New light microscope to pinpoint single proteins
powerful ituses a nanometre-scale resolution to spot individual
proteins in a cell byusing a fluorescent labelling method.
The new technique, called photoactivated localisation microscopy (PALM),allows scientists to identify and locate molecules that are only two to 25nanometres apart.
Currently used optical microscopes are limited by the wavelength of lightand therefore cannot distinguish molecules separated by less than 200nanometres, a resolution insufficient to locate individual proteins - whichmeasure one to two nanometres in diameter.
To create this more powerful microscope, researchers at the Howard HughesMedical Institute (HHMI) in Virginia combined optical microscopy with a newmethod of fluorescent labels developed by a biology lab.
"In the world of biology there is a new generation of fluorescent proteins that you can switch on at will with the use of violet light," said Harald Hess, co-developer of PALM.
This fluorescent technique allowed the researchers to develop PALM, byattaching a label to each copy of a protein. Studied molecules are labelledwith a photoactivatable probe, and then exposed to a small amount of violetlight, which activates fluorescence in a small percentage of molecules.
The researchers continue imaging until bleaching of the fluorescent proteins removed many of them from view. Repeating this process many thousands of times, a computer image is eventually created in which the positions of all the molecules are determined.
"Because the number of molecules captured in each image is small, they are far enough apart to see each molecule individually and thereby localise its centre," said Hess.
"This level of detail was previously only obtainable with an electronmicroscope. However the contrast in electron microscopy is moreindiscriminate, whereas we can limit our contrast to only specific proteinsof interest."
The use of PALM in conjunction with electron microscopy is thereforeparticularly interesting and, in fact, the new technique brings together the advantages of light and electron microscopy, producing a new approach for looking at the cell in molecular detail.
One drawback of PALM, however, is that it takes a long time - typicallyseveral hours - to take all the snapshots needed for a complete image.
"It's very slow and difficult to use at the moment but we are working todesign a second generation that could be commercialised and used bybiologists," Eric Betzig, leader researcher at the HHMI and co-developer of PALM, told Labtechnologist.com.
The researchers think that they could speed this up both by increasing thenumber of molecules 'switched on' in each image and by making them brighter.
"Now if we can begin to refine this technique, we should really be able to take it to a lot of new applications in cell biology," Betzig concluded.
The research was published on 10 August 2006 in Science Express.
