Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

Total Internal Reflection Fluorescence Microscopy

Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Unsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cells.

Methods and applications in fluorescence·2026

Same author

Resolving fluorescently labeled species using highly multiplexed spectral FLIM.

Scientific reports·2026

Same author

A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development.

Methods and applications in fluorescence·2026

Same author

Expanded applications of bioluminescence microscopy with phasor analysis.

Cell reports methods·2026

Same author

Bioluminescent Probes for Multiplexed RNA Imaging.

Journal of the American Chemical Society·2026

Same author

Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer.

bioRxiv : the preprint server for biology·2026

Same journal

An assay to quantify sexual commitment and stage conversion in the human malaria parasite Plasmodium falciparum.

Nature protocols·2026

Same journal

Author Correction: Direct inoculation of bioreactor-controlled stirred suspension culture with cryopreserved human pluripotent stem cells.

Nature protocols·2026

Same journal

High-throughput measurements of protein domain functions using magnetic separation.

Nature protocols·2026

Same journal

Inducing physiological polarity and performing gene editing using CRISPR-Cas9 in human trophoblast organoids.

Nature protocols·2026

Same journal

Photocatalytic low-temperature defluorination of PTFE.

Nature protocols·2026

Same journal

Multimodal imaging and quantification of lanthanide chelate-labeled micro- and nanoplastics in plants.

Nature protocols·2026

See all related articles

Search research articles

Related Experiment Video

Updated: Jun 7, 2026

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Published on: August 2, 2018

Raster image correlation spectroscopy in live cells.

Molly J Rossow¹, Jennifer M Sasaki, Michelle A Digman

¹Department of Biomedical Engineering, University of California Irvine, Irvine, California, USA.

Nature Protocols

|October 30, 2010

Summary

This summary is machine-generated.

Raster image correlation spectroscopy (RICS) is a noninvasive live-cell imaging technique. This method quantifies molecular concentration and diffusion, enabling indirect detection of molecular binding events.

More Related Videos

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Published on: December 17, 2015

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Published on: December 11, 2021

Related Experiment Videos

Last Updated: Jun 7, 2026

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Published on: August 2, 2018

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Published on: December 17, 2015

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Published on: December 11, 2021

Area of Science:

Cell biology
Biophysics
Microscopy

Background:

Raster Image Correlation Spectroscopy (RICS) is a powerful technique for analyzing molecular dynamics in live cells.
It offers a unique niche by providing spatial information and functioning with conventional confocal microscopes.

Purpose of the Study:

To detail a protocol for performing Raster Image Correlation Spectroscopy (RICS) using specific microscopy and software.
To demonstrate the application of RICS for quantifying molecular diffusion and concentration in live cells.

Main Methods:

Acquisition of raster scanned images using an Olympus FluoView FV1000 confocal laser scanning microscope and Olympus FluoView software.
Analysis of acquired data using SimFCS software for RICS measurements.
Calibration of the focal volume with a known diffusion coefficient standard.

Main Results:

The protocol enables the measurement of diffusion coefficients for cytosolic enhanced green fluorescent protein (EGFP) and EGFP-paxillin.
RICS allows for the quantification of molecular concentration and diffusion coefficients in live cells.
The entire RICS procedure, including calibration, can be completed in approximately 2 hours.

Conclusions:

RICS is a versatile, noninvasive technique for live-cell analysis of molecular concentration and diffusion.
The described protocol provides a reproducible method for applying RICS with standard confocal microscopy.
RICS offers a unique capability to measure a broad range of diffusion coefficients not accessible by other optical correlation techniques.