Optical Superresolution Microscopy (Nanoscopy)

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

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 has been devoted to STED and STED-like methods. We had constructed our own STED microscope around existing CARS lasers and FCS detection electronics (from other prior projects). We 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). For widespread STED use, a general calibration scheme developed for STED dyes that enables nanoscopists to compensate for the quirks in their individual optics .. The "Saturation Intensity" calibration manuscript was published previously. In past years 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. 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 design a microscope using inexpensive diode lasers to achieve our STAQ nanoscopy, , 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. Finally, combining STAQ or STED with pulse-modulated donut time profiles has been theoretically examined to suggest even lower powers for encoding, and we have designed a "STEN" ("SpatioTemporal Encoding Nanoscopy") microscopy scheme for the newer STED microscopes. The software to accomplish this is still in early development stages. this year, we rebuilt some legacy global code to adapt to this task and altered "IRF" (Instrument Response Function) reconvolution approaches. Additional programming resources will be diverted to it in 2026. This year we focused on the numerical methods to extract IRF information from fluorescent transients with unique numerical algorithms (using Laplace inversion). A further development based on photobleaching/shelving with multiple photons similar to the "PIM" methods of others is also in process, requiring our prototyping designs for a new multibeam - timed interleaved illumination instrument, previously delayed by budget and limited on-site presence. We have also acquired building blocks for a [Moku lock-in amplifier/NI-DAQ] based SE (Stimulated Emission) microscope, it will need new scanning software development in 2026; when complete, it should help us refine our STAQ method and choice of probes, by sensing currently confounding dark states of TAQ antennae. .