Since its invention in 2006, STORM microscopy has become the most widely used super-resolution microscopy technique for single-molecule imaging. STORM stands for "Stochastic Optical Reconstruction Microscopy" and it relies on the stochastic activation of individual fluorophores with photoactivatable properties.
During STORM, single fluorophores "blink" by a process of random activation from an off or dark state, to an on or emission state, quickly followed by a switch back to a dark state or photobleaching. This process is sequentially repeated many times until most fluorophores have been imaged. For a successful STORM imaging, individual fluorophores must be sparse enough, so that only one molecule is activated within a diffraction-limited region at any given time.
During the STORM reconstruction process, individual molecules can be precisely localized by determining their position coordinates from the photons detected for each activation event (often as a Gaussian function).
dSTORM image of rabbit psoas myofibrils labeled with the titin antibody T12-AF647 (blue). The antibody localizes in a pair of lines, one to on either side of the Z-disk, separated by 160-200nm. Sample provided by Mathias Gautel, Pauline Bennett, and Susan Cox (scale bar: 1um)
There are two variants of STORM. The first one, known as direct STORM or dSTORM microscopy, is compatible with many commonly used fluorophores, which can be converted to an off state using specific excitation parameters and in combination with specialized oxygen-scavenging imaging buffers. Ideal fluorophores for STORM should be very bright, have a high rate of photoswitching and exhibit minimal photobleaching in thiol-containing buffers.
The second type of STORM microscopy uses a pair of activator-reporter dyes; the "activator" induces a switching and the "reporter" emits the signal detected. While the multiple activator-report pairs available allow multicolor STORM applications, this system requires dual labelling of the same target antibody and relies on the proximity of the two fluorophores. STORM without the activator dye, dSTORM, is currently widely used with different report dyes, as the photoswitching induced is efficient enough with substantial laser power.
By stochastically imaging small subsets of photoswitchable fluorophores over time, STORM microscopy allows to spatially resolve the localization of individual molecules with high precision even in dense populations. STORM microscopes can produce high-quality images of cellular components resolved to under 20nm, changing the way scientists visualize molecular structures, interactions within organelles and biological processes.
While STORM is an excellent localization-based super-resolution technique, its use for live imaging is limited, mainly due to phototoxicity issues. In this case, PALM microscopy, which uses different photoactivatable fluorophores, is better suited for live imaging and single-particle tracking. PALM allows to track the movement of molecules over time, calculate diffusion coefficients and describe particle behavior.
dSTORM image of nuclear pores (labelled NuP98-AF647, red) in a U2OS cell, sample prepared by S. Tyagi and U. Manor, Salk Institute, USA (scale bar: 5um)
The Nanoimager offers several super-resolution modes for imaging cells or purified components at the nanoscale level. The Nanoimager's extraordinary sensitivity, stability and precision make it an excellent tool for precise measurements using STORM and PALM microscopy. STORM microscopy will produce images with an enhanced resolution to about 20nm and can provide additional image information by performing single molecule visualization or cluster analysis.
The Nanoimager has four different laser lines and offers the possibility of imaging two fluorophores simultaneously on a single STORM microscope sample, allowing single molecule localizations registered in one channel to be assigned to cellular markers in the second channel.
The Nanoimager has been used to quantitatively analyze mitochondria in neuroblastoma cells and better understand protein complex assembly in E.coli using dSTORM microscopy. In combination with TIRF microscopy or HILO microscopy, the Nanoimager can perform STORM imaging in thinner samples with an improved signal to noise ratio, opening up the possibilities for scientists to study membrane associated complexes and small vesicles.
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