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A method improving the accuracy of fluorescence recovery after photobleaching analysis.

Jönsson, Peter LU ; Jonsson, Magnus LU ; Tegenfeldt, Jonas LU and Höök, Fredrik LU (2008) In Biophysical Journal 95(11). p.5334-5348
Abstract
Fluorescence recovery after photobleaching, FRAP, has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under non-ideal experimental conditions the current methods of analysis still suffer from occasional problems, for example, when the signal to noise ratio is low, when there are temporal fluctuations in the illumination or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under non-ideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires... (More)
Fluorescence recovery after photobleaching, FRAP, has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under non-ideal experimental conditions the current methods of analysis still suffer from occasional problems, for example, when the signal to noise ratio is low, when there are temporal fluctuations in the illumination or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under non-ideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental FRAP data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within ~1 %, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with less than 10 % error. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biophysical Journal
volume
95
issue
11
pages
5334 - 5348
publisher
Cell Press
external identifiers
  • wos:000260999500033
  • pmid:18567628
  • scopus:58149340610
ISSN
1542-0086
DOI
10.1529/biophysj.108.134874
language
English
LU publication?
yes
id
e2bcf462-d63b-4d24-8c8b-ae944d1e1fb7 (old id 1168643)
date added to LUP
2009-03-02 15:07:35
date last changed
2017-10-01 03:40:40
@article{e2bcf462-d63b-4d24-8c8b-ae944d1e1fb7,
  abstract     = {Fluorescence recovery after photobleaching, FRAP, has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under non-ideal experimental conditions the current methods of analysis still suffer from occasional problems, for example, when the signal to noise ratio is low, when there are temporal fluctuations in the illumination or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under non-ideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental FRAP data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within ~1 %, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with less than 10 % error.},
  author       = {Jönsson, Peter and Jonsson, Magnus and Tegenfeldt, Jonas and Höök, Fredrik},
  issn         = {1542-0086},
  language     = {eng},
  number       = {11},
  pages        = {5334--5348},
  publisher    = {Cell Press},
  series       = {Biophysical Journal},
  title        = {A method improving the accuracy of fluorescence recovery after photobleaching analysis.},
  url          = {http://dx.doi.org/10.1529/biophysj.108.134874},
  volume       = {95},
  year         = {2008},
}