Fluorescence Correlation Spectroscopy Measurement Based on Fiber Optics for Biological Materials

Johtaro Yamamoto,Akira Sasaki

doi:10.3390/app11156744

Abstract

A robust fluorescence correlation spectroscopy system called fiber-optic based fluorescence correlation spectroscopy (FB-FCS) was developed; this system enables the measurement of diffusion dynamics and concentration of fluorescent molecules based on the principle of fluorescence correlation spectroscopy without any mechanical adjustment of the experimental setup. The system consisted of fiber optics and a water-immersion objective lens. The hydrodynamic diameters and concentrations of organic fluorescent dyes and fluorescently labeled proteins were successfully measured. Because of the fiber-optic-based setup, the FB-FCS system is compact and inexpensive. We expect FB-FCS to be suitable for use in laboratories, medical diagnosis, and environmental measurements.

Highlights

Fluorescence correlation spectroscopy (FCS) is a technique used to quantitatively measure the concentration and diffusion coefficient of fluorescently labeled molecules [1,2,3].Because FCS measurement can be performed inside living cells, FCS has been mainly used in biology to analyze changes in apparent molecular size due to intermolecular interactions and the expression of specific proteins, even in cells
The time In FCS, background intensity lowers the amplitude of the autocorrelation function (ACF), resulting in an overresolution of the correlator is variable in the range of 10-1,280 ns because it is constructed estimation of concentration
Correction from the excitation laser, which is reflected at the end In FCS, background intensity lowers the amplitude of the ACF, resulting in an overestimation of concentration

Summary

Introduction

Fluorescence correlation spectroscopy (FCS) is a technique used to quantitatively measure the concentration and diffusion coefficient of fluorescently labeled molecules [1,2,3].Because FCS measurement can be performed inside living cells, FCS has been mainly used in biology to analyze changes in apparent molecular size due to intermolecular interactions and the expression of specific proteins, even in cells. The diffusion coefficient of fluorescently labeled probe molecules can be changed by intermolecular interactions because the apparent size of the probe and interactant complex becomes larger than that of the probe. The dissociation constants of the GFP-GR homodimer were successfully determined based on the concentration and fluorescence brightness of the GFP-GR monomer and homodimer measured using FCS [5]. In FCS, background intensity lowers the amplitude of the ACF, resulting in an overresolution of the correlator is variable in the range of 10-1,280 ns because it is constructed estimation of concentration. In FCS, background intensity lowers the amplitude of the ACF, resulting in an overestimation of concentration. The background intensity of conventional FCS can be considered negligible in most cases because it is sufficiently lower than the fluorescence signal intensity

Methods

Results

Conclusion