Saturation effects in fluorescence correlation spectroscopy

Lloyd M. Davis; Guoqing Shen; David A. Ball

doi:10.1117/12.591099

30 March 2005 Saturation effects in fluorescence correlation spectroscopy

Lloyd M. Davis, Guoqing Shen, David A. Ball

Proceedings Volume 5700, Multiphoton Microscopy in the Biomedical Sciences V; (2005) https://doi.org/10.1117/12.591099
Event: SPIE BiOS, 2005, San Jose, CA, United States

Abstract

Fluorescence correlation spectroscopy (FCS) could provide a more useful tool for intracellular studies and biological sample characterization if measurement times could be reduced. While an increase in laser power can enable an autocorrelation function (ACF) with adequate signal-to-noise to be acquired within a shorter measurement time, excitation saturation then leads to distortion of the ACF and systematic errors in the measurement results. An empirical method for achieving reduced systematic errors by employing a fitting function with an additional adjustable parameter has been previously introduced for two-photon FCS. Here we provide a unified physical explanation of excitation saturation effects for the three cases of continuous-wave, pulsed one-photon excitation, and two-photon excitation FCS. When the time between laser pulses is longer than the fluorescence lifetime, the signal rate at which excitation saturation occurs is lower for pulsed excitation than for cw excitation, and due to the disparate timescales of the photophysical processes following excitation, it is lower still for two-photon excitation. We use a single-molecule description of FCS to obtain improved analytical ACF fitting functions for the three cases. The fitting functions more accurately account for saturation effects than those previously employed without the need for an additional empirical parameter. Use of these fitting functions removes systematic errors and enables measurements to be acquired more quickly by use of higher laser powers. Increase of background, triplet photophysics, and the cases of scanning FCS and fluorescence cross-correlation spectroscopy are also discussed. Experimental results acquired with a custom built apparatus are presented.

Citation Download Citation

Lloyd M. Davis, Guoqing Shen, and David A. Ball "Saturation effects in fluorescence correlation spectroscopy", Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); https://doi.org/10.1117/12.591099

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