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Ground state depletion followed by individual molecule return (GSDIM) and stochastic optical reconstruction microscopy (STORM) are single-molecule based super-resolution localization microscopy (SRLM) techniques. They surpass the diffraction-limited resolution barrier and have been vital in the understanding of biological structures and systems. Both share the same methodology of creating a super-resolution image by individually localizing single fluorophores and separating them temporally. The difference between these two techniques is how the fluorophores are switched on and off.
The key to overcoming the 200 nm diffraction limit with GSDIM microscopy is sequentially turning on and off and temporally separating fluorophores. The electrons of fluorophores are accumulated in a metastable, non-fluorescent dark state (triplet state and others) by a high-power laser beam. Molecules return stochastically to the ground state from the dark state for re-entering the fluorescence cycles, which are called "blinking". These fluorescent blinking events are captured over time. The diffraction-limited events are read out by applying a fitting algorithm to get a high-resolution image. Eventually, the ultimate high-resolution image is reconstructed by plotting the measured positions of thousands of recorded events.
Typically, in STORM, fluorophores will be excited to the emission state and the dark state, where they will bind to free oxygen species to enter the bleached state. The fluorophores are prevented from entering the bleached state by depriving the free oxygen species. However, strong laser power may cause the dark-state fluorophores to enter the emission state again. This constant movement from the dark state to the emission state gives the impression that fluorophores are blinking. Importantly, the blinking is stochastic, so for each frame of an acquisition, a different subset of fluorophore molecules will be emitting. Many of the emitting molecules will be apart far enough that they could be individually localized. After collecting many thousands of frames, a single, super-resolution image is reconstructed to show each fluorophore molecule's position.
Single-molecule based techniques provide unprecedented resolutions to break the fundamental limits of light microscopy that stood for hundreds of years. More so than any other fluorescence microscopy, GSDIM and STORM require stringent attention in choosing fluorophores, labeling methods, and buffers for successful imaging. Creative Biostructure has taken care of these details to provide comprehensive services for our customers.
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