Continuous fluorescence depletion anisotropy (CFDA) measurement of protein rotation

B. George Barisas; Hongyan Zhang

doi:10.1117/12.426757

10 May 2001 Continuous fluorescence depletion anisotropy (CFDA) measurement of protein rotation

B. George Barisas, Hongyan Zhang

Proceedings Volume 4260, Optical Diagnostics of Living Cells IV; (2001) https://doi.org/10.1117/12.426757
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

Fluorescence depletion anisotropy (FDA) measurements of protein rotation combine the long lifetime of chromophore triplet states with the sensitivity of the fluorescence excitation and detection. Frequency domain (FDA) addresses certain practical limitations of time-domain procedures, such as the need for detector gating, but presents its own difficulties. We have combined time- and frequency-domain FDA methods into an efficient continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repetitive waveform and fluorescence signals are averaged over recurring waveform periods by a low rearm time signal averager. Methods for extracting triplet decay and absorption anisotropy decay kinetics from data traces generated by arbitrary waveforms have been developed. For a sample of eosin-BSA in 86% glycerol at 9 degree(s)C, rotational correlation times of 77 micrometers and 137 microsecond(s) , initial anisotropies of 0.109 and 0.125 and limiting anisotropies of 0.017 and 0.022, were obtained by CFDA and cuvet FDA, respectively. Differences in results apparently arise from the weighing of decay components in CFDA by triplet lifetimes of individual components.

Citation Download Citation

B. George Barisas and Hongyan Zhang "Continuous fluorescence depletion anisotropy (CFDA) measurement of protein rotation", Proc. SPIE 4260, Optical Diagnostics of Living Cells IV, (10 May 2001); https://doi.org/10.1117/12.426757

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available