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.
Unlike PALM and STORM which are widefield imaging methods, STED microscopy is a deterministic scanning approach. It uses the non linearity of Stimulated Emission to overcome the Abbediffraction 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 (phase shift) 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 has recently been completed in the Imaging Core facility. It is an all-pulsed, laser-scanning STED microscope that is unique in that it uses a Spatial Light Modulator to more controllably create the donut pattern required for depletion.
The STED microscope:
640nm pulsed excitation
775nm (~1ns) pulsed depletion
200 micron fast piezo with position feedback for precise sectioning
100x 1.4 NA lens
STED: Stimulated Emission Depletion
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.
Coherent Obis lasers are used for direct modulation
60x 1.49 and 100x 1.49 lenses are available around a microscope with active focus maintenance
PALM: Photo-Activated Localization Microscopy
STORM: Stochastic Optical Reconstruction Microscopy