Photoactivated Localization Microscopy
Photoactivated Localization Microscopy (PALM, German localization microscopy after photoactivation or photoactivated localization microscopy ) or Stochastic Optical Reconstruction Microscopy (STORM) are special methods of light microscopy , more precisely of fluorescence microscopy . They are based on a light-controlled switching on and off of fluorescence in individual molecules. Switching on and off takes place over a certain period of time over which several individual images can be recorded. A subsequent computer calculation can determine the position of individual molecules with a resolution beyond theoptical resolution limitdescribedby Ernst Abbe .
The technology was developed in parallel by three groups in 2006 and given different names. Eric Betzig and colleagues at the Howard Hughes Medical Institute called it PALM, ST Hess and colleagues at FPALM and Xiaowei Zhuang and colleagues at Harvard University called it STORM. The resolution achieved was given as 2 to 25 nm or 20 nm. It is now possible with this technique to follow individual enzyme molecules in individual bacteria at their work.
In classic fluorescence microscopy, fluorescent molecules that are too close together can no longer be resolved: They appear as a single structure.
PALM circumvents this problem by the special characteristics of photoactivatable fluorescent proteins (English: photoactivatable fluorescent proteins PA-FPs) makes Use. These special variants of the Green Fluorescent Protein (GFP) can be activated and deactivated in a targeted manner using light of a certain wavelength and intensity. Like other GFPs, they can be fused molecularly to proteins whose position in the cell is to be investigated.
First of all, all PA-FPs are inactive, i.e. not fluorescent. A short flash of light with light of a suitable wavelength randomly activates a few PA-FPs. This switching is to a high degree non-linear, since molecules can only be activated or not activated. Afterwards they can be excited to fluorescence with light of a “query wavelength” and the emitted fluorescence photons can be detected. As the exposure progresses, these fluorescent molecules bleach out, which means that the ability to fluoresce in this molecule is irretrievably lost. More pictures are taken continuously. The test conditions are chosen in such a way that the probability that two molecules lying close to one another are activated at the same time in the event of an activation flash remains very small. Since fluorescent molecules that are too close together cannot be distinguished from one another, this is a prerequisite for the high resolution in the nanometer range.
Another flash of light randomly activates other PA-FPs and the process is repeated. After a lot of rounds, all PA-FPs are recorded once. You can record until all PA-FPs are ultimately bleached. Instead of using individual activation flashes, the preparation can also be illuminated continuously with the activation light. Such a low brightness is used that only individual - randomly distributed - PA-FPs are activated.
The fluorescent molecules initially appear blurred due to the diffraction of the microscope. However , the exact position of each molecule can be calculated by a mathematical algorithm using the point spread function . The idea is based on the fact that each molecular image was recorded spatially isolated and its position can therefore be determined with a higher resolution, for example as the center of gravity of the light spot obtained. However, this only works if adjacent molecules are not active at the same time. A computer program then determines the positions of the molecules active therein for all partial images and generates the final image from this.
The principle of high-resolution localization microscopy is not limited to fluorescent, switchable proteins. Flashing dyes, which have a longer-lasting dark state, can also be used.
Non-switchable, fluorescent proteins ( GFP ) can also be made to blink and thus used for high-resolution microscopy.
Properties, distribution and alternatives
One advantage is the comparatively simple structure of the microscope. Since the optics used essentially consist of normal microscope parts, a classic fluorescence microscope with a fast camera can in principle be used for PALM.
- Harald Hess: Super-Resolution Microscopy Takes on a Third Dimension . Howard Hughes Medical Institute, February 2, 2009, accessed May 12, 2010 (More information and multimedia on PALM; English).
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