Fluorescence Recovery After Photobleaching Method Research Articles

Diffusivity of Human Cartilage Endplates in Healthy and Degenerated Intervertebral Disks.

The cartilage endplates (CEPs) on the superior and inferior surfaces of the intervertebral disk (IVD), are the primary nutrient transport pathways between the disk and the vertebral body. Passive diffusion is responsible for transporting small nutrient and metabolite molecules through the avascular CEPs. The baseline solute diffusivities in healthy CEPs have been previously studied, however alterations in CEP diffusion associated with IVD degeneration remain unclear. This study aimed to quantitatively compare the solute diffusion in healthy and degenerated human CEPs using a fluorescence recovery after photobleaching (FRAP) approach. Seven healthy CEPs and 22 degenerated CEPs were collected from five fresh-frozen human cadaveric spines and 17 patients undergoing spine fusion surgery, respectively. The sodium fluorescein diffusivities in CEP radial and vertical directions were measured using the FRAP method. The CEP calcification level was evaluated by measuring the average X-ray attenuation. No difference was found in solute diffusivities between radial and axial directions in healthy and degenerated CEPs. Compared to healthy CEPs, the average solute diffusivity was 44% lower in degenerated CEPs (Healthy: 29.07 μm2/s (CI: 23.96-33.62 μm2/s); degenerated: 16.32 μm2/s (CI: 13.84-18.84 μm2/s), p < 0.001). The average solute diffusivity had an inverse relationship with the degree of CEP calcification as determined by the normalized X-ray attenuation values (ß = -22.19, R2 = 0.633; p < 0.001). This study suggests that solute diffusion through the disk and vertebral body interface is significantly hindered by CEP calcification, providing clues to help further understand the mechanism of IVD degeneration.

Fluorescence recovery after photobleaching: analyses of cyanobacterial phycobilisomes reveal intrinsic fluorescence recovery.

Fluorescence recovery after photobleaching (FRAP) has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997. Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome (PBS) diffusion on the surface of the thylakoid membrane. The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion. However, in red algae, another PBS-containing group, the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS, which raises questions concerning the role of FRAP in red algal PBS. Therefore, it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria. In the present study, four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics. Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains, in which the contribution of PBS diffusion to the fluorescence recovery was not possible. Moreover, the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery. These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.

Closed-form formulas vs. PDE based numerical solution for the FRAP data processing: Theoretical and practical comparison

Fluorescence recovery after photobleaching (FRAP) is a widely used method to analyze (usually using fluorescence microscopy) the mobility of either fluorescently tagged or autofluorescent (e.g., photosynthetic) proteins in living cells. The FRAP method resides in imaging the recovery of fluorescence intensity over time in a region of interest previously bleached by a high-intensity laser pulse. While the basic principles of FRAP are simple and the experimental setup is usually fixed, quantitative FRAP data analysis is not well developed. Different models and numerical procedures are used for the underlying model parameter estimation without knowledge of how robust the methods are, i.e., the parameter inference step is not currently well established.In this paper we rigorously formulate the inverse problem of model parameter estimation (including the sensitivity analysis), making possible the comparison of different FRAP parameter inference methods. Then, in a study on simulated data, we focus on how three different methods for inference influence the error in parameter estimation. We demonstrate both theoretically and empirically that our new method based on a solution of a general initial–boundary value problem for the Fick diffusion partial differential equation exhibits less bias and narrower confidence intervals of the estimated diffusion parameter, than two closed formula methods.

RNA imaging: seeing is believing.

RNA imaging: seeing is believing.

Single- and two-photon fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.

Measuring F-actin properties in dendritic spines.

During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines.

Parameters that affect estimation of nucleolar proteins’ mobility in living cells by the FRAP method with the example of protein fibrillarin

The fluorescence recovery after photobleaching (FRAP) method, in combination with confocal laser scanning microscopy, represents one of the basic approaches to studying the properties of proteins in living mammalian cells. However, the data of different authors on the dynamic properties of the same protein and even in cells of the same type can differ greatly. Until now, the reasons for such discrepancies have not been specifically analyzed. In the present work, using the example of nucleolar protein fibrillarin fused with EGFP, we studied the effect of the area of the irradiated region (the region of interest (ROI)) and temperature conditions of experiments on the main dynamic characteristics of the protein—the portion of the mobile fraction of protein and the half-recovery time of fluorescence after photobleaching (t 1/2). The obtained results have shown that both parameters affect markedly the estimation of the fibrillarin-EGFP mobility in HeLa cells. It was concluded that, in FRAP experiments the ROI area can be standardized and, where possible, minimized. In addition, when analyzing the dynamic characteristics of the nucleolar proteins, which participate in the temperature-dependent enzymatic reactions, it is necessary to maintain standard temperature conditions.

Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules

This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies. Initially, we discuss the lateral diffusion of membrane proteins, as measured by FRAP. Then, we talk about the use of FRAP to probe interactions between membrane proteins by obtaining fundamental information such as geometry and stoichiometry of the interacting complex. Afterwards, we discuss some applications of FRAP at the cellular level as well as the level of organisms. We conclude by comparing diffusion coefficients obtained by FRAP and several other alternative methods.

Conditions for using FRAP as a quantitative technique—Influence of the bleaching protocol

Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent way of data analysis.

Enlarged extracellular space of aquaporin-4–deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers

Aquaporin-4 (AQP4) water channels expressed on glia have been implicated in maintaining the volume of extracellular space (ECS). A previous diffusion study employing small cation tetramethylammonium and a real-time iontophoretic (RTI) method demonstrated an increase of about 25% in the ECS volume fraction (α) in the neocortex of AQP4 −/− mice compared to AQP4 +/+ mice but no change in the hindrance imposed to diffusing molecules (tortuosity λ). In contrast, other diffusion studies employing large molecules (dextran polymers) and a fluorescence recovery after photobleaching (FRAP) method measured a decrease of about 10%–20% in λ in the neocortex of AQP4 −/− mice. These conflicting findings on λ would imply that large molecules diffuse more readily in the enlarged ECS of AQP4 −/− mice than in wild type but small molecules do not. To test this hypothesis, we used integrative optical imaging (IOI) to measure tortuosity with a small Alexa Fluor 488 (molecular weight [MW] 547, λ AF) and two large dextran polymers (MW 3000, λ dex3 and MW 75,000, λ dex75) in the in vitro neocortex of AQP4 +/+ and AQP4 −/− mice. We found that λ AF=1.59, λ dex3=1.76 and λ dex75=2.30 obtained in AQP4 −/− mice were not significantly different from λ AF=1.61, λ dex3=1.76, and λ dex75=2.33 in AQP4 +/+ mice. These IOI results demonstrate that λ measured with small and large molecules each remain unchanged in the enlarged ECS of AQP4 −/− mice compared to values in AQP4 +/+ mice. Further analysis suggests that the FRAP method yields diffusion parameters not directly comparable with those obtained by IOI or RTI methods. Our findings have implications for the role of glial AQP4 in maintaining the ECS structure.

Analysis of receptor oligomerization by FRAP microscopy

Here we describe an approach to investigate di- or oligomerization of transmembrane receptors in living cells with fluorescence recovery after photobleaching (FRAP). We immobilized a defined fraction of receptors with antibodies and then measured lateral mobility of the nonimmobilized fraction by FRAP. We validated this approach with CD86 and CD28 as monomeric and dimeric reference proteins, respectively. Di- or oligomerization of G protein-coupled receptors is strongly debated. We studied human beta-adrenergic receptors as prototypical G protein-coupled receptors and found that beta(1)-AR shows transient interactions whereas beta(2)-AR can form stable oligomers. We propose that this FRAP method can be widely applied to study di- or oligomerization of cell-surface proteins.

Proposal of a Set of Model Polymer Additives Designed for Confocal FRAP Diffusion Experiments

The migration of additives from food packaging to food stuffs is kinetically governed by the diffusion coefficient (D) of the additive within the polymer. Food safety authorities have recently allowed the use of mathematical models to predict D, with the additive molecular weight as a single entry parameter. Such models require experimental values to feed the databases, but these values are often scattered. To deal with this issue, a fluorescent chemically homologous series of model additives was synthesized with molecular weights (MW) ranging from 236 g.mol (-1) to 1120 g.mol (-1). This set was then used to collect diffusion coefficients D through confocal fluorescence recovery after photobleaching (FRAP). This microscopic technique allows in situ packaging micro migration tests. The FRAP method was tested against results from the literature before being applied to two different model polystyrenes in a preliminary study to investigate the relationship D = f(MW). Our intermediate objective was to compare various experimental D = f(MW) from our method with predictions from other mathematical or semiempirical models.

From macroscopic to molecular scale investigations of mass transfer of small molecules through edible packaging applied at interfaces of multiphase food products

Abstract This paper presents a multi-scale approach to investigate mass transfer properties of edible films that integrates some published data and new original results. The transport of small molecules, such as water, between the different parts of multiphase food products leads to quality deterioration and thus requires the use of barrier edible films or coatings. Therefore, it is necessary to characterise the properties of both food and edible barriers, like the diffusivities of their respective migrants. Surface properties characterisation of edible films, composed of an iota-carrageenan matrix in association with a high melting point fat was investigated by goniometry and allows the determination of wetting and water absorption, with emphasis on the constituents functionality. Diffusion of a model probe using the FRAP (Fluorescence Recovery After Photobleaching) method is characterised by a critical water content threshold, inducing an increase of the molecular mobility, and better explains the role of glycerol, used as the plasticizer. This study brings information on the diffusion of a small molecule in the film. Further NMR (Nuclear Magnetic Resonance) spectroscopy investigation contributes to a better understanding and identification of the interactions between the polymer and the diffusant. Finally, FTIR-ATR (Fourier Transform InfraRed–Attenuated Total Reflectance) analysis enables to obtain the diffusion coefficient of the liquid water in the film. Such a multi-scale investigation highlights the permeation mechanism within edible barriers. Industrial relevance Synthetic food packaging is usually used to prevent mass transfers of some molecules, such as moisture, gases, flavor compounds or solutes, between a food and its surrounding medium. In the case of multiphase food products, the development of edible films and coatings, applied between the different phases of this food, has a great potential to increase its shelf life. This article examines the different scale levels of investigation in order to determine the diffusivity of small molecules through these edible packaging.

Line FRAP with the Confocal Laser Scanning Microscope for Diffusion Measurements in Small Regions of 3-D Samples

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.

Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes.

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.

Age-dependence of the lateral mobility of lipids in hepatocyte plasma membrane of male rats and the effect of life-long dietary restriction.

The lateral diffusion constant of lipids (D(1)) in hepatocyte plasma membranes was measured in liver smears by means of the fluorescence recovery after photobleaching (FRAP) method, applying the label, N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE). Nineteen ad libitum fed, male Fischer-344 rats in four age groups (2.1-29.8 months of age) were studied. A highly significant negative linear age-correlation of D(1) (cc = 0.958) was found. D(1) values were 1.39 x 10(-9) cm2/s in the young rats, and only 6.77 x 10(-10) cm2/s in the oldest rats. Lipid lateral mobility is changing in parallel with that of proteins, having been measured previously also with the FRAP method by the authors. Fractional recovery values (FR%) of the lipids were lower than those of proteins even in the young ages, but also decreased linearly with age, therefore, the parameter, D, x FR decreased even steeper with age than D(1) itself. D(1) was also measured in a group of six male Fischer 344 rats having been kept on dietary restriction (DR) since their age of 1 month until 30 months of age (applying the every-other-day (EOD) feeding). DR caused an increase of D(1), compared with the age-matched ad libitum fed animals: the mean was 9.24 x 10(-10) cm(2)/s. FR% and D(I), x FR again increased considerably under DR. The results are interpreted in terms of the increased protein and lipid turnover under DR.

Lateral mobility of phospholipid molecules in thin liquid films

The Fluorescence Recovery After Photobleaching (FRAP) method was applied to measure the lateral mobility of the fluorescent lipid analog, dioctadecylindocarbocyanine perchlorate (Dil-C18), in microscopic thin liquid films (Foam Films (FFs)). The foam film structures were comprised of two phosphatidylcholine monolayers adsorbed at air/water interfaces which sandwiched a thin liquid core. Lateral diffusion of the DiI molecules in the plane of the monolayers was determined as a function of the thickness of the thin liquid core of the film between the FF monolayers. The results obtained indicated that the diffusion coefficient was strongly dependent both on the distance between the FF monolayers in the range 4 nm to 85 nm (corresponding to the FF thickness) and on the film type. The applicability of the FRAP method for studying the molecular mobility in phospholipid FFs was demonstrated. Considerable differences in the surface diffusion coefficient of Dil were observed, ranging between 2 × 10−8 cm2/s and 22 × 10−8 cm2/s in so called yellow, gray, common black and Newton black FFs. The effect of the presence of polyethylene glycol (PEG-400) in the liquid core of lecithin FFs on surface diffusion was also studied. The surface diffusion results from the FF studies were compared with data from black lipid membranes (BLMs). These structures are related in thickness terms but the molecular orientation in FFs is the reverse of that in BLMs.

Developmental changes in the lateral diffusion of Leydig cell membranes measured by the FRAP method

A simple method for isolation and fluorescence labelling of Leydig cells (L-cells) from rat testes was developed. Lateral diffusion coefficients of both lipid and protein membrane fluorescent probes were measured by the method of fluorescence recovery after photobleaching (FRAP). Age-dependent changes in diffusibility of membrane lipids and proteins were discovered.