Fluorescence Recovery After Photobleaching Measurements Research Articles

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

Membrane dynamics are slowed for Alexa594-labeled membrane proteins due to substrate interactions

The addition of fluorescent dyes to proteins, lipids and other biological molecules can affect a range of processes such as mobility, molecular interactions, localization, and, ultimately, function. The dynamics of a protein can be dramatically affected if the label interacts non-specifically with the substrate or with other molecules in the system. To test how dye-substrate interactions affect protein diffusion, fluorescence recovery after photobleaching (FRAP) measurements were designed to explicitly determine the role of the dye on the diffusion of a transmembrane protein, Syntaxin1a, expressed on the cell surface. Syntaxin1a, was tagged with EGFP on the extracellular side and an EGFP nanobody with or without a dye label was attached. FRAP was performed on Syx1a-EGFP and the choice of cell growth substrate affected mobility in the presence of a dye labeled nanobody. This work provides evidence for choosing fibronectin (Fn) over poly-L-lysine (PLL) in FRAP and single molecule tracking measurements when using Alexa594, a common probe for red fluorescent measurements. Alexa594-labeled nanobody but not unlabeled nanobody, dramatically reduced the mobility of Syx1a-EGFP when cells were cultured on PLL. However, when Fn was used, the mobility returned. Mobility measured by single molecule tracking measurements align with the FRAP measurements with Fn coated surfaces being more mobile than PLL.

Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP

Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a two-dimensional (2D) plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping both intranuclear diffusion of NLS-GFP and recovery of 53BP1-mCherry, a marker for DNA damage, in live MDA-MB-231 cells. SPIM-FRAP proves to be an order of magnitude faster than fluorescence-correlation-spectroscopy-based techniques for such measurements. We observe large length-scale (>∼500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of NLS-GFP recovery times showed no pixel-by-pixel correlation with histone density, although slower diffusion was observed in nucleoli. Additionally, recovery of 53BP1-mCherry was observed to be slowed at sites of DNA damage. We finally developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques. Our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a straightforward means of quantifying the spatial distribution of protein recovery or diffusion in living cells.

Improved FRAP Measurements on Biofilms

We expand the standard fluorescence recovery after photobleaching (FRAP) model introduced by Axelrod et al. in 1976. Our goal is to capture some of the following common artifacts observed in the fluorescence measurements obtained with a confocal laser scanning microscope in biofilms: 1) linear drift, 2) exponential decrease (due to bleaching during the measurements), 3) stochastic Gaussian noise, and 4) uncertainty in the exact time point of the onset of fluorescence recovery. To fit the resulting stochastic model to data from FRAP measurements and to estimate all unknown model parameters, we apply a suitably adapted Metropolis-Hastings algorithm. In this way, a more accurate estimation of the diffusion coefficient of the fluorophore is achieved. The method was tested on data obtained from FRAP measurements on a cultivated biofilm.

Probing the microenvironment of polyacrylamide hydrogel matrix using turbidity and fluorescence recovery after photobleaching: One versus Two phases

The microstructure of polyacrylamide hydrogel matrices was probed by turbidity measurements, and by fluorescence recovery after photobleaching (FRAP) of fluorescently labeled bovine serum albumin (fBSA), as a function of polymer concentration and crosslink density. Turbidity increased with increasing polymer and/or crosslinker concentration. At low polymer and crosslinker concentrations, FRAP dynamics were well described by diffusion through a single polymer phase. However for higher concentrations of polymer and crosslinker, the diffusion model was inadequate, and a second mode, likely corresponding to release of fBSA from regions of relatively dense polymer, was observed. At low network concentrations, probe diffusivity (inversely related to FRAP relaxation time) decreased with increasing polymer and crosslinker concentrations. Following onset of the second mode, however, diffusivity increased with increasing monomer concentration, suggesting that the network through which fBSA diffused became looser. The optical and FRAP measurements, while showing qualitatively similar trends, were not directly correlated, and they likely reflected different modes of network phase separation.

Silica-based diffusion probes for use in FRAP and NMR-diffusometry

Development of multi-purpose probes for mass transport measurements is of importance to gain knowledge in diffusional behaviour in heterogeneous structures such as food, hygiene or pharamceuticals. By combining different techniques, such as Fluorescence Recovery After Photobleaching (FRAP) and Nuclear Magnetic Resonance Diffusometry (NMR-d), information of both local and global diffusion can be collected and used to gain insights on for example material heterogeneities and probe-material interactions. To obtain a FRAP-responsive probe, fluorescent silica particles were produced using fluorescent preconjugates added in a modified Stöber process. A NMR-d responsive moiety was introduced by derivatizing the fluorescent silica particles with polyethylene glycol. The particle size distributions were determined by dynamic light scattering and transmission electron microscopy and these measurements were compared to value extrapolated from diffusion measurements using FRAP and NMR-d. The good agreement between the FRAP and NMR-d measurements demonstrates the potential of multi-purpose probes for future applications concerning mass transport at local and global scale simultaneously.

Exploring accessibility of pretreated poplar cell walls by measuring dynamics of fluorescent probes

BackgroundThe lignocellulosic cell wall network is resistant to enzymatic degradation due to the complex chemical and structural features. Pretreatments are thus commonly used to overcome natural recalcitrance of lignocellulose. Characterization of their impact on architecture requires combinatory approaches. However, the accessibility of the lignocellulosic cell walls still needs further insights to provide relevant information.ResultsPoplar specimens were pretreated using different conditions. Chemical, spectral, microscopic and immunolabeling analysis revealed that poplar cell walls were more altered by sodium chlorite-acetic acid and hydrothermal pretreatments but weakly modified by soaking in aqueous ammonium. In order to evaluate the accessibility of the pretreated poplar samples, two fluorescent probes (rhodamine B-isothiocyanate–dextrans of 20 and 70 kDa) were selected, and their mobility was measured by using the fluorescence recovery after photobleaching (FRAP) technique in a full factorial experiment. The mobility of the probes was dependent on the pretreatment type, the cell wall localization (secondary cell wall and cell corner middle lamella) and the probe size. Overall, combinatory analysis of pretreated poplar samples showed that even the partial removal of hemicellulose contributed to facilitate the accessibility to the fluorescent probes. On the contrary, nearly complete removal of lignin was detrimental to accessibility due to the possible cellulose–hemicellulose collapse.ConclusionsEvaluation of plant cell wall accessibility through FRAP measurement brings further insights into the impact of physicochemical pretreatments on lignocellulosic samples in combination with chemical and histochemical analysis. This technique thus represents a relevant approach to better understand the effect of pretreatments on lignocellulose architecture, while considering different limitations as non-specific interactions and enzyme efficiency.

FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.

Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.

On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements

Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations.

Diffusion of Immunoglobulin G in Shed Vaginal Epithelial Cells and in Cell-Free Regions of Human Cervicovaginal Mucus.

Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments.

Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques.

The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments.

Fluorescence Recovery after Photobleaching and Single-Molecule Tracking Measurements of Anisotropic Diffusion within Identical Regions of a Cylinder-Forming Diblock Copolymer Film.

This work demonstrates ensemble and single-molecule diffusion measurements within identical regions of a cylinder-forming polystyrene-poly(ethylene oxide) diblock copolymer (PS-b-PEO) film using fluorescence recovery after photobleaching (FRAP) and single-molecule tracking (SMT). A PS-b-PEO film (∼4 μm thick) with aligned cylindrical PEO microdomains containing 10 μM sulforhodamine B (SRB) was prepared by directional solvent-vapor penetration (SVP) of 1,4-dioxane. The ensemble diffusion behavior of SRB in the microdomains was assessed in FRAP studies of circular photobleached regions (∼7 μm in diameter). The SRB concentration was subsequently reduced by additional photobleaching, and the diffusion of individual SRB molecules was explored using SMT in the identical area (∼16 × 16 μm(2)). The FRAP data showed anisotropic fluorescence recovery, yielding the average microdomain orientation. The extent of fluorescence recovery observed (∼90%) demonstrated long-range microdomain connectivity, while the recovery time dependence provided an ensemble measurement of the SRB diffusion coefficient within the cylindrical microdomains. The SMT data exhibited one-dimensional diffusion of individual SRB molecules along the SVP direction across the entire film thickness, as consistent with the FRAP results. Importantly, the average of the single-molecule diffusion coefficients was close to the value obtained from FRAP in the identical area. In some cases, SMT offered smaller diffusion coefficients than FRAP, possibly due to contributions from SRB molecules confined within short PEO microdomains. The implementation of FRAP and SMT measurements in identical areas provides complementary information on molecular diffusion with minimal influence of sample heterogeneity, permitting direct comparison of ensemble and single-molecule diffusion behavior.

A FRAP-based evaluation of protein diffusion in polyelectrolyte multilayers

The evaluation of molecular diffusion by fluorescence recovery after photobleaching (FRAP) gives an option to examine properties of various soft materials aiming at material design and development. Biofunctional polyelectrolyte multilayer films can be fabricated using the layer-by-layer technique that offers easy tunability of the film chemical composition, thickness, and mechanical properties. Here the mobility of a model protein cytochrome C loaded into the multilayer film consisting of hyaluronic acid and poly-l-lysine is investigated. An approach based on analytical solution of Fick’s law is used for evaluation of the protein diffusion coefficient from FRAP measurements. The FRAP measurement procedure has been optimised to ensure a high quality of the recorded data and to improve sensitivity of the measurements. For this purpose, various settings of the confocal laser scanning microscope have been examined and changing of the pinhole size has been found the most appropriate way to reduce signal noise and improve sensitivity. This study opens up a possibility of the fundamental understanding of intermolecular interactions within the polyelectrolyte multilayers as well as applicability of FRAP for the evaluation of diffusion of various molecules.

In vivo and in vitro Studies of Myosin-XXI Dynamics

Supported by Deutsche Forschungsgemeinschaft, SFB 863, B6Myosin-XXI is one of only two myosin isoforms found in the genome of the Leishmania parasite. It is involved in the assembly and elongation of the flagellum and in intracellular trafficking. Using mammalian cell transfections, FRAP (fluorescence recovery after photobleaching) measurements and single particle tracking, we elucidated myosin-XXI dynamics in vitro and in vivo. Transfections with myosin-XXI resulted in an accumulation of myosin at the plasma membrane and inside the tips of filopodia. A minimal construct encoding the N-terminal SH3 domain, motor domain, and parts of the tail upto aa 808 was required for this localization pattern. Notably, the myosin-XXI tail contains several lipid binding sites. Live imaging revealed that filopodial myosin accumulations endured length changes of filopodia, pointing toward the possibility of myosin-XXI being stably bound to the plasma membrane. Additionally, we observed an increase in the length and density of filopodia in transfected cells, indicating that the myosin might tether the actin cytoskeleton to the plasma membrane thus stabilizing the architecture of membrane protrusions. To investigate the dynamic behavior of this myosin when bound to membranes, we anchored myosin-XXI to planar supported lipid bilayers via a biotin-streptavidin-biotin link and investigated its diffusion by FRAP and single particle imaging. Furthermore, we studied the myosin's capability of moving filamentous actin when bound to lipids. In filament gliding assays on lipid vesicles, vesicle size appeared to influence filament motility. Accordingly, we tested myosin-XXI for curvature sensitivity employing SLiC (single liposome curvature) assays.

Organizational Interplay of Golgi N-Glycosyltransferases Involves Organelle Microenvironment-Dependent Transitions between Enzyme Homo- and Heteromers

Glycosylation of proteins and lipids takes place in the Golgi apparatus by the consecutive actions of functionally distinct glycosidases and glycosyltransferases. Current evidence indicates that they function as enzyme homomers and/or heteromers in the living cell. Here we investigate their organizational interplay and show that glycosyltransferase homomers are assembled in the endoplasmic reticulum. Upon transport to the Golgi, the majority of homomers are disassembled to allow the formation of enzyme heteromers between sequentially acting medial-Golgi enzymes GnT-I and GnT-II or trans-Golgi enzymes GalT-I and ST6Gal-I. This transition is driven by the acidic Golgi environment, as it was markedly inhibited by raising Golgi luminal pH with chloroquine. Our FRAP (fluorescence recovery after photobleaching) measurements showed that the complexes remain mobile Golgi membrane constituents that can relocate to the endoplasmic reticulum or to the scattered Golgi mini-stacks upon brefeldin A or nocodazole treatment, respectively. During this relocation, heteromers undergo a reverse transition back to enzyme homomers. These data unveil an unprecedented organizational interplay between Golgi N-glycosyltransferases that involves dynamic and organelle microenvironment-driven transitions between enzyme homomers and heteromers during their trafficking within the early secretory compartments.

Dynamics of mitochondrial RNA-binding protein complex in Trypanosoma brucei and its petite mutant under optimized immobilization conditions.

There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei. Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo. In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi. The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

The characterization of the nuclear dynamics of syntenin‐2, a PIP2 binding PDZ protein

Cellular signaling is largely dependent on the presence, that is, assembly/disassembly, of supramolecular complexes. Postsynaptic density protein, Discs-large, Zona occludens (PDZ) domains play important roles in the assembly of various signaling complexes. Syntenin-2 (S2) is a PDZ protein that interacts with nuclear phosphatidylinositol 4,5-bisphosphate (PIP2 ). Although nuclear lipids emerge as key players in nuclear processes, the global significance of nuclear phosphoinositide-protein interactions is still poorly understood. Those phosphoinositide-protein interactions that have been studied in detail appear to have profound physiological effects. To our knowledge none of these were investigated by dynamic studies such as Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), or Fluorescence Recovery After Photobleaching (FRAP). Although the exact function of S2 is unknown, siRNA experiments suggest that this PDZ protein plays a role in the organization of nuclear PIP2 , cell division, and cell survival. As a consequence of its PIP2 interaction, its reported self-association in a yeast two-hybrid study and its speculated interaction with many, yet unidentified, proteins one can hypothesize that S2 plays an important role in cell signaling. Therefore, we studied the dynamics of S2 using FCS, FCCS, and FRAP, utilizing an active truncated form deleted for the first 94 amino acids (S2-ΔN). We showed that S2-ΔN self-associates and is distributed in three groups. One immobile group, one slow diffusing group, which interacts with the nuclear environment and one fast diffusing group, which is not incorporated in high molecular weight complexes. In addition, our FCS and FRAP measurements on S2-ΔN mutants affected in their PIP2 binding showed that PIP2 plays an important role in the distribution of S2-ΔN among these groups, and favors the enrichment of S2-ΔN in the slow diffusing and immobile group. This work indicates that S2 relies on nuclear PIP2 to interact with practically immobile structures, possibly chromatin.

Probing the Interior of a Secretory Granule

Although a great deal is known about events leading to the release of secretory granule contents into the extracellular space, little is known about the physical state of the granule lumenal contents prior to exocytosis. We have used TIRFM as our core technique and combined it with either Fluorescent Correlation Spectroscopy (FCS) or Fluorescence Recovery After Photobleaching (FRAP) to assess the mobility of either Neuropeptide Y (NPY) or tissue plasminogen activator (tPA) inside chromaffin granules (300 nm diameter). Overall granule motion was taken into account by viewing simultaneously in the same granule one subset of protein molecules labeled with cerulean and the other with mCherry. Theories for separating out the effects of granule motion for both FCS and FRAP measurements were derived. The rapidly decaying evanescent field (decay constant ∼ 110 nm) enables a small measurement volume in FCS. The fluorescence fluctuations of the fluorophore diffusing into and out of the measurement volume had a characteristic correlation time of several hundred milliseconds. The rapidly decaying evanescent field also allows a spatially restricted bleaching of the granule fluorophore in FRAP, thereby permitting recovery by diffusion from unbleached volume within the granule. FRAP and FCS results were consistent. These initial measurements suggest a diffusion coefficient of these proteins of the order of 10-10 cm2/s, at least two orders of magnitude slower than soluble proteins in cytosol. Our initial results, therefore, indicate highly restricted protein mobility within secretory granules. Research support from NIH Grant R21NS073686 to RWH and DA.

The effect of ligand affinity on integrins’ lateral diffusion in cultured cells

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

A generalized approach for measuring microcapsule permeability with Fluorescence Recovery After Photobleaching

Measurement of the permeability of microcapsules with Fluorescence Recovery After Photobleaching (FRAP) enables analysis of individual microcapsules, the study of their batch heterogeneity, and the survey of changes in an individual capsule’s transport properties under different environmental conditions. We present a modified protocol and analysis which expands the parameter space of microcapsule sizes and permeabilities that can be accurately measured with FRAP. Simulations and experiments show that the hybrid analysis, which accounts for both permeation through the shell and free diffusion, precisely captures the kinetics of typical fluorescence recovery curves. The modified approach eliminates the need for painstaking controls to determine the FRAP measurement volume and the spatial distribution of bleached tracers, while accurately extracting the permeability of the microcapsule from FRAP data acquired using a straightforward procedure.