Total Internal Reflection Fluorescence Imaging Research Articles

Plasma Membrane Diffusion Modes of FcεRI Receptor for Immunoglobin E Measured with Imaging Fluorescence Correlation Spectroscopy

Spatio-temporal organization of the plasma membrane plays a significant role in cell signaling. A prevailing view is that the plasma membrane spatially segregates into ordered lipid (Lo-like) nanodomains co-existing with disordered lipid (Ld-like) regions. Our laboratory, using super-resolution fluorescence imaging, previously showed that immunoglobin E bound to its receptor, FceRI, (IgE-FceRI complex) undergoes time-dependent redistribution on the membrane surface after multivalent ligand stimulation, upon which the receptor is phosphorylated by membrane-anchored Lyn kinase. A large body of evidence shows that this interaction of IgE-FceRI and Lyn occurs in the ordered regions of the plasma membrane enriched in cholesterol. We are examining the nature and dynamics of plasma membrane organization as experienced by IgE-FceRI in resting state and after antigen stimulation. We employ imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS), which maps lateral diffusion with pixel resolution, to investigate the diffusion distribution of IgE-FceRI complexes on the ventral surface of live RBL cells in native and experimentally modulated (e.g., cholesterol depleted) conditions. We also conduct spot variation FCS (svFCS), which analyzes space-dependence of diffusion coefficients from the same set of ITIR-FCS data, to reveal the existence of nanoscopic obstacles (such as nanodomains) that affect the diffusion of IgE-FceRI complexes. These observations are compared with the diffusion behavior of protein markers that are known to prefer ordered or disordered regions of plasma membrane. These results, in conjunction with super-resolution imaging, which determines the size and density of these complexes, will provide a new level of insight into plasma membrane organization and its dynamic remodeling upon ligand-receptor stimulation.

Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization.

Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

Clearer view for TIRF and oblique illumination microscopy.

In Total Internal Reflection Fluorescence (TIRF) microscopy, the sample is illuminated with an evanescent field that yields a thin optical section. However, its widefield detection has no rejection mechanism against out-of-focus blur from scattered light that can compromise TIRF images. Here I demonstrate that via structured illumination, out-of-focus blur can be effectively suppressed in TIRF microscopy, yielding strikingly clearer images. The same mechanism can also be applied to oblique illumination schemes that extend the reach of TIRF microscopy beyond the basal surface of the cell. The two imaging modes are used to image a biosensor, clathrin coated vesicles and the actin cytoskeleton in different cell types with improved contrast.

Simultaneous Manipulation and Super-Resolution Fluorescence Imaging of Individual Kinetochores Coupled to Microtubule Tips.

Kinetochores are large multiprotein complexes that drive mitotic chromosome movements by mechanically coupling them to the growing and shortening tips of spindle microtubules. Kinetochores are also regulatory hubs, somehow sensing when they are erroneously attached and, in response, releasing their incorrect attachments and generating diffusible wait signals to delay anaphase until proper attachments can form. The remarkable ability of a kinetochore to sense and respond to its attachment status might stem from attachment- or tension-dependent changes in the structural arrangement of its core subcomplexes. However, direct tests of the relationship between attachment, tension, and core kinetochore structure have not previously been possible because of the difficulties of applying well-controlled forces and determining unambiguously the attachment status of individual kinetochores in vivo. The recent purification of native yeast kinetochores has enabled in vitro optical trapping-based assays of kinetochore tip-coupling and, in separate experiments, fluorescence imaging of single kinetochore particles. Here we introduce a dual instrument, combining optical trapping with multicolor total internal reflection fluorescence (TIRF) imaging, to allow kinetochore structure to be monitored directly with nanometer precision while mechanical tension is simultaneously applied. Our instrument incorporates differential interference contrast (DIC) imaging as well, to minimize the photo-bleaching of fluorescent tags during preparative bead and microtubule manipulations. A simple modification also allows the trapping laser to be easily converted into a real-time focus detection and correction system. Using this combined instrument, the distance between specific subcomplexes within a single kinetochore particle can be measured with 2-nm precision after 50s observation time, or with 11-nm precision at 1s temporal resolution. While our instrument was constructed specifically for studying kinetochores, it should also be useful for studying other filament-binding protein complexes, such as spindle poles, cortical microtubule attachments, focal adhesions, or other motor-cytoskeletal junctions.

Are Filopodia Privileged Signaling Structures in Migrating Cells?

Filopodia are thin, fingerlike structures that contain bundled actin filaments and project from the cell periphery. These structures are dogmatically endowed with the ability to sense cues in the microenvironment, implying that filopodia foster local signal transduction, yet their small diameter hampers the imaging of dynamic processes therein. To overcome this challenge, we analyzed total internal reflection fluorescence images of migrating fibroblasts coexpressing either a plasma membrane marker or tagged AktPH domain, a translocation biosensor for signaling through the phosphoinositide 3-kinase pathway, along with a cytosolic volume marker. We devised a scheme to estimate the radii of filopodia using either the membrane marker or volume marker data, and we used that information to account for geometry effects in the biosensor data. With conservative estimates of relative target molecule abundance, it is revealed that filopodia typically harbor higher densities of 3′ phosphoinositides than adjacent regions at the cell periphery. In this context at least, the analysis supports the filopodial signaling hypothesis.

Base-Excision-Repair-Induced Construction of a Single Quantum-Dot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity.

DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually time-consuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin-streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10(-6) U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.

Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy.

Total internal reflection fluorescence (TIRF) microscopy allows visualization of biological events at the single-molecule level by restricting excitation to a precise focal plane near the coverslip and eliminating out-of-focus fluorescence. The quality of TIRF imaging relies on a high signal-to-noise ratio and therefore it is imperative to prevent adherence of molecules to the glass coverslip. Nonspecific interactions can make it difficult to distinguish true binding events and may also interfere with accurate quantification of background noise. In addition, nonspecific binding of the fluorescently tagged protein will lower the effective working concentration, thereby altering values used to calculate affinity constants. To prevent spurious interactions, we thoroughly clean the surface of the coverslip and then functionalize the glass either by applying a layer of silane or by coating with a lipid bilayer.

Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching

We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.

Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy

We recently proposed a straightforward fluorescence microscopy technique to study adhesion of Giant Unilamellar Vesicles. This technique is based on dual observations which combine epi-fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy: TIRF images are normalized by epi-fluorescence ones. By this way, it is possible to map the membrane/substrate separation distance with a nanometric resolution, typically ~20nm, with a maximal working range of 300–400nm. The purpose of this paper is to demonstrate that this technique is useful to quantify vesicle adhesion from ultra-weak to strong membrane–surface interactions. Thus, we have examined unspecific and specific adhesion conditions. Concerning unspecific adhesion, we have controlled the strength of electrostatic forces between negatively charged vesicles and various functionalized surfaces which exhibit a positive or a negative effective charge. Specific adhesion was highlighted with lock-and-key forces mediated by the well defined biotin/streptavidin recognition.

Mapping Epidermal Growth Factor Receptor (EGFR) Dimerization by Imaging Fluorescence Cross-Correlation Spectroscopy

The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, plays a role in a number of important cellular processes such as migration, differentiation and proliferation. Although EGFR has been studied intensively for many years, some aspects of its activation and function have not been satisfactorily explained. For example, the dimerization of EGFR in the absence of its ligand remains controversial. Predominantly monomeric to predominantly dimeric and possibly higher oligomeric states have been reported in literature. In this study we have used a novel imaging modality called dual-colour imaging total internal reflection fluorescence cross-correlation spectroscopy (DC-ITIR-FCCS) to map EGFR dimerization in the membranes of CHO-K1 cells. The cells were expressing EGFR analogues tagged by eGFP and mRFP, respectively, and for each image pixel we measured the cross-correlation between fluorescence intensity fluctuations in the green and the red channel. High cross-correlation means co-diffusion of molecules tagged with eGFP and mRFP, which indicates presence of dimers or possibly higher oligomers. Our results show levels of cross-correlation consistent with considerable fractions of EGFR in the form of dimers. There exist, however, considerable differences between individual cells, some displaying negligible while others almost complete dimerization. The imaging FCCS modality allows us to evaluate not only the differences between individual cells but also the distribution of EGFR dimers within the membrane of each individual cell, showing for example increased dimerization close to the bounderies of the cells. Furthermore, the method allows us to map the differences in EGFR dimerization and its spatial distribution following EGFR activation or extraction of cholesterol from the membrane.

The Dynamics and Localization of the Epidermal Growth Factor Receptor (EGFR) on Live Cell Plasma Membrane Studied by ITIR-FCS

The Epidermal Growth Factor Receptor (EGFR), a prototypical receptor tyrosine kinase, plays an important role in cellular processes such as proliferation, migration and apoptosis. EGFR is known to be strongly regulated by its localization and the state of activation on the plasma membrane. However, the spatial distribution of EGFR on the plasma membrane has not been resolved in detail.Here, we use Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) to investigate EGFR behavior on live cells before and after stimulation. ITIR-FCS allows monitoring EGFR dynamics on cell membranes and in combination with the FCS diffusion laws provides information on the nanoscopic EGFR organization.Our results indicate that EGFR is partly trapped in cholesterol sensitive domains and also couples to the cytoskeleton. Stimulation by EGF activates EGFR in a dose dependent manner. At low-dose stimulation (10 ng/ml), the membrane diffusion and confinement of EGFR remain unchanged and EGFR internalization is cholesterol independent and EGFR recycles back to the membrane. At high-dose EGF treatment (100 ng/ml), EGFR endocytosis is cholesterol dependent and cholesterol-depletion impairs receptor internalization. In addition, cholesterol-depletion with concomitant EGF stimulation leads to stronger confinement of the receptor, independent of the EGF dose, as clusters accumulate on the membrane. Furthermore, the actin cytoskeleton promotes EGFR internalization after ligand stimulation. Disruption of the actin cytoskeleton inhibits EGFR endocytosis and induces clustering of EGFR on the cell membrane. This study demonstrates the capabilities of ITIR-FCS to follow receptor stimulation events on live cells. It demonstrates not only that EGFR activation is cholesterol and cytoskeleton dependent but also that the activation pathway is EGF dose dependent, thus providing new insights into receptor dynamics and organization on live cells.

The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking.

In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies “M-type” currents, a slowly-activating voltage-gated K+ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.

Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis.

Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.

Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes.

Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, β-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.

Role of Na+/H+ exchanger regulatory factor 1 in forward trafficking of the type IIa Na+-Pi cotransporter.

Na+/H+ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na+-Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-Pi medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-Pi medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pi medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.

Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells.

Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.

Total internal reflection fluorescence quantification of receptor pharmacology.

Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown. However, TIRF microscopy has found little use in high content screening due to its complexity in instrumental setup and experimental procedures. Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling. This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

Orai1–Orai2 complex is involved in store-operated calcium entry in chondrocyte cell lines

Ca2+ influx via store-operated Ca2+ entry (SOCE) plays critical roles in many essential cellular functions. The Ca2+ release-activated Ca2+ (CRAC) channel complex, consisting of Orai and STIM, is one of the major components of store-operated Ca2+ (SOC) channels. Our previous study demonstrated that histamine can cause sustained Ca2+ entry through SOC channels in OUMS-27 cells derived from human chondrosarcoma. This SOCE was increased by low- and decreased by high-concentrations of 2-aminoethoxydiphenyl borate. Quantitative reverse transcription PCR and Western blot analyses revealed abundant expressions of Orai1, Orai2 and STIM1. Introduction of dominant negative mutant of Orai1, or siOrai1 knockdown significantly attenuated SOCE. Following histamine application, single molecule imaging using total internal reflection fluorescence (TIRF) microscopy demonstrated punctate Orai1–STIM1 complex formation in plasma membrane. In contrast, knockdown or over-expression of Orai2 resulted in an increase or a decrease in SOCE, respectively. Finally, TIRF imaging revealed direct coupling between Orai1 and Orai2, and suggested that Orai2 reduces Orai1 function by formation of a hetero-tetramer. These results provide substantial evidence that Orai1, Orai2 and STIM1 form functional CRAC channels in OUMS-27 cells and that these complexes are responsible for sustained Ca2+ entry in response to agonist stimulation.

Tracking Ceacam1 Interactions and Dynamics with homo-FRET and Image Correlation Techniques

The carcinoembryonic antigen (CEA) family plays an important role in numerous normal and pathogenic processes related to cellular growth and differentiation. Crucial roles of various CEA-family members are well established while the effect of carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on immune-cell function has only recently been recognized. CEACAM1 is known to exist in both monomeric and dimeric states that are heterogeneously distributed at the cell surface. However, which form participates in cell-cell interactions and its related dynamics remain unknown. We report here details of CEACAM1's spatial distribution, oligomeric state, diffusional characteristics and dynamics as determined by live cell homo-fluorescence energy resonance transfer (homo-FRET) microscopy and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS).

Imaging sub-plasma membrane cAMP dynamics with fluorescent translocation reporters.

Imaging cAMP dynamics in single cells and tissues can provide important insights into the regulation of a variety of cellular processes. In recent years, a large number of tools for cAMP measurements have been developed. While most cAMP reporters are designed to undergo changes in fluorescence resonance energy transfer (FRET), there are alternative techniques with advantages for certain applications. Here, we describe protocols for cAMP measurements in the sub-plasma membrane space based on the detection of the cAMP-induced translocation of engineered fluorescent protein-tagged subunits of protein kinase A between the cytoplasm and the plasma membrane. Total internal reflection fluorescence (TIRF) imaging of the changes in reporter localization yields robust signal changes and has contributed to the discovery of cAMP oscillations in the sub-plasma membrane space of insulin-secreting β-cells stimulated with glucose and gluco-incretin hormones. We also demonstrate how the technique can be combined with measurements of the cytosolic Ca(2+) concentration or with recordings of the subcellular localization of the cAMP effector protein Epac2. The translocation reporter approach provides a valuable complement to other methods for imaging sub-membrane cAMP dynamics in various types of cells.