Dual-color two-photon fluorescence correlation spectroscopy

Keith M. Berland

doi:10.1117/12.424564

24 April 2001 Dual-color two-photon fluorescence correlation spectroscopy

Keith M. Berland

Proceedings Volume 4262, Multiphoton Microscopy in the Biomedical Sciences; (2001) https://doi.org/10.1117/12.424564
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a research tool in biological and biophysical research. Under favorable conditions, FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. However, interpretation of FCS data quickly becomes complicated as the heterogeneity of a molecular system increases, as well as when there is significant non-stationery fluorescence background (e.g. intracellular autofluorescence). Use of multi-parameter correlation measurements is one promising approach that can improve the fidelity of FCS measurements in complex systems. In particular, the use of dual-color fluorescence assays, in which different interacting molecular species are labeled with unique fluorescent indicators, can "tune" the sensitivity of FCS measurements in favor of particular molecular species of interest, while simultaneously minimizing the contribution of other molecular species to the overall fluorescence correlation signal. Here we introduce the combined application of two-photon fluorescence excitation and dual-color cross-correlation analysis for detecting molecular interactions in solution. The use of two-photon excitation is particularly advantageous for dual-color FCS applications due to the uncomplicated optical alignment and the superior capabilities for intracellular applications. The theory of two-photon dual-color FCS is introduced, and initial results quantifying hybridization reactions between three independent single stranded DNA molecules are presented.

Citation Download Citation

Keith M. Berland "Dual-color two-photon fluorescence correlation spectroscopy", Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); https://doi.org/10.1117/12.424564

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