Due to the wave-like nature of light, performance of an optical microscope is limited by diffraction which causes an infinitely small light source to be visible only as an extended spot. This resolution limit, known as the Abbe limit, has recently been overcome enabling unprecedented observations of biological structures and processes.
PALM and STORM are two conceptually related approaches breaking the Abbe limit. Both rely on stochastic switching of individual fluorophores. The method known as STED exploits the non-linearity of the phenomenon of Stimulated Emission to break the diffraction barrier.
PALM and STORM are implemented in the facility on an inverted TIRF microscope. A STED microscope is under construction.
PALM / STORM
The PALM and STORM methods result from two separate realizations. First, single fluorescent molecules can be localized with high accuracy by fitting their image formed on a camera with a Gaussian profile. Next, the engineering of photo-convertible fluorescent proteins and the switching properties of particular dyes provides a way to turn on the specimen fluorescence one molecule at a time. By an iterative process in which a few molecules are activated, imaged and then switch-off or photo-bleached, a sub-diffraction image can be reconstructed after determining the position of all individual molecules.
PALM: Photo-Activated Localization Microscopy
STORM: Stochastic Optical Reconstruction Microscopy
Unlike PALM and STORM which are widefield imaging methods, STED microscopy is a scanning approach. It uses the non linearities of the phenomenon of Stimulated Emission to overcome the Abbe limit.
STED uses two laser beams. The first one, the excitation beam, excites the fluorescent molecules. The second one, the depletion beam, is shaped as a ring surrounding the first focused beam. Using intense depletion light causes almost all of the excited molecules to return to the ground state, leaving only the region of the sample very close to the center of the excitation spot excited. Fluorescence from the remaining excited dye molecules is then detected by the microscope.
The size of the spot where molecules are still allowed to fluoresce gets smaller with increasing intensity of the depletion light. This size corresponds to the achievable resolution.
A STED microscope is under construction in the facility and will be available soon.
STED: Stimulated Emission Depletion