ImpRes: A robust FRAP framework to quantify fast diffusion of cytoplasmic probes
Abstract
Diffusion within the cytoplasm is fundamental to numerous biological processes. Fluorescence recovery after photobleaching (FRAP) is one of the most common method for quantifying molecular diffusivity in living cells using standard laser scanning confocal microscopy (LSCM). However, accurately measuring fast cytoplasmic diffusion (typically >10 µm 2 /s) is challenging due to rapid recovery kinetics, weak signal-to-noise ratios, post-bleach signal artifacts, and spatial restrictions affecting normalization. While individual challenges have been addressed in specific contexts, a simple and robust framework to quantify cytoplasmic diffusivity remains elusive. Here, we present a FRAP methodology specifically designed to overcome these obstacles. By utilizing the Gaussian function – the impulse response (ImpRes) of the diffusion equation in an infinite medium – our approach leverages the full spatiotemporal dataset through a single-equation three-parameter fitting procedure, thus releasing restrictions to small regions of interest and arbitrary initial time-points. The methodology was validated on three datasets of increasing complexity: in silico simulated recovery profiles, in vitro data from FITC-dextran in glycerol solution, and live-cell imaging of cytoplasmic free-GFP. Systematic comparison with existing models demonstrates that the ImpRes approach significantly reduces sensitivity to noise and imperfect fluorescence normalization, while remaining robust against short-term biases, such as transient probe photo-activation. Given its robustness under realistic experimental conditions and its ease of implementation, the proposed FRAP methodology provides a reliable tool for quantitative cytoplasmic analysis.
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BibTeX
@misc{destrian2026impres,
title = {{ImpRes: A robust FRAP framework to quantify fast diffusion of cytoplasmic probes}},
author = {Olivier Destrian and René-Marc Mège and Benoît Goyeau and Morgan Chabanon},
howpublished = {HAL},
year = {2026},
month = aug,
doi = {10.64898/2026.08.14.744877},
url = {https://hal.science/hal-05797541v1},
note = {CC BY-NC 4.0},
}
RIS
TY - UNPB
TI - ImpRes: A robust FRAP framework to quantify fast diffusion of cytoplasmic probes
AU - Olivier Destrian
AU - René-Marc Mège
AU - Benoît Goyeau
AU - Morgan Chabanon
PY - 2026
DA - 2026/08/19
PB - HAL
DO - 10.64898/2026.08.14.744877
UR - https://hal.science/hal-05797541v1
AB - Diffusion within the cytoplasm is fundamental to numerous biological processes. Fluorescence recovery after photobleaching (FRAP) is one of the most common method for quantifying molecular diffusivity in living cells using standard laser scanning confocal microscopy (LSCM). However, accurately measuring fast cytoplasmic diffusion (typically >10 µm 2 /s) is challenging due to rapid recovery kinetics, weak signal-to-noise ratios, post-bleach signal artifacts, and spatial restrictions affecting normalization. While individual challenges have been addressed in specific contexts, a simple and robust framework to quantify cytoplasmic diffusivity remains elusive. Here, we present a FRAP methodology specifically designed to overcome these obstacles. By utilizing the Gaussian function – the impulse response (ImpRes) of the diffusion equation in an infinite medium – our approach leverages the full spatiotemporal dataset through a single-equation three-parameter fitting procedure, thus releasing restrictions to small regions of interest and arbitrary initial time-points. The methodology was validated on three datasets of increasing complexity: in silico simulated recovery profiles, in vitro data from FITC-dextran in glycerol solution, and live-cell imaging of cytoplasmic free-GFP. Systematic comparison with existing models demonstrates that the ImpRes approach significantly reduces sensitivity to noise and imperfect fluorescence normalization, while remaining robust against short-term biases, such as transient probe photo-activation. Given its robustness under realistic experimental conditions and its ease of implementation, the proposed FRAP methodology provides a reliable tool for quantitative cytoplasmic analysis.
ER -