Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.     

A guide to accurate fluorescence microscopy colocalization measurements

Biomolecular interactions are fundamental to the vast majority of cellular processes, and identification of the major interacting components is usually the first step toward an understanding of the mechanisms that govern various cell functions. Thus, statistical image analyses that can be performed on fluorescence microscopy images of fixed or live cells have been routinely applied for biophysical and cell biological studies. These approaches measure the fraction of interacting particles by analyzing dual color fluorescence images for colocalized pixels. Colocalization algorithms have proven to be effective, although the dynamic range and accuracy of these measurements has never been well established. Spatial image cross-correlation spectroscopy (ICCS), which cross-correlates spatial intensity fluctuations recorded in images from two detection channels simultaneously, has also recently been shown to be an effective measure of colocalization as well. Through simulations, imaging of fluorescent antibodies adsorbed on glass and cell measurements, we show that ICCS performs much better than standard colocalization algorithms at moderate to high densities of particles, which are often encountered in cellular systems. Furthermore, it was found that the density ratio between the two labeled species of interest plays a major role in the accuracy of the colocalization analysis. By applying a direct and systematic comparison between the standard, fluorescence microscopy colocalization algorithm and spatial ICCS, we show regimes where each approach is applicable, and more importantly, where they fail to yield accurate results.     

Advances in Image Correlation Spectroscopy: Measuring Number Densities,Aggregation States,and Dynamics of Fluorescently labeled Macromolecules in Cells

A brief historical outline of fluorescence fluctuation correlation techniques is presented, followed by an in-depth review of the theory and development of image correlation techniques, including: image correlation spectroscopy (ICS), temporal ICS (TICS), image cross-correlation spectroscopy (ICCS), spatiotemporal ICS (STICS), k-space ICS (kICS), raster ICS (RICS), and particle ICS (PICS). These techniques can be applied to analyze image series acquired on commercially available laser scanning or total internal reflection fluorescence microscopes, and are used to determine the number density, aggregation state, diffusion coefficient, velocity, and interaction fraction of fluorescently labeled molecules or particles. A comprehensive review of the application of ICS techniques to a number of systems, including cell adhesion, membrane receptor aggregation and dynamics, virus particle fusion, and fluorophore photophysics, is presented.     

Accurate measurements of protein interactions in cells via improved spatial image cross-correlation spectroscopy

The sensitive detection of protein interactions in living cells is an important first step toward understanding each of the multitude of cellular processes that are regulated by such interactions. Spatial image cross-correlation spectroscopy (ICCS) is one method used to measure protein-protein interactions from the analysis of two-channel fluorescence microscopy images. In spatial ICCS, cross-correlation of fluctuations in fluorescence intensity recorded as images from two independent wavelength detection channels in a fluorescence microscope is used to determine the average number of interacting particles in the imaged region. Even in situations where the particle number density is relatively high, ICCS provides an accurate measure of molecular interactions. However, it was shown previously that the method suffers from relatively high detection limits of interacting particles (approximately 20%) and can be perturbed by heterogeneous spatial distributions of the fluorescent particles within the images. Here, we demonstrate new approaches to circumvent some of the limitations of ICCS. Spatial scrambling of pixel blocks within fluorescence images was investigated as a way of extending the detection of spatial ICCS to measure lower interaction fractions as well as colocalization within cells. We also show that 'mean-intensity-padding' of regions of interest within fluorescence images is a feasible method of applying ICCS to arbitrarily selected areas of the cell with boundaries or edge morphologies that would be impossible to analyze with conventional ICCS. Using these newly developed strategies we were able to measure the fraction of actin that interacts with alpha-actinin in the leading edge of a migrating cell.     

Studies of distribution,location and dynamic properties of EGFR on the cell surface measured by image correlation spectroscopy

In this work, we have studied the distribution and dynamic properties of Epidermal Growth Factor (EGF) receptors in the plasma membrane of fixed and live cells as well as the extent of co-localization of this transmembrane protein with proteins specific for three-membrane microdomains: membrane rafts, caveolae and clathrin-coated pits. This was achieved using a family of image-processing tools called image correlation spectroscopy (ICS), image cross-correlation spectroscopy (ICCS) and dynamic image correlation spectroscopy (DICS). Our results indicate that EGFR is diffusely distributed on the cell surface at 37°C and aggregates as the temperature is lowered to 4°C. This aggregation takes place within 15 min and is reversible. Changes in temperature also affect the diffusion of EGFR by two orders of magnitude. The dynamic properties of EGFR are similar to the dynamic properties of a GPI-anchored protein known to be present in membrane rafts, which motivated us to explore the extent of co-localization of EGFR with this membrane raft protein using ICCS. Our results indicate that more than half of the EGFR population is present in membrane rafts and smaller percentages are present in caveolae and clathrin-coated pits.     

Measuring fast dynamics in solutions and cells with a laser scanning microscope

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.     

Detecting Protein Complexes in Living Cells from Laser Scanning Confocal Image Sequences by the Cross Correlation Raster Image Spectroscopy Method

We describe a general method for detecting molecular complexes based on the analysis of single molecule fluorescence fluctuations from laser scanning confocal images. The method detects and quantifies complexes of two different fluorescent proteins noninvasively in living cells. Because in a raster scanned image successive pixels are measured at different times, the spatial correlation of the image contains information about dynamic processes occurring over a large time range, from the microseconds to seconds. The correlation of intensity fluctuations measured simultaneously in two channels detects protein complexes that carry two molecules of different colors. This information is obtained from the entire image. A map of the spatial distribution of protein complexes in the cell and their diffusion and/or binding properties can be constructed. Using this cross correlation raster image spectroscopy method, specific locations in the cell can be visualized where dynamics of binding and unbinding of fluorescent protein complexes occur. This fluctuation imaging method can be applied to commercial laser scanning microscopes thereby making it accessible to a large community of scientists.     

Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells.

Previously we introduced image correlation spectroscopy (ICS) as an imaging analog of fluorescence correlation spectroscopy (FCS). Implementation of ICS with image collection via a standard fluorescence confocal microscope and computer-based autocorrelation analysis was shown to facilitate measurements of absolute number densities and determination of changes in aggregation state for fluorescently labeled macromolecules. In the present work we illustrate how to use ICS to quantify the aggregation state of immunolabeled plasma membrane receptors in an intact cellular milieu, taking into account background fluorescence. We introduce methods that enable us to completely remove white noise contributions from autocorrelation measurements for individual images and illustrate how to perform background corrections for autofluorescence and nonspecific fluorescence on cell population means obtained via ICS. The utilization of photon counting confocal imaging with ICS analysis in combination with the background correction techniques outlined enabled us to achieve very low detection limits with standard immunolabeling methods on normal, nontransformed human fibroblasts (AG1523) expressing relatively low numbers of platelet-derived growth factor-beta (PDGF-beta) receptors. Specifically, we determined that the PDGF-beta receptors were preaggregated as tetramers on average with a mean surface density of 2.3 clusters micrometer(-2) after immunolabeling at 4 degreesC. These measurements, which show preclustering of PDGF-beta receptors on the surface of normal human fibroblasts, contradict a fundamental assumption of the ligand-induced dimerization model for signal transduction and provide support for an alternative model that posits signal transduction from within preexisting receptor aggregates.     

Imaging molecular interactions in living cells

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.     

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application.

Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.     

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.     

In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal microscopy

Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells. The fluorescent molecules are often tagged proteins participating in a biological function. The high spatial resolution of confocal microscopy compared to wide field imaging comes from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions. Confocal microscopy at the optical diffraction limit images volumes on the order of approximately 0.5 femtoliter (10(-15) L). Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM). With TIRM, an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that, when combined with conventional confocal microscopy, permits image formation from <7 attoL (10(-18) L) volumes [Borejdo et al. (2006) Biochim. Biophys. Acta, in press]. Demonstrated here is a new variation of TIRM, focused TIRM (fTIRM) that decreases the volume element to approximately 3 attoL. These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements. A novel application for TIRM is in situ single-molecule fluorescence spectroscopy. Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging. In situ, proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro. The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ. Muscle tissue contains myosin at a approximately 120 microM concentration. Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.     

Measuring Actin Flow in 3D Cell Protrusions

Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast.     

Measuring Actin Flow in 3D Cell Protrusions

Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast.     

Sendai Virus Binds to a Dispersed Population of NBD-GD1a

Receptor aggregation is believed to be an important step in the attachmentof membrane enveloped virus' to target cell membranes. A likely receptorfor Sendai virus is the ganglioside GD1a. In this work we have studied themembrane diffusion of the fluorescent ganglioside NBD-GD1a on the surfaceof CV-1 cells with standard photobleaching techniques. Using confocallaser scanning microscopy (CLSM) and Image Correlation Spectroscopy(ICS) NBD-GD1a is shown to exist in at least two populations: dispersedand aggregated. By quantifying the distribution of NBD-GD1a pre- andpost-incubation with Sendai virus it is shown that the virus inducesa dose-dependent clustering of NBD-GD1a. Image cross-correlationspectroscopy (ICCS) is used to further quantitatively characterizethis clustering by demonstrating that it occurs due to binding ofvirus to the dispersed population of NBD-GD1a.     

Pin-Hole Array Correlation Imaging: Highly Parallel Fluorescence Correlation Spectroscopy

In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.     

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.     

Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromolecules

Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation analysis of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coefficients can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and analysis of computer simulated image time series verified that the effect we observe is caused by fluorescence intermittency. We show that reciprocal space image correlation analysis can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. We also demonstrate application of the image correlation methods for measurement of the diffusion coefficient of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts.     

Localization of protein-protein interactions in live cells using confocal and spectral imaging FRET microscopy

Microscopy has become an essential tool for cellular protein investigations. The development of new fluorescent markers such as green fluorescent proteins generated substantial opportunities to monitor protein-protein interactions qualitatively and quantitatively using advanced fluorescence microscope techniques including wide-field, confocal, multiphoton, spectral imaging, lifetime, and correlation spectroscopy. The specific aims of the investigation of protein dynamics in live specimens dictate the selection of the microscope methodology. In this article confocal and spectral imaging methods to monitor the dimerization of alpha enhancer binding protein (C/EBPalpha) in the pituitary GHFT1-5 living cell nucleus have been described. Also outline are issues involved in protein imaging using light microscopy techniques and the advantages of lifetime imaging of protein-protein interactions.     

Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex

Live-cell microscopy imaging of fluorescent-tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable-angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high-signal, low-background and near real-time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion-tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.