Validation of Normalizations, Scaling, and Photofading Corrections for FRAP Data Analysis.

Minchul Kang,Anne K Kenworthy,Manuel Andreani,Jörg Langowski

doi:10.1371/journal.pone.0127966

Minchul Kang, Anne K Kenworthy + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0127966

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Journal: PloS one	Publication Date: May 27, 2015
Citations: 28	License type: CC BY 4.0

Affiliation: St. Thomas University, Vanderbilt University

Abstract

Fluorescence Recovery After Photobleaching (FRAP) has been a versatile tool to study transport and reaction kinetics in live cells. Since the fluorescence data generated by fluorescence microscopy are in a relative scale, a wide variety of scalings and normalizations are used in quantitative FRAP analysis. Scaling and normalization are often required to account for inherent properties of diffusing biomolecules of interest or photochemical properties of the fluorescent tag such as mobile fraction or photofading during image acquisition. In some cases, scaling and normalization are also used for computational simplicity. However, to our best knowledge, the validity of those various forms of scaling and normalization has not been studied in a rigorous manner. In this study, we investigate the validity of various scalings and normalizations that have appeared in the literature to calculate mobile fractions and correct for photofading and assess their consistency with FRAP equations. As a test case, we consider linear or affine scaling of normal or anomalous diffusion FRAP equations in combination with scaling for immobile fractions. We also consider exponential scaling of either FRAP equations or FRAP data to correct for photofading. Using a combination of theoretical and experimental approaches, we show that compatible scaling schemes should be applied in the correct sequential order; otherwise, erroneous results may be obtained. We propose a hierarchical workflow to carry out FRAP data analysis and discuss the broader implications of our findings for FRAP data analysis using a variety of kinetic models.

Highlights

Over the past few decades, Fluorescence Recovery After Photobleaching (FRAP) has become an indispensable biophysical tool for tracking cellular organelles, proteins, and lipids in cells in a spatio-temporal manner [1,2,3,4,5,6,7]
Scalings that are widely used in FRAP analysis fall into three mathematical categories: (i) exponential scaling, (ii) affine scaling, and (iii) linear scaling
Since several of these scalings should be combined to account for various factors, verifying the compatibilities of these scalings is important to guarantee the accuracy of FRAP analysis

Summary

Introduction

Over the past few decades, Fluorescence Recovery After Photobleaching (FRAP) has become an indispensable biophysical tool for tracking cellular organelles, proteins, and lipids in cells in a spatio-temporal manner [1,2,3,4,5,6,7]. In diffusion FRAP, fluorescently tagged molecules in a small region of interest (ROI) are irreversibly photobleached using a high intensity laser source for a short period of time, and the exchange of fluorescent and photobleached molecules in and out of the bleached region is monitored using low intensity laser excitation to follow fluorescence recovery. In this process, the microscope system records the fluorescence intensity in a relative scale (for example 8 bit images: 0 * 256 scale) and generates a series of fluorescence images (Fig 1A). The immobile fraction is formally defined as 1−Mf, where Mf is given by

À F0 Fi À F0

Methods and materials

Fi À F0

Findings

Discussion