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

Kang M, Andreani M, Kenworthy AK - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Science, Technology & Engineering Management, St. Thomas University, Miami Gardens, Florida, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Representative photofading rates obtained from the whole image fluorescence for a variety of different fluorescently tagged molecules.A photofading model, f(t) = e−κt was fitted to normalized whole image fluorescence data FData(t)/Fi averaged over multiple data sets (n = 10) for each of the indicated fluorescent proteins or fluorescent lipid probes. (A–B) Best fitting photofading model applied to whole image fluorescence. Solid lines show the best fitting curves. (C) Photofading rate constants obtained from the fits shown in A and B are shown in descending order. Error bar represents the standard deviation (n = 14).
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pone.0127966.g002: Representative photofading rates obtained from the whole image fluorescence for a variety of different fluorescently tagged molecules.A photofading model, f(t) = e−κt was fitted to normalized whole image fluorescence data FData(t)/Fi averaged over multiple data sets (n = 10) for each of the indicated fluorescent proteins or fluorescent lipid probes. (A–B) Best fitting photofading model applied to whole image fluorescence. Solid lines show the best fitting curves. (C) Photofading rate constants obtained from the fits shown in A and B are shown in descending order. Error bar represents the standard deviation (n = 14).

Mentions: To estimate the order of photofading rates (κ) observed under typical experimental conditions, we analyzed FRAP data for a series of proteins and lipids previously collected using confocal FRAP [2]. Fluorescently tagged molecules considered were Alexa488-CTxB, YFP-GLGLPI, EGFP, mEmerald-Cav1, Flot-RFP, and DiIC16, which have diffusion coefficients ranging from 0.1 ˜ 40 μm2/s. These FRAP datasets were obtained using different filter sets and laser excitation optimized for each fluorescent molecule, and also were obtained using different time intervals to allow for differences in the kinetics of recovery. In all these cases, photofading during image acquisition could be well described by an exponential decay photofading model with κ values between 10−3 ∼ 10−2/s (Eq 28) as shown in Fig 2A and 2B.


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

Kang M, Andreani M, Kenworthy AK - PLoS ONE (2015)

Representative photofading rates obtained from the whole image fluorescence for a variety of different fluorescently tagged molecules.A photofading model, f(t) = e−κt was fitted to normalized whole image fluorescence data FData(t)/Fi averaged over multiple data sets (n = 10) for each of the indicated fluorescent proteins or fluorescent lipid probes. (A–B) Best fitting photofading model applied to whole image fluorescence. Solid lines show the best fitting curves. (C) Photofading rate constants obtained from the fits shown in A and B are shown in descending order. Error bar represents the standard deviation (n = 14).
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Related In: Results  -  Collection

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pone.0127966.g002: Representative photofading rates obtained from the whole image fluorescence for a variety of different fluorescently tagged molecules.A photofading model, f(t) = e−κt was fitted to normalized whole image fluorescence data FData(t)/Fi averaged over multiple data sets (n = 10) for each of the indicated fluorescent proteins or fluorescent lipid probes. (A–B) Best fitting photofading model applied to whole image fluorescence. Solid lines show the best fitting curves. (C) Photofading rate constants obtained from the fits shown in A and B are shown in descending order. Error bar represents the standard deviation (n = 14).
Mentions: To estimate the order of photofading rates (κ) observed under typical experimental conditions, we analyzed FRAP data for a series of proteins and lipids previously collected using confocal FRAP [2]. Fluorescently tagged molecules considered were Alexa488-CTxB, YFP-GLGLPI, EGFP, mEmerald-Cav1, Flot-RFP, and DiIC16, which have diffusion coefficients ranging from 0.1 ˜ 40 μm2/s. These FRAP datasets were obtained using different filter sets and laser excitation optimized for each fluorescent molecule, and also were obtained using different time intervals to allow for differences in the kinetics of recovery. In all these cases, photofading during image acquisition could be well described by an exponential decay photofading model with κ values between 10−3 ∼ 10−2/s (Eq 28) as shown in Fig 2A and 2B.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Science, Technology & Engineering Management, St. Thomas University, Miami Gardens, Florida, USA.

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.

No MeSH data available.


Related in: MedlinePlus