"This level of detail was previously only obtainable with an electron microscope," said Harald Hess of NuQuest Research. "However, the contrast in electron microscopy is more indiscriminate, whereas we can limit our contrast to only specific proteins of interest."
Hess and colleagues made mammal cells express target proteins tagged with the fluorescent protein molecules by a process of transient transfection. Applying laser light with a wavelength of 561 nm excited the activated form of the expressed fluorescent proteins. The team then imaged the result via total internal reflection fluorescence microscopy onto a single-photon-sensitive electron multiplying CCD camera.
The researchers continued imaging until bleaching of the fluorescent proteins removed many of them from view. Then the team used laser light with a wavelength of 405 nm to activate more of the proteins and repeated the measurement process. They performed this cycle many times, taking from around 10,000 to more than 100,000 image frames, until almost no inactivated, unbleached fluorescent molecules remained.
Analysis of the resulting image enabled the team to determine the centre of each fluorescence emission and to resolve – in the best cases – molecules that were around 10 nm apart.
In this way the team has imaged sections of lysosomes (structures inside the cell which digest molecules) and mitochondria (cell "power plants"). Inside whole cells the team has imaged vinculin (a cytoskeletal protein) at focal adhesions, actin within a lamellipodium (a projection on the mobile edge of a cell) and the distribution of the retroviral protein Gag at the plasma membrane.
"A great feature of PALM is that it can readily be used with electron microscopy, which produces a detailed image of very small structures, but not proteins, in cells," said Jennifer Lippincott-Schwartz of the National Institute of Child Health and Human Development. "By correlating a PALM image showing protein distribution with an electron microscope image showing cell structure of the same sample, it becomes possible to understand how molecules are individually distributed in a cellular structure at the molecular scale. Correlative PALM unites the advantages of light and electron microscopy, producing a revolutionary new approach for looking at the cell in molecular detail."
The camera took one or two pictures each second, so that it could take between two and 12 h to image one sample. The researchers hope to speed up the technique, perhaps by activating more molecules per frame or by making the molecules brighter, so that the time needed to take each image is less.
"Right now the resolution is wonderful, but it's all single-label stuff," said Eric Betzig of the Howard Hughes Medical Institute. "Knowing where one protein is located is nice but what you really want to know is where multiple proteins are in relation to one another."
The team is now investigating the use of additional labelling molecules, which could enable them to view how proteins interact inside cells. "As the PALM technology advances, it may prove to be a key factor in unlocking at the molecular level secrets of intracellular dynamics that are unattainable by other methods," said Michael Davidson of Florida State University.
The researchers reported their work in Sciencexpresss.