MOTIVATION: Fluorescence recovery after photobleaching (FRAP) is a functional live cell imaging technique that permits the exploration of protein dynamics in living cells. To extract kinetic parameters from FRAP data, a number of analytical models have been developed. Simplifications are inherent in these models, which may lead to inexhaustive or inaccurate exploitation of the experimental data. An appealing alternative is offered by the simulation of biological processes in realistic environments at a particle level. However, inference of kinetic parameters using simulation-based models is still limited. RESULTS: We introduce and demonstrate a new method for the inference of kinetic parameter values from FRAP data. A small number of in silico FRAP experiments is used to construct a mapping from FRAP recovery curves to the parameters of the underlying protein kinetics. Parameter estimates from experimental data can then be computed by applying the mapping to the observed recovery curves. A bootstrap process is used to investigate identifiability of the physical parameters and determine confidence regions for their estimates. Our method circumvents the computational burden of seeking the best-fitting parameters via iterative simulation. After validation on synthetic data, the method is applied to the analysis of the nuclear proteins Cdt1, PCNA and GFPnls. Parameter estimation results from several experimental samples are in accordance with previous findings, but also allow us to discuss identifiability issues as well as cell-to-cell variability of the protein kinetics. IMPLEMENTATION: All methods were implemented in MATLAB R2011b. Monte Carlo simulations were run on the HPC cluster Brutus of ETH Zurich. CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SUMMARY: We present easyFRAP, a versatile tool that assists quantitative and qualitative analysis of fluorescence recovery after photobleaching (FRAP) data. The user can handle simultaneously large data sets of raw data, visualize fluorescence recovery curves, exclude low quality data, perform data normalization, extract quantitative parameters, perform batch analysis and save the resulting data and figures for further use. Our tool is implemented as a single-screen Graphical User Interface (GUI) and is highly interactive, as it permits parameterization and visual data quality assessment at various points during the analysis. AVAILABILITY: easyFRAP is free software, available under the General Public License (GPL). Executable and source files, supplementary material and sample data sets can be downloaded at: ccl.med.upatras.gr/easyfrap.html.