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Updated: Jun 19, 2026

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
16:10

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Published on: March 22, 2012

Protocol for total internal reflection fluorescence microscopy image preprocessing and radial analysis of LFA-1 in T

Lewis Murugu1, Johan Henriksson2, Hudson Pace3

  • 1Department of Molecular Biology, Umeå University, 90187 Umeå, Sweden.

STAR Protocols
|June 17, 2026
PubMed

Here, we present a protocol for total internal reflection fluorescence (TIRF) microscopy image preprocessing and radial analysis of lymphocyte function-associated antigen-1 (LFA-1) in T cell immune synapses. We describe steps for combining customizable Fiji/ImageJ macros for image background subtraction and segmentation with flexible R-based scripts for peripheral supramolecular activation cluster (pSMAC) center identification, center-based cropping, and radial analysis of synaptic LFA-1 in T cells. This workflow supports established analyses, including radial averaging/spinning and fluorescence intensity measurements across the synapse.

Keywords:
BioinformaticsCell BiologyImmunologyMicroscopyMolecular Biology

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Total Internal Reflection Fluorescence Microscopy01:05

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.