Photoactivated Localization Microscopy
Photoactivated Localization Microscopy ( PALM, German localization microscopy after photoactivation or photoactivated localization microscopy ) and Stochastic Optical Reconstruction Microscopy ( STORM ) are special methods of light microscopy, accurate fluorescence microscopy. They are based on a light-controlled switching on and off of fluorescence in individual molecules. The switching on and off is carried out over a period of time, can be absorbed by the plurality of individual images. A subsequent computer calculations, the position of individual molecules with a resolution beyond the optical resolution limit described by Ernst Abbe can be determined.
History
The technique was developed in parallel by three groups in 2006 and named differently. Eric Betzig and colleagues at the Howard Hughes Medical Institute called PALM, ST Hess and colleagues FPALM Xiaowei Zhuang and colleagues at Harvard University and called them STORM. The resolution achieved was reported to be 2 to 25 nm and 20 nm respectively. It is now possible to pursue with this technique, single enzyme molecules in individual bacteria in their work.
Principle of operation
In classical fluorescence microscopy, fluorescent molecules that are too close together, are no longer resolved: they appear as a single structure.
PALM circumvents this problem by the special properties of photoactivatable fluorescent proteins (English: photoactivatable fluorescent proteins, PA - FPs ) makes advantage. These special variants of the green fluorescent protein ( GFP) can be selectively activated and deactivated by light of a specific wavelength and intensity. Like other GFPs also they can be fused to molecular biology such proteins, whose position is to be examined in the cell.
First of all PA - FPs are inactive, ie not fluorescent. A short flash of light with light of a suitable wavelength few PA - FPs are activated randomly. This switch is non-linear to a high degree, because only molecules activated or non- activated may be present. Then they can with light of a " query " wavelength excited to fluorescence and the fluorescence emitted photons are detected. With progressive exposure, these fluorescent molecules bleach, that is, the ability to fluoresce in this molecule is lost irretrievably. Here, more pictures are continuously made . The experimental conditions are chosen such that the probability that, in a flash activation two molecules close to each other to be activated at the same time, is very small. Since too close lying fluorescent molecules can not be distinguished from each other, this is a prerequisite for the high resolution in the nanometer range.
The next flash of light activated again by chance other PA - FPs and the process is repeated. After very many passages all PA - FPs are recorded once. You can keep recording until all PA - FPs are bleached ultimatum. Instead of using individual activation flashes, the product can also be continuously illuminated with the light activation. Here, such a low brightness is used that only individual - randomly distributed - PA - FPs are activated.
The fluorescent molecules first appear blurry due to the diffraction of the microscope. By a mathematical algorithm using the point spread function, however, the exact position of each molecule can be calculated. The idea is based on that each molecule picture was taken spatially isolated and therefore its position can be determined, for example, as the focus of the resulting light spot with higher resolution. However, this only works if adjacent molecules are not at the same time are active. A computer program then determines for all partial images in the positions of the active molecules and generates the final image.
The principle of high-resolution localization microscopy is not limited to fluorescent, switchable proteins. And flashing colors, which may have a longer- dark state to be used.
Even non-switchable fluorescent proteins ( GFP) can be made to blink and thus used for high-resolution microscopy.
Properties, distribution and alternatives
One advantage is the relatively simple design of the microscope. Since the optics of the microscope consists essentially of normal parts, the use of a conventional fluorescence microscope is possible, in principle, to camera PALM.
Other options in light microscopy to achieve a very high resolution close STED microscopy and Nahfeldmikroskopien (TIRF and SNOM) with a.
Commercial microscope systems that work with the PALM principle are announced.