A Method Improving the Accuracy of Fluorescence Recovery after Photobleaching Analysis
2008 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 95, no 11, 5334-5348 p.Article in journal (Refereed) Published
Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/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 nonideal 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 fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within similar to 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.
Place, publisher, year, edition, pages
Biophysical Society , 2008. Vol. 95, no 11, 5334-5348 p.
Computer Vision and Robotics (Autonomous Systems)
IdentifiersURN: urn:nbn:se:liu:diva-118829DOI: 10.1529/biophysj.108.134874ISI: 000260999500033PubMedID: 18567628OAI: oai:DiVA.org:liu-118829DiVA: diva2:817137
Funding Agencies|Swedish Research Council for Engineering Sciences [2005-3140]2015-06-042015-06-042015-06-18