Ground-state depletion microscopy

About this technique

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Confocal microscopy invented in the 1980s generated a new era of optical microscopy. By scanning the light beam, or a focal point of the beam, and collecting signals filtered by geometric pinholes, it offers optical sectioning ability. Combining with fluorescent labeling techniques, confocal microscopy offers a great visualization tool to biologists.

Resolution of light microscopy, whether wide-field or confocal, has been limited to the diffraction nature of light. In other words light bends when it come across an object small enough. This sets the highest optical resolution to somewhere 220nm in lateral dimension in short visible wavelength and about 500nm in the axial dimension.

GSD stands for Ground State Depletion. The way this technique breaks the barriers of diffraction limit lies fundamentally in fluorescent markers.  In most fluorescent microscopies, fluorophore molecules can freely be excited from ground state and returns spontaneously via emission of a photon. In order to reach, in theory, molecular resolution, GSD limits the number of molecules that is “bright” for each image and adds thousands of high-resolution low-signal images to generate a super resolution image for the whole frame. One way of “silencing” majority of the fluorescent molecules is by pumping them into a long-lived dark state, i.e. a state where no fluorescence occurs. As long as the molecule is in the long-lived dark state (e.g. a triplet state), it cannot be excited from the ground state and hence ground state depleted. Understandably, GSD microscopy requires either special fluorophores with high triplet yield, or removal of oxygen by use of various mounting medium like monoethanolamine.

Users can do super-resolution imaging in EPI and TIRF modes. A wide range of standard fluorochromes have been proved working including Alexa Fluor 647 Phalloidin, Alexa Fluor 488 IgG, etc.