TY - CHAP
T1 - Fluorescence Imaging with One Nanometer Accuracy
T2 - In Vitro and In Vivo Studies of Molecular Motors
AU - Hoffman, Melinda Tonks
AU - Sheung, Janet
AU - Selvin, Paul R.
N1 - Publisher Copyright:
© 2011, Springer Science+Business Media, LLC.
PY - 2011
Y1 - 2011
N2 - Traditional microscopy techniques are limited by the wave-like characteristics of light, which dictate that about 250 nm (or roughly half the wavelength of the light) is the smallest distance by which two identical objects can be separated while still being able to distinguish between them. Since most biological molecules are much smaller than this limit, traditional light microscopes are generally not sufficient for single-molecule biological studies. Fluorescence Imaging with One Nanometer Accuracy (FIONA) is a technique that makes possible localization of an object to approximately one nanometer. The FIONA technique is simple in concept; it is built upon the idea that, if enough photons are collected, one can find the exact center of a fluorophore’s emission to within a single nanometer and track its motion with a very high level of precision. The center can be localized to approximately (λ/2)/Ö—N, where λ is the wavelength of the light and N is the number of photons collected. When N = 10,000, FIONA achieves an accuracy of 1–2 nm, assuming the background is sufficiently low. FIONA, thus, works best with the use of high-quality dyes and fluorescence stabilization buffers, sensitive detection methods, and special microscopy techniques to reduce background fluorescence. FIONA is particularly well suited to the study of molecular motors, which are enzymes that couple ATP hydrolysis to conformational change and motion. In this chapter, we discuss the practical application of FIONA to molecular motors or other enzymes in biological systems.
AB - Traditional microscopy techniques are limited by the wave-like characteristics of light, which dictate that about 250 nm (or roughly half the wavelength of the light) is the smallest distance by which two identical objects can be separated while still being able to distinguish between them. Since most biological molecules are much smaller than this limit, traditional light microscopes are generally not sufficient for single-molecule biological studies. Fluorescence Imaging with One Nanometer Accuracy (FIONA) is a technique that makes possible localization of an object to approximately one nanometer. The FIONA technique is simple in concept; it is built upon the idea that, if enough photons are collected, one can find the exact center of a fluorophore’s emission to within a single nanometer and track its motion with a very high level of precision. The center can be localized to approximately (λ/2)/Ö—N, where λ is the wavelength of the light and N is the number of photons collected. When N = 10,000, FIONA achieves an accuracy of 1–2 nm, assuming the background is sufficiently low. FIONA, thus, works best with the use of high-quality dyes and fluorescence stabilization buffers, sensitive detection methods, and special microscopy techniques to reduce background fluorescence. FIONA is particularly well suited to the study of molecular motors, which are enzymes that couple ATP hydrolysis to conformational change and motion. In this chapter, we discuss the practical application of FIONA to molecular motors or other enzymes in biological systems.
KW - FIONA
KW - Molecular motors
KW - Single-molecule tracking
KW - TIRF microscopy
UR - http://www.scopus.com/inward/record.url?scp=80054723783&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80054723783&partnerID=8YFLogxK
U2 - 10.1007/978-1-61779-261-8_4
DO - 10.1007/978-1-61779-261-8_4
M3 - Chapter
C2 - 21809199
AN - SCOPUS:80054723783
SN - 9781617792601
T3 - Methods in Molecular Biology
SP - 33
EP - 56
BT - Single Molecule Enzymology
PB - Humana Press Inc.
ER -