STORM Microscopy

STORM stands for 'STochastic Optical Reconstruction Microscopy'. Together with PALM (Photoactivated Localization Micrsocopy) and GSDIM (Ground-State Depletion and Individual Molecule Return) and many other similar methods, these are single-molecule localization methods operating in the far-field mode.

By Dr. Jakob Bierwagen, AHF analyentechnik AG

In nearly all methods the vast majority of the fluorescent markers in the images are dark (or previously brought to a dark state). Only a few molecules can fluoresce. These must be farther apart than the diffraction limit. The emitted photons are detected on a fast camera. Subsequently, the molecules go back to a dark state or bleach.

Since one assumes that one observes only individual molecules, one can calculate the center of the emission by unfolding the diffraction pattern of the individual points with appropriate functions (for example, the 2D bell curve). The recording and reconstruction processes are repeated many hundreds to several thousand times, so that many molecules were detected individually. The individual localization images are then superimposed to form a complete picture.

In order to achieve a good resolution, it is crucial to be able to separate the individual fluorescent molecules from the background. To exclude background light very good excitation and detection filters must be used. To achieve a resolutions of 10–20 nm, it is also important that the beam splitter is as flat as possible, otherwise it comes to distortions that change the image so that it no longer corresponds to the reality within the subdiffractional resolution. Therefore, beam splitters with a flatness of <λ / 5 per inch are preferred to use in these methods.