Equipment Setup for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This article describes procedures for setting up the equipment necessary for FIONA and achieving total internal reflection (TIR).

Data Analysis Methods for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. Data analysis of a FIONA experiment requires the use of several software programs. Data must be acquired and exported into a proper format before analysis can take place. This article describes various options for data analysis.

Constructing Sample Chambers for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides details on constructing an inexpensive sample chamber for use in single-molecule FIONA experiments and two methods for cleaning slides and coverslips.

Fluorescence Imaging with One-Nanometer Accuracy (FIONA) of Cy3-DNA under Deoxygenation Conditions.

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides the steps necessary to run a control experiment, using DNA labeled with Cy3, to assess the efficacy of the FIONA setup and the level of attainable resolution. The Cy3-DNA is immobilized on a coverslip and imaged under deoxygenation conditions. It is important that the fluorophores remain photostable throughout the experiment. This requires an oxygen-scavenging system (e.g., glucose oxidase and catalase) in the medium.

Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

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.