STORM | STORM
Key Words: Super resolution, SIM, Perfect focus system (PFS), fluorescence, fluorophores
Definition:STORM is a super-resolution microscope imaging technique based on the random activation and deactivation of fluorophore pairs.
TECHNOLOGY:
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In STORM specimen preparation requires the use of photoswitchable fluorophore pairs, which are randomly activated to identify the position of each fluorophore. For example, in a Cy3-Cy5 pair where the Cy5 is the reporter and Cy3 is the activator, Cy5 can be cycled hundreds of times between a dark and fluorescent state by using light of different wavelenghts Each cycle randomly illuminates just a few Cy5 fluorophores, each of which can be resolved cleanly allowing position to be determined accurately. By overlaying fluorescence images from many cycles, an overall image of Cy5 fluorophores can be reconstructed. Its exceptional high resolution is made possible by the construction of a fluorescence image from the highly accurate localisation (to 1nm) of fluorescent molecules attached to nucleic acids or proteins in a sample. Image resolution is determined by the number of photons emitted per cycle and not the wavelength of light. Several fluorophore pairs have been characterised enabling powerful and highly versatile high contrast imaging.
IMAGE:
Comparison of conventional light microscopy (left) and N-STORM imaging (right) of microtubules.
APPLICATION:
STORM reveals nanoscale details in biological samples. Because of the need for successive image capture, STORM is most suited to the study of fixed specimens.
MICROSCOPE CONFIGURATION:
The Nikon STORM system (N-STORM), available through a license agreement with Harvard University, is based on the Eclipse Ti research level inverted microscope. The system provides multi-spectral 2-dimensional and 3-dimensional nanoscale imaging, and in principle, imaging at the molecular scale. Three-dimensional imaging can be achieved with a simple optical switchover device built in the microscope, eliminating the need for time consuming serial sectioning. N-STORM is managed through NIS-Elements for easy operation and seamless integration into workflow.
