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A Discontinuous Galerkin Model for Fluorescence Loss in Photobleaching.

ABSTRACT: Fluorescence loss in photobleaching (FLIP) is a modern microscopy method for visualization of transport processes in living cells. This paper presents the simulation of FLIP sequences based on a calibrated reaction-diffusion system defined on segmented cell images. By the use of a discontinuous Galerkin method, the computational complexity is drastically reduced compared to continuous Galerkin methods. Using this approach on green fluorescent protein (GFP), we can determine its intracellular diffusion constant, the strength of localized hindrance to diffusion as well as the permeability of the nuclear membrane for GFP passage, directly from the FLIP image series. Thus, we present for the first time, to our knowledge, a quantitative computational FLIP method for inferring several molecular transport parameters in parallel from FLIP image data acquired at commercial microscope systems.

SUBMITTER: Hansen CV

PROVIDER: S-EPMC5780497 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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A Discontinuous Galerkin Model for Fluorescence Loss in Photobleaching.

Hansen Christian V CV Schroll Hans J HJ Wüstner Daniel D

Scientific reports 20180123 1

Fluorescence loss in photobleaching (FLIP) is a modern microscopy method for visualization of transport processes in living cells. This paper presents the simulation of FLIP sequences based on a calibrated reaction-diffusion system defined on segmented cell images. By the use of a discontinuous Galerkin method, the computational complexity is drastically reduced compared to continuous Galerkin methods. Using this approach on green fluorescent protein (GFP), we can determine its intracellular dif ...[more]

PMID: 29362364

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Project description:BackgroundIntracellular phase separation and aggregation of proteins with extended poly-glutamine (polyQ) stretches are hallmarks of various age-associated neurodegenerative diseases. Progress in our understanding of such processes heavily relies on quantitative fluorescence imaging of suitably tagged proteins. Fluorescence loss in photobleaching (FLIP) is particularly well-suited to study the dynamics of protein aggregation in cellular models of Chorea Huntington and other polyQ diseases, as FLIP gives access to the full spatio-temporal profile of intensity changes in the cell geometry. In contrast to other methods, also dim aggregates become visible during time evolution of fluorescence loss in cellular compartments. However, methods for computational analysis of FLIP data are sparse, and transport models for estimation of transport and diffusion parameters from experimental FLIP sequences are missing.ResultsIn this paper, we present a computational method for analysis of FLIP imaging experiments of intracellular polyglutamine protein aggregates also called inclusion bodies (IBs). By this method, we can determine the diffusion constant and nuclear membrane transport coefficients of polyQ proteins as well as the exchange rates between aggregates and the cytoplasm. Our method is based on a reaction-diffusion multi-compartment model defined on a mesh obtained by segmentation of the cell images from the FLIP sequence. The discontinuous Galerkin (DG) method is used for numerical implementation of our model in FEniCS, which greatly reduces the computing time. The method is applied to representative experimental FLIP sequences, and consistent estimates of all transport parameters are obtained.ConclusionsBy directly estimating the transport parameters from live-cell image sequences using our new computational FLIP approach surprisingly fast exchange dynamics of mutant Huntingtin between cytoplasm and dim IBs could be revealed. This is likely relevant also for other polyQ diseases. Thus, our method allows for quantifying protein dynamics at different stages of the protein aggregation process in cellular models of neurodegeneration.

Project description:BackgroundFluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings.ResultsWe present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction-diffusion simulations, that the StrExp function can describe both, binding/barrier-limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB's). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB's to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments.ConclusionsWe present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport.

Dataset Information

A Discontinuous Galerkin Model for Fluorescence Loss in Photobleaching.

Publications

A Discontinuous Galerkin Model for Fluorescence Loss in Photobleaching.

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