European Biophysics Journal

, Volume 38, Issue 5, pp 649–661

Convolution-based one and two component FRAP analysis: theory and application

  • Astrid Tannert
  • Sebastian Tannert
  • Steffen Burgold
  • Michael Schaefer
Original Paper

DOI: 10.1007/s00249-009-0422-4

Cite this article as:
Tannert, A., Tannert, S., Burgold, S. et al. Eur Biophys J (2009) 38: 649. doi:10.1007/s00249-009-0422-4

Abstract

The method of fluorescence redistribution after photobleaching (FRAP) is increasingly receiving interest in biological applications as it is nowadays used not only to determine mobility parameters per se, but to investigate dynamic changes in the concentration or distribution of diffusing molecules. Here, we develop a new simple convolution-based approach to analyze FRAP data using the whole image information. This method does not require information about the timing and localization of the bleaching event but uses the first image acquired directly after photobleaching to calculate the intensity distributions, instead. Changes in pools of molecules with different velocities, which are monitored by applying repetitive FRAP experiments within a single cell, can be analyzed by means of a global model by assuming two global diffusion coefficients with changing portions. We validate the approach by simulation and show that translocation of the YFP-fused PH-domain of phospholipase Cδ1 can be quantitatively monitored by FRAP analysis in a time-resolved manner. The new FRAP data analysis procedure may be applied to investigate signal transduction pathways using biosensors that change their mobility. An altered mobility in response to the activation of signaling cascades may result either from an altered size of the biosensor, e.g. due to multimerization processes or from translocation of the sensor to an environment with different viscosity.

Keywords

Fluorescence recovery after photobleaching Mobility-based biosensors Diffusion Multiple diffusion coefficients Global data analysis 

Abbreviations

AOI

Area of interest

AOTF

Acousto-optic tunable filter

CCD

Charge-coupled device

CLSM

Confocal laser scanning microscopy

FRAP

Fluorescence redistribution after photobleaching

HBS

HEPES-buffered solution

HEK

Human embryonic kidney

PH

Pleckstrin homology

PI(4,5)P2

Phosphatidylinositol-4,5-bisphosphate

PLCδ1(PH)

PH domain of phospholipase Cδ1

RSS

Residual sum of squares

TIR

Total internal reflection

YFP

Yellow fluorescent protein

Supplementary material

249_2009_422_MOESM1_ESM.pdf (39 kb)
Supplementary Figure (PDF 39 KB)
249_2009_422_MOESM2_ESM.pdf (34 kb)
Supplementary Table (PDF 34 KB)

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Astrid Tannert
    • 1
  • Sebastian Tannert
    • 2
  • Steffen Burgold
    • 3
    • 4
  • Michael Schaefer
    • 1
  1. 1.Rudolf-Boehm-Institut für Pharmakologie und ToxikologieUniversität LeipzigLeipzigGermany
  2. 2.PicoQuant GmbHBerlinGermany
  3. 3.Department of Molecular Pharmacology and Cell Biology, Neurowissenschaftliches ForschungszentrumCharité, Universitätsmedizin BerlinBerlinGermany
  4. 4.Institut für Neuropathologie, Zentrum für Neuropathologie und Prionforschung (ZNP)Ludwig-Maximilians-Universität MünchenMunichGermany