is aimed at bridging the gap between the resolution achieved by FRET in the 1-10 nm range and that of conventional diffraction limited microscopy beyond 300 nm.

“Our method is completely different from FRET,” he says. “It does not rely on the physical effect – the transfer of energy from one fluorophore to the other. It simply uses fluorescence filters to look first at one fluorophore and then the other one, thus avoiding the overlap of their image.

“By using the right image processing algorithms, it is possible to extend the useful range for making these distance measurements from 0-10 nm to 0-1 micron, in principle.”

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