Selvin Paul R, Lougheed Tyler, Tonks Hoffman Melinda, Park Hyokeun, Balci Hamza, Blehm Benjamin H, Toprak Erdal
Biophysics Center and Physics Department, University of Illinois, Urbana, IL 61801, USA.
CSH Protoc. 2007 Oct 1;2007:pdb.top27. doi: 10.1101/pdb.top27.
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.
引言
单纳米精度荧光成像（FIONA）是一种将单个染料或一组染料定位到约1纳米精度范围内的技术。使用全内反射荧光显微镜、脱氧剂和高量子产率、低噪声探测器可实现这种高精度。FIONA有多种变体，包括一些分辨率优于10纳米的变体。其中一种变体是光漂白单分子高分辨率成像（SHRIMP），它只需要一种染料，例如两种绿色荧光蛋白（GFP）或两种罗丹明。然而，SHRIMP只能在静态系统上实现高分辨率。另一方面，单分子高分辨率共定位（SHREC）是一种FIONA变体，能够在动态系统上实现高分辨率。散焦取向和位置成像（DOPI）可确定三维取向，它本身或与FIONA结合使用，可将染料结合分子定位到几纳米范围内。最后，单纳米精度明场成像（bFIONA）实现了FIONA的时间和光谱定位，但使用的是明场显微镜，从而避免了荧光的使用。
