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 $1.1M) has the potential for video nanoscopy but applies large laser powers (in the erase donut beam) that damages living cells. 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, 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 new prototype dyes, finding we could squeeze down to under 60nm resolution using only 12mW of 783nm erase beam power at the focal plane. Multicolor tubulin fibrils and beads have been imaged in the same, single-frame image. We also began exploiting the nanosecond nature of our dyes to prototype a microscope using inexpensive diode lasers to achieve our STAQ nanoscopy, both CW and ns-pulsed, and we worked toward adding external TAQ antennae to the popular GFP family of fluorescent protein paint molecules. The latter are popular because they can be genetically connected to the structure of interest in cells. Combining STAQ or STED with modulated donut time profiles has been theoretically examined to suggest even lower powers for encoding, and we have begun assembling this STEN (SpatioTemporal Encoding Nanoscopy microscope.
