ArticleBiophysical journal2009
A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes.
Article in Biophysical journal, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 50 papers.
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Who cites it
50 citing papers in PubMed, 98 citations in OpenAlex.
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- Beyond analytic solution: Analysis of FRAP experiments by spatial simulation of the forward problem.Biophysical journal · 2023Article
- The Utility of Fluorescence Recovery after Photobleaching (FRAP) to Study the Plasma Membrane.Membranes · 2023Review
- Inferences from FRAP data are model dependent: A subdiffusive analysis.Biophysical journal · 2022Article
- Regression Analysis of Confocal FRAP and its Application to Diffusion in Membranes.Journal of fluorescence · 2022Article
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- Parameter estimation in fluorescence recovery after photobleaching: quantitative analysis of protein binding reactions and diffusion.Journal of mathematical biology · 2021Article
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- A Simple Derivation of Diffusion Fluorescence Correlation Spectroscopy Equations.Journal of fluorescence · 2020Article
- Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching.Biophysical journal · 2019Article
- Mechanical regulation of nucleocytoplasmic translocation in mesenchymal stem cells: characterization and methods for investigation.Biophysical reviews · 2019Review
- The Development and Enhancement of FRAP as a Key Tool for Investigating Protein Dynamics.Biophysical journal · 2018Review
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.
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