Abstract
Nanoscopy has been a rapidly expanding field over the last 5 years. Different modalities have been developed based on optical, computational and combined optical/computational techniques that can be used to circumvent the diffraction barrier of visible light. Stimulated emission depletion (STED) microscopy is a purely optical technique that was originally developed by Stephane Hell and has been recently commercialized by Leica Microsystems. This technique uses two superimposed laser beams. One beam excites a point of fluorescence while the second beam – a donut shape – depletes the fluorescence emission around the spot through stimulated emission. The result is a point source of excitation which can be scanned across a fluorescence sample giving resolutions on the order of 20–40 nm. Single molecule localization microscopy is a purely computation technique that comes under many flavors (PALM, F-PALM, STORM, dSTORM). Each technique relies on the repetitive excitation and localization of a small subset of fluorescent molecules within a sample. Single molecules are imaged as Gaussian spots and with sufficient signal-to-noise can each be fit with a precision approaching 20 nm. Finally, structured illumination uses a combination of optics and image processing. Grid patterns are super-imposed on fluorescent samples with 15–25 different angles and z-axis positions. The result is a translation of high frequency sample information into low frequency Moire patterns. The 15–25 images are post processed based on the imposed patterns to de-convolve out fine sample details with at least twice the resolution limit of a conventional light microscope. This overview talk will introduce these various nanoscopy techniques.