Fluorescence Imaging with One Nanometer Accuracy (FIONA) is a method for localizing single fluorophores (or single groups of fluorophores) in the X-Y plane with high spatial resolution. In the October issue of CSH Protocols, Paul Selvin and colleagues provide a series of articles explaining the concepts behind FIONA (and several related techniques) as well as protocols for FIONA equipment set-up, chamber construction, imaging and data analysis.

Fluorescence Imaging with One-Nanometer Accuracy (FIONA) is a Topic Introduction that covers the concepts behind FIONA, as well as its variants, including SHRIMP (Single-molecule High-Resolution IMaging with Photobleaching), SHREC (Single-molecule High-Resolution Colocalization), DOPI (Defocused Orientation and Positional Imaging) and bFIONA (Bright-Field Imaging with One-Nanometer Accuracy). These variants allow you to resolve and measure two dyes separated by distances of only 10 nm (standard imaging methods have a limit closer to 250 nm).

Equipment Setup for Fluorescence Imaging with One-Nanometer Accuracy (FIONA) details how to set up a system for FIONA as well as how to achieve Total Internal Reflection (TIR).

Constructing Sample Chambers for Fluorescence Imaging with One-Nanometer Accuracy (FIONA) gives instructions on constructing an inexpensive sample chamber and two methods for preparing slides.

A protocol is given for Fluorescence Imaging with One-Nanometer Accuracy (FIONA) of Cy3-DNA under Deoxygenation Conditions and Data Analysis Methods are discussed as well.

The authors have used these techniques in detailed studies of various molecules, including kinesin, myosin V and myosin VI, showing that they walk in a hand-over-hand mechanism, rather than in an inchworm fashion. The lead author on these studies, Ahmet Yildiz, won a GE & Science Award for Young Life Scientists for an essay describing the work.