Fluorescence Recovery After Photobleaching Research Articles

Super-Resolution Imaging Reveals Dynamic Reticular Cytoophidia.

CTP synthase (CTPS) can form filamentous structures termed cytoophidia in cells in all three domains of life. In order to study the mesoscale structure of cytoophidia, we perform fluorescence recovery after photobleaching (FRAP) and stimulated emission depletion (STED) microscopy in human cells. By using an EGFP dimeric tag as a tool to explore the physical properties of cytoophidia, we find that cytoophidia are dynamic and reticular. The reticular structure of CTPS cytoophidia may provide space for other components, such as IMPDH. In addition, we observe CTPS granules with tentacles.

Nasal epithelial cell culture fluorescence recovery after photobleaching predicts cystic fibrosis therapeutic response.

Human nasal epithelial (HNE) cells can be sampled noninvasively and cultured to provide a model of the airway epithelium that reflects cystic fibrosis (CF) pathophysiology. We hypothesised that in vitro measures of HNE cell physiology would correlate directly with in vivo measures of lung physiology and therapeutic response, providing a framework for using HNE cells for therapeutic development and precision medicine. We sampled nasal cells from participants with CF (CF group, n=26), healthy controls (HC group, n=14) and single CF transmembrane conductance regulator (CFTR) mutation carrier parents of the CF group (CR group, n=16). Participants underwent lung physiology and sweat chloride testing, and nuclear imaging-based measurement of mucociliary clearance (MCC) and small-molecule absorption (ABS). CF participants completed a second imaging day that included hypertonic saline (HS) inhalation to assess therapeutic response in terms of MCC. HNE measurements included Ussing chamber electrophysiology, small-molecule and liquid absorption rates, and particle diffusion rates through the HNE airway surface liquid (ASL) measured using fluorescence recovery after photobleaching (FRAP). Long FRAP diffusion times were associated with increased MCC response to HS in CF. This implies a strong relationship between inherent factors affecting ASL mucin concentration and therapeutic response to a hydrating therapy. MCC decreased with age in the CR group, which had a larger range of ages than the other two groups. Likely this indicates a general age-related effect that may be accentuated in this group. Measures of lung ABS correlated with sweat chloride in both the HC and CF groups, indicating that CFTR function drives this measure of paracellular small-molecule probe absorption. Our results demonstrate the utility of HNE cultures for assessing therapeutic response for hydrating therapies. In vitro measurements of FRAP were particularly useful for predicting response and for characterising important properties of ASL mucus that were ultimately reflected in lung physiology.

Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics.

Fluorescence Recovery After Photobleaching (FRAP) has been extensively used to understand molecular dynamics in cells. This technique when applied to soluble, globular molecules driven by diffusion is easily interpreted and well understood. However, the classical methods of analysis cannot be applied to anisotropic structures subjected to directed transport, such as cytoskeletal filaments or elongated organelles transported along microtubule tracks. A new mathematical approach is needed to analyze FRAP data in this context and determine what information can be obtain from such experiments. To address these questions, we analyze fluorescence intensity profile curves after photobleaching of fluorescently labelled intermediate filaments anterogradely transported along microtubules. We apply the analysis to intermediate filament data to determine information about the filament motion. Our analysis consists of deriving equations for fluorescence intensity profiles and developing a mathematical model for the motion of filaments and simulating the model. Two closed forms for profile curves were derived, one for filaments of constant length and one for filaments with constant velocity, and three types of simulation were carried out. In the first type of simulation, the filaments have random velocities which are constant for the duration of the simulation. In the second type, filaments have random velocities which instantaneously change at random times. In the third type, filaments have random velocities and exhibit pausing between velocity changes. Our analysis shows: the most important distribution governing the shape of the intensity profile curves obtained from filaments is the distribution of the filament velocity. Furthermore, filament length which is constant during the experiment, had little impact on intensity profile curves. Finally, gamma distributions for the filament velocity with pauses give the best fit to asymmetric fluorescence intensity profiles of intermediate filaments observed in FRAP experiments performed in polarized migrating astrocytes. Our analysis also shows that the majority of filaments are stationary. Overall, our data give new insight into the regulation of intermediate filament dynamics during cell migration.

Mechanisms of UV-B light-induced photoreceptor UVR8 nuclear localization dynamics.

Light regulates the subcellular localization of plant photoreceptors, a key step in light signaling. Ultraviolet-B radiation (UV-B) induces the plant photoreceptor UV RESISTANCE LOCUS 8 (UVR8) nuclear accumulation, where it regulates photomorphogenesis. However, the molecular mechanism for the UV-B-regulated UVR8 nuclear localization dynamics is unknown. With fluorescence recovery after photobleaching (FRAP), cell fractionation followed by immunoblotting and co-immunoprecipitation (Co-IP) assays we tested the function of UVR8-interacting proteins including CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 in the regulation of UVR8 nuclear dynamics in Arabidopsis thaliana. We showed that UV-B-induced rapid UVR8 nuclear translocation is independent of COP1, which previously was shown to be required for UV-B-induced UVR8 nuclear accumulation. Instead, we provide evidence that the UV-B-induced UVR8 homodimer-to-monomer photo-switch and the concurrent size reduction of UVR8 enables its monomer nuclear translocation, most likely via free diffusion. Nuclear COP1 interacts with UV-B-activated UVR8 monomer, thereby promoting UVR8 nuclear retention. Conversely, RUP1and RUP2, whose expressions are induced by UV-B, inhibit UVR8 nuclear retention via attenuating the UVR8-COP1 interaction, allowing UVR8 to exit the nucleus. Collectively, our data suggest that UV-B-induced monomerization of UVR8 promotes its nuclear translocation via free diffusion. In the nucleus, COP1 binding promotes UVR8 monomer nuclear retention, which is counterbalanced by the major negative regulators RUP1 and RUP2.

Inferences from FRAP data are model dependent: A subdiffusive analysis

Fluorescence recovery after photobleaching (FRAP) is a widely used biological experiment to study the kinetics of molecules that react and move randomly. Since the development of FRAP in the 1970s, many reaction-diffusion models have been used to interpret FRAP data. However, intracellular molecules are widely observed to move by anomalous subdiffusion instead of normal diffusion. In this article, we extend a popular reaction-diffusion model of FRAP to the case of subdiffusion modeled by a fractional diffusion equation. By analyzing this reaction-subdiffusion model, we show that FRAP data are consistent with both diffusive and subdiffusive motion in many scenarios. We illustrate this general result by fitting our model to FRAP data from glucocorticoid receptors in a cell nucleus. We further show that the assumed model of molecular motion (normal diffusion or subdiffusion) strongly impacts the biological parameter values inferred from a given experimentally observed FRAP curve. We additionally analyze our model in three simplified parameter regimes and discuss parameter identifiability for varying subdiffusion exponents.

The centrosomal recruitment of γ-tubulin and its microtubule nucleation activity is α-fodrin guided

ABSTRACT The regulation and recruitment of γ-TuRCs, the prime nucleator of microtubules, to the centrosome are still thrust areas of research. The interaction of fodrin, a sub-plasmalemmal cytoskeletal protein, with γ-tubulin is a new area of interest. To understand the cellular significance of this interaction, we show that depletion of α-fodrin brings in a significant reduction of γ-tubulin in neural cell centrosomes making it functionally under-efficient. This causes a loss of nucleation ability that cannot efficiently form microtubules in interphase cells and astral microtubules in mitosis. Fluorescence Recovery after Photobleaching (FRAP) experiment implies that α-fodrin is important in the recruitment of γ-tubulin to the centrosome resulting in the aforementioned effects. Further, our experiments indicate that the interaction of α-fodrin with certain pericentriolar matrix proteins such as Pericentrin and CDK5RAP2 are crucial for the recruitment of γ-tubulin to the centrosome. Earlier we reported that α-fodrin limits the nucleation potential of γ-TuRC. In that context, this study suggests that α-fodrin is a γ-tubulin recruiting protein to the centrosome thus preventing cytoplasmic microtubule nucleation and thereby compartmentalizing the process to the centrosome for maximum efficiency. Summary statementα-fodrin is a γ-tubulin interacting protein that controls the process of γ-tubulin recruitment to the centrosome and thereby regulates the microtubule nucleation capacity spatially and temporally.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.

Targeting cell-matrix interface mechanobiology by integrating AFM with fluorescence microscopy

Mechanosensing at the interface of a cell and its surrounding microenvironment is an essential driving force of physiological processes. Understanding molecular activities at the cell-matrix interface has the potential to provide novel targets for improving tissue regeneration and early disease intervention. In the past few decades, the advancement of atomic force microscopy (AFM) has offered a unique platform for probing mechanobiology at this crucial microdomain. In this review, we describe key advances under this topic through the use of an integrated system of AFM (as a biomechanical testing tool) with complementary immunofluorescence (IF) imaging (as an in situ navigation system). We first describe the body of work investigating the micromechanics of the pericellular matrix (PCM), the immediate cell micro-niche, in healthy, diseased, and genetically modified tissues, with a focus on articular cartilage. We then summarize the key findings in understanding cellular biomechanics and mechanotransduction, in which, molecular mechanisms governing transmembrane ion channel-mediated mechanosensing, cytoskeleton remodeling, and nucleus remodeling have been studied in various cell and tissue types. Lastly, we provide an overview of major technical advances that have enabled more in-depth studies of mechanobiology, including the integration of AFM with a side-view microscope, multiple optomicroscopy, a fluorescence recovery after photobleaching (FRAP) module, and a tensile stretching device. The innovations described here have contributed greatly to advancing the fundamental knowledge of extracellular matrix biomechanics and cell mechanobiology for improved understanding, detection, and intervention of various diseases.

BOK controls ER proteostasis and physiological ER stress responses in neurons.

The Bcl-2 family proteins BAK and BAX control the crucial step of pore formation in the mitochondrial outer membrane during intrinsic apoptosis. Bcl-2-related ovarian killer (BOK) is a Bcl-2 family protein with a high sequence similarity to BAK and BAX. However, intrinsic apoptosis can proceed in the absence of BOK. Unlike BAK and BAX, BOK is primarily located on the endoplasmic reticulum (ER) and Golgi membranes, suggesting a role for BOK in regulating ER homeostasis. In this study, we report that BOK is required for a full ER stress response. Employing previously characterized fluorescent protein-based ER stress reporter cell systems, we show that BOK-deficient cells have an attenuated response to ER stress in all three signaling branches of the unfolded protein response. Fluo-4-based confocal Ca2+ imaging revealed that disruption of ER proteostasis in BOK-deficient cells was not linked to altered ER Ca2+ levels. Fluorescence recovery after photobleaching (FRAP) experiments using GRP78/BiP-eGFP demonstrated that GRP78 motility was significantly lower in BOK-deficient cells. This implied that less intraluminal GRP78 was freely available and more of the ER chaperone bound to unfolded proteins. Collectively, these experiments suggest a new role for BOK in the protection of ER proteostasis and cellular responses to ER stress.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Abstract P1058: Whole-cell Mechanical Loading And Unloading Triggers More Post-translational Modifications In α-actinin Than Myosin Activators And Inhibitors

The hypothesis tested is that unloading of mechanical forces affects acetylation, ubiquitination, and phosphorylation of the actin-binding protein α-actinin located in the Z-disc. We applied targeted proteomics to interrogate the post-translational modification changes during loading and unloading. Cell morphology and post-translational modifications were determined in a mechanical intervention consisting of 1 Hz cyclic strain for 24 hr (loaded) followed by 6 hr rest (unloaded) of cultured neonatal rat ventricular myocytes (NRVMs). This was compared to a chemical intervention consisting of treating NRVMs with the myosin inhibitor Mavacamten (1 μM, 6hr) or activator Omecamtiv Mecarbil (0.5 μM, 6hr). Quantitative immunofluorescence showed both chemical and mechanical loading increased α-actinin content while Mavacamten decreased the α-actinin content. Mass spectrometry analysis of affinity-purified α-actinin revealed that the mechanical intervention led to increased levels of post-translational modifications compared with the chemical interventions. Specifically, α-actinin ubiquitination increased with mechanical loading-unloading; acetylation decreased with mechanical loading-unloading and increased with mechanical loading; and phosphorylation remained unchanged with mechanical loading-unloading but increased with mechanical loading. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that Mavacamten increased the dynamics of overexpressed YFP-tagged α-actinin and a GFP-tagged CapZ in NRVMs when compared to Omecamtiv Mecarbil treatments and controls. Overall, the results suggest a link between sarcomere homeostasis and mechanical forces via mechanisms involving acetylation, phosphorylation and ubiquitination of α-actinin and a second Z-disc protein, CapZ. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis. Funded by NIH grants HL151825 (CS) and HL62426 (RJS, BR, and CMW).

Reconstitution of Phase-Separated Signaling Clusters and Actin Polymerization on Supported Lipid Bilayers.

Liquid–liquid phase separation driven by weak interactions between multivalent molecules contributes to the cellular organization by promoting the formation of biomolecular condensates. At membranes, phase separation can promote the assembly of transmembrane proteins with their cytoplasmic binding partners into micron-sized membrane-associated condensates. For example, phase separation promotes clustering of nephrin, a transmembrane adhesion molecule, resulting in increased Arp2/3 complex-dependent actin polymerization. In vitro reconstitution is a powerful approach to understand phase separation in biological systems. With a bottom-up approach, we can determine the molecules necessary and sufficient for phase separation, map the phase diagram by quantifying de-mixing over a range of molecular concentrations, assess the material properties of the condensed phase using fluorescence recovery after photobleaching (FRAP), and even determine how phase separation impacts downstream biochemical activity. Here, we describe a detailed protocol to reconstitute nephrin clusters on supported lipid bilayers with purified recombinant protein. We also describe how to measure Arp2/3 complex-dependent actin polymerization on bilayers using fluorescence microscopy. These different protocols can be performed independently or combined as needed. These general techniques can be applied to reconstitute and study phase-separated signaling clusters of many different receptors or to generally understand how actin polymerization is regulated at membranes.

In preprints: morphogens in motion.

In preprints: morphogens in motion.

UV LIGHT INDUCED FLUORESCENCE RECOVERY OF GFP AFTER PHOTOBLEACHING IN MICROSCOPY IMAGING

Fluorescence microscopy has become one of the most important tools for biologists to visualize and study organelles and molecules in a cell. Fluorescent markers are used to visualize specific molecules. One of the most used markers is green fluorescent protein (GFP), which can be expressed along with a protein of interest. However, it is known that the intensity of fluorescence decreases with observation time. To combat this problem, researchers and companies have developed protocols and additives to mitigate photobleaching. In this study, we tested the effects of the three most used culture media on photobleaching and developed a new approach of short-wavelength fluorescence recovery after photobleaching (FRAP). Photobleaching was analyzed by comparing pixel brightness on images taken with a fluorescence microscope. We determined photobleaching of GFP-expressing cells from images taken with a fluorescence microscope by comparing pixel brightness. Statistical analysis was performed to determine the average bleaching for specific culture media. The culture media analyzed had no significant effect on the photobleaching of GFP. However, a brief UV burst (15 sec) restores 50% of the original fluorescence and neither increases ROS nor decreases cell viability. To avoid artifacts in image analysis and interpretation, our study suggests using this simple method of GFP fluorescence recovery with UV light induced FRAP to extend the fluorescence lifetime and imaging of GFP molecules.

Pepsin diffusion in complex food matrices

Pepsin diffusion in food particles during gastric digestion is one of the main factors limiting proteolysis kinetics. Diffusion coefficients of pepsin are needed as input parameters for in silico models of digestion, but no values are currently available in real foods. The challenge of this study was to apply the Fluorescent Recovery After Photobleaching (FRAP) technique to determine diffusion coefficients of fluorescently labeled (FITC)-pepsin in four realistic food matrices with complex and heterogeneous structures (Custard, Pudding, Sponge cake and Biscuit), but an identical composition on a dry matter basis. The effective diffusion coefficients determined for FITC-pepsin at 37 °C ranged from 48 ± 14 to 2 ± 1 μm 2 . s -1 for Custard and Biscuit, respectively. A modelling approach based on the stretched exponential equation generated a very good fit of the experimental dataset as a function of dry matter content of the matrix. • First quantification of pepsin diffusivity inside complex food matrices. • Food matrices of same dry matter but different structures from liquid to dry solid. • Pepsin diffusivity is requested as input parameter for in silico modelling of digestion. • Pepsin diffusivity within solid food is mainly conditioned by dry matter content. • Diffusivity followed the exponential stretched equation in line with theoretical diffusion model.

Analysis of chemomechanical behavior of stress fibers by continuum mechanics-based FRAP

Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently labeled β-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate, which represents effective off-rate of β-actin, was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the rate of displacement or velocity along stress fibers but, interestingly, not with the absolute magnitude of strain. Moreover, stress fibers are subjected to centripetal flow that is facilitated by the circulation of actin molecules. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.

Endogenous slow and fast myosin dynamics in myofibers isolated from mice expressing GFP-Myh7 and Kusabira Orange-Myh1.

Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFP-Myh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging.

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM‐FRAP (Small Methods 6/2022)

Front Cover In article number 2200149, Colin-York, Fritzsche, and co-workers found that by combining lattice light sheet microscopy (LLSM), fluorescence recovery after photo-bleaching (FRAP), and numerical simulations, LLSM-FRAP provides a means of quantifying molecular dynamics within complex 3D cell morphologies.

Temperature-Dependent Constrained Diffusion of Micro-Confined Alkylimidazolium Chloride Ionic Liquids.

Alkylimidazolium chloride ionic liquids (ILs) have many uses in a variety of separation systems, including micro-confined separation systems. To understand the separation mechanism in these systems, the diffusion properties of analytes in ILs under relevant operating conditions, including micro-confinement dimension and temperature, should be known. For example, separation efficiencies for various IL-based microextraction techniques are dependent on the sample volume and temperature. Temperature-dependent (20-100 °C) fluorescence recovery after photobleaching (FRAP) was utilized to determine the diffusion properties of a zwitterionic, hydrophilic dye, ATTO 647, in alkylimidazolium chloride ILs in micro-confined geometries. These micro-confined geometries were generated by sandwiching the IL between glass substrates that were separated by ∼1 to 100 μm. From the measured temperature-dependent FRAP data, we note alkyl chain length-, thickness-, and temperature-dependent diffusion coefficients, with values ranging from 0.021 to 46 μm2/s. Deviations from Brownian diffusion are observed at lower temperatures and increasingly less so at elevated temperatures; the differences are attributed to alterations in intermolecular interactions that reduce temperature-dependent nanoscale structural heterogeneities. The temperature- and thickness-dependent data provide a useful foundation for efficient design of micro-confined IL separation systems.