The extension of optical spectroscopy below the "diffraction limit" (about a third of the wavelength of light;e.g.,230nm) has been realized in recent years by two different classes of microscope: "PALM/STORM" and "RESOLFT/STED". The former recreates a biological scene in a 'pontillist'manner;centers of individual fluorescent 'paint'dots are located with 20nm precision on the scene, a few at a time, until the full picture emerges. It is precise but painstakingly slow. The second method, STED (STimulated Emission Depletion) superposes the normal spot illuminating the scene with another diffuse "donut" beam whose job is to erase fluorescence around the edges. This leaves a smaller spot at the center of the donut to sweep across the image, revealing it in 50nm detail. Both sorts of microscope are commercially available. The PALM version is inexpensive but slow, best for acquiring still images. The STED version (over $2M) has the potential for video nanoscopy but applies large laser powers (in the "erase" donut beam) that damages living cells. We are attacking the nanoscopy field in two directions. First, in PALM, we are collaborating with George Patterson to identify fluorescent proteins appropriate for a sort of "Sequential PALM";i.e. a picture with both superresolution and velocity information, akin to time-lapse photography. About 5% of our nanoscopy effort is expended in this direction. Most of our nanoscopy effort is devoted to STED and STED-like methods. We have constructed our own STED microscope around existing CARS lasers and FCS detection electronics (from other prior projects). We have designed (and provisionally patented) an 'azicon'(azimuthal polarizer axicon) to make the central spot of the donut beam very dark (preserving central brightness in the image, allowing for stronger erase beams and hence finer resolution). Most important, we have designed, provisionally patented, and begun testing a new class of fluorescent dyes that provide two key features: 1. lower power requirements for erase beam. This allows finer resolution and longer observations in living cells, making video nanoscopy more practical. 2. Simultaneous multicolor erase beam. STED had previously been limited to two colors, but the mechanism inherent in our dyes expands the available palette. This is important in providing biological context to the image of macromolecules one will paint. This year we tested several prototype dyes, finding we could squeeze down to 53nm resolution using only 50mW of 783nm "erase" beam power. This year we found the actual power requirement could be reduced to 12mW at the focal plane. Multicolor tubulin fibrils and beads have been imaged in the same, single-frame image. Ms. submitted. We also began exploiting the nanosecond nature of our dyes to prototype a microscope using inexpensive diode lasers to achieve our STAQ nanoscopy.

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National Heart, Lung, and Blood Institute
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Combs, C A; Smirnov, A; Glancy, B et al. (2014) Compact non-contact total emission detection for in vivo multiphoton excitation microscopy. J Microsc 253:83-92