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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Protein Diffusion in the Membrane01:24

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Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...
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Related Experiment Video

Updated: Dec 12, 2025

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer
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Anomalous Diffusion Characterization by Fourier Transform-FRAP with Patterned Illumination.

Andreas C Geiger1, Casey J Smith1, Nita Takanti1

  • 1Department of Chemistry, Purdue University, West Lafayette, Indiana.

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|August 11, 2020
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Fourier transform fluorescence recovery after photobleaching (FT-FRAP) offers a robust method for quantifying diffusion. This technique enhances signal-to-noise and simplifies analysis for both normal and anomalous diffusion studies.

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Area of Science:

  • Biophysics
  • Materials Science
  • Chemical Engineering

Background:

  • Diffusion characterization is crucial in biology, pharmacology, and food science.
  • Conventional fluorescence recovery after photobleaching (FRAP) faces limitations like signal-to-noise issues and sample heterogeneity bias.
  • Conventional FRAP is incompatible with multiphoton excitation due to local heating.

Purpose of the Study:

  • To theorize and demonstrate Fourier transform fluorescence recovery after photobleaching (FT-FRAP) for quantitative diffusion evaluation.
  • To overcome limitations of conventional FRAP, including signal-to-noise, mathematical complexity, and multiphoton excitation compatibility.
  • To enable simultaneous measurement of diffusion at multiple length scales and characterize anomalous diffusion.

Main Methods:

  • Utilized patterned illumination for photobleaching via two-photon excitation with a custom nonlinear optical beam-scanning microscope.
  • Performed measurements in the spatial Fourier domain to eliminate dependence on the photobleach profile and reduce bias.
  • Analyzed multiple spatial harmonics of the photobleaching pattern for diffusion measurement at various length scales.

Main Results:

  • FT-FRAP demonstrated significant improvements in signal-to-noise, mathematical simplicity, and multiphoton compatibility.
  • Normal diffusion showed a single-exponential decay in the spatial Fourier domain, aligning with theoretical predictions.
  • Anomalous diffusion was characterized by nonlinear fitting to multiple spatial harmonics, with constrained fits improving parameter confidence.

Conclusions:

  • FT-FRAP provides a superior method for quantitative diffusion analysis compared to conventional FRAP.
  • The technique accurately characterizes both normal and anomalous diffusion across multiple length scales.
  • FT-FRAP's phase analysis offers insights into sample flow and translation.