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Related Experiment Video

Updated: May 10, 2026

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

Published on: April 25, 2025

Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments.

Lior Turgeman¹, Dror Fixler

¹Faculty of Engineering and Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Ramat Gan, 52900, Israel ; Tur.Lior@gmail.com.

Biomedical Optics Express

|June 14, 2013

Summary

This summary is machine-generated.

This study analyzes fluorescence intensity fluctuations to measure molecular rotation. The method improves signal-to-noise ratio for accurately determining rotational correlation times in various media.

Keywords:

(000.5490) Probability theory, stochastic processes, and statistics (110.4280) Noise in imaging systems (180.2520) Fluorescence microscopy (260.5430) Polarization

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Area of Science:

Physical Chemistry
Spectroscopy
Biophysics

Background:

Fluorescence fluctuation polarization sensitive experiments face limitations in detecting rotational timescales.
Fluorescence Correlation Spectroscopy (FCS) is commonly used to overcome these limitations.

Purpose of the Study:

To analyze the variance of time-averaged fluorescence intensity in the short time limit.
To develop a method for extracting rotational correlation times of fluorescent molecules, especially when these times are faster than system temporal resolution.

Main Methods:

Analysis of the variance of time-averaged fluorescence intensity from the second moment of measured intensity.
Ensemble averaging over time-averaged trajectories to construct the time-averaged intensity distribution.
Application to fluorescein molecules in media of varying viscosities.

Main Results:

The proposed method effectively extracts rotational correlation times in the anti-bunching time range (1-10 ns).
Improved signal-to-noise ratio was achieved through ensemble averaging.
The technique is applicable to measuring molecular dynamics in different viscosity environments.

Conclusions:

The developed method overcomes limitations in measuring fast rotational dynamics using fluorescence fluctuation polarization.
Accurate determination of rotational correlation times is possible even when they are below the system's temporal resolution.
This approach enhances the capability of fluorescence-based techniques for studying molecular motion.