Fluorescence Imaging with One-Nanometer Accuracy (FIONA): Figure 1.

Abstract

INTRODUCTIONFluorescence imaging with one-nanometer accuracy (FIONA) is a technique for localizing a single dye, or a single group of dyes, to within ~1-nm accuracy. This high degree of precision is achieved using total internal reflection fluorescence microscopy, deoxygenation agents, and a high quantum yield, low-noise detector. There are several variations of FIONA, including some capable of better than 10-nm resolution. One such variant is single-molecule high-resolution imaging with photobleaching (SHRIMP), which requires only one type of dye, e.g., two green fluorescent proteins (GFPs), or two rhodamines. However, SHRIMP can only achieve high resolution on static systems. Single-molecule high-resolution colocalization (SHREC), on the other hand, is a FIONA variant that is capable of high resolution with dynamic systems. Defocused orientation and positional imaging (DOPI) enables the three-dimensional orientation to be determined, and either by itself or in combination with FIONA can localize the dye-bound molecules to within a few nanometers. Finally, bright-field imaging with one-nanometer accuracy (bFIONA) achieves the temporal and spectral localization of FIONA but with bright-field microscopy, thus avoiding the use of fluorescence.