Scanning Microphotolysis: Three-Dimensional Diffusion Measurement and Optical Single-Transporter Recording

Scanning microphotolysis (SCAMP) is a combination of fluorescence microphotolysis and confocal laser scanning microscopy. A laser scanning microscope is equipped with an optical switch able to modulate the power or/and wavelength of the laser beam in less than a microsecond while a dedicated computer program is employed to precisely coordinate scanning process and laser beam modulation. By these means it becomes possible to vary the power or/and wavelength of the laser beam during scanning at a precision of one resolution element. Patterns of almost arbitrary design can be written into the object by photolysis, e.g., photobleaching or photoactivation. The dissipation of the photolysis pattern by diffusion or other types of molecular transport can be followed at confocal resolution and used to characterize the transport process. SCAMP can be employed in conjunction with single-photon or multiphoton excitation. Furthermore, it can be easily installed on virtually any confocal laser scanning microscope. We summarize at first the conceptual and practical basis of SCAMP. Then, two novel applications are discussed: (i) measurements of translational diffusion coefficients in truly three-dimensional systems at diffraction-limited resolution, and (ii) optical recording of single transporters in membrane patches.     

k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or "blinking". Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of alpha5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection.     

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

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

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples

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

Fluorescence pattern photobleaching recovery for samples with multi-component diffusion

The translational mobility of proteins and lipids in phospholipid bilayers is often not well described as ideal self diffusion. One of the best methods for characterizing such non-ideal diffusion is to use fluorescence pattern photobleaching recovery. In this method, the spatial gradient of the monitoring and bleaching intensity is created by using epi-fluorescence and an expanded Gaussian-shaped laser beam which passes though a Ronchi ruling placed at the back image plane of a microscope. A difficulty arises when the fluorescence recovery from the exchange of slowly diffusing molecules between illuminated and non-illuminated stripes temporally overlaps with the recovery from the exchange of more rapidly diffusing molecules through the gradient produced by the broad Gaussian shape of the illumination. In the work presented here, a general theory is developed that describes the shape of the resulting fluorescence recovery curve for these typical experimental conditions. Approximate expressions amenable to non-linear curve fitting are also given. The new theoretical formalism has been demonstrated on data for the translational mobility of a fluorescent lipid probe in phospholipid bilayers deposited on planar-fused silica substrates.     

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.     

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.     

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.     

Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP.

In eukaryotes, protein trafficking to and from the nucleus, or shuttling, has been demonstrated to be an important function for proteins that have vital roles in one or both subcellular compartments. Current techniques of detecting protein nuclear shuttling are extremely labor intensive and only statically visualize evidence of shuttling. Fluorescence recovery after photobleaching (FRAP), or fluorescence microphotolysis, has proven to be an effective method of analyzing protein dynamics in live cells, especially when coupled to GFP technology. Here, we describe a relatively simple in vivo protein nuclear shuttling assay that utilizes red fluorescent and green fluorescent protein fusions as substrates for FRAP using a laser confocal microscope. This technique is less time consuming than established shuttle assays, is internally controlled, and visualizes nucleocytoplasmic shuttling in living cells of the same species and cell type. This technique can be potentially used to detect the ability of any nuclear protein to shuttle from the nucleus to any other subcellular compartment for any eukaryotic species in which GFP or dsRed1 fusion protein can be expressed.     

Raster image correlation spectroscopy in live cells

Raster image correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in a live cell, including concentration of molecules and diffusion coefficients of molecules; in addition, by measuring changes in diffusion coefficients, RICS can indirectly detect binding. Any specimen containing fluorophores that can be imaged with a laser scanning microscope can be analyzed using RICS. There are other techniques to measure diffusion coefficients and binding; however, RICS fills a unique niche. It provides spatial information and can be performed in live cells using a conventional confocal microscope. It can measure a range of diffusion coefficients that is not accessible with any other single optical correlation-based technique. In this article we describe a protocol to obtain raster scanned images with an Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Each RICS measurement takes several minutes. The entire procedure can be completed in ～2 h. This procedure includes focal volume calibration using a solution of fluorophores with a known diffusion coefficient and measurement of the diffusion coefficients of cytosolic enhanced green fluorescent protein (EGFP) and EGFP-paxillin.     

Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion.

Fluorescence microphotolysis was combined with confocal laser-scanning microscopy to yield a method, herein referred to as line-scanning microphotolysis (LINESCAMP), for the measurement of molecular transport at a lateral resolution of approximately 0.34 microns and a temporal resolution of approximately 0.5 ms. A confocal microscope was operated in the line scan mode, while the laser beam power could be switched during scanning between low monitoring and high photolysing levels in less then a microsecond. The number and location of line segments to be photolysed could be freely determined. The length of the photolysed segments could be also chosen and was only limited by diffraction. Together with instrumentation a new, completely general, theoretical framework for the evaluation of diffusion measurements was developed. Based on the numerical simulation of diffusion processes employing a modified Crank-Nicholson scheme, the theory could be applied to any photobleaching geometry and profile as the initial condition and took into account the convolution with the microscope point spread function. With small diffraction-limited areas, the method yielded accurate values for diffusion coefficients in the range between approximately 10(-4) and 1 micron2 s-1. A first application of the method to the diffusion of a fluorescently labeled tracer inside the cell nucleus showed the potential of the method for the study of complex biological systems.     

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.     

Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy

Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50-100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules.     

Steady state dynamics of intermediate filament networks

We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.     

A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.     

A nonfitting method using a spatial sine window transform for inhomogeneous effective-diffusion measurements by FRAP

Determining averaged effective diffusion constants from experimental measurements of fluorescent proteins in an inhomogeneous medium in the presence of ligand-receptor interactions poses problems of analytical tractability. Here, we introduced a nonfitting method to evaluate the averaged effective diffusion coefficient of a region of interest (which may include a whole nucleus) by mathematical processing of the entire cellular two-dimensional spatial pattern of recovered fluorescence. Spatially and temporally resolved measurements of protein transport inside cells were obtained using the fluorescence recovery after photobleaching technique. Two-dimensional images of fluorescence patterns were collected by laser-scanning confocal microscopy. The method was demonstrated by applying it to an estimation of the mobility of green fluorescent protein-tagged heterochromatin protein 1 in the nuclei of living mouse embryonic fibroblasts. This approach does not require the mathematical solution of a corresponding system of diffusion-reaction equations that is typical of conventional fluorescence recovery after photobleaching data processing, and is most useful for investigating highly inhomogeneous areas, such as cell nuclei, which contain many protein foci and chromatin domains.     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

Spatial Fourier analysis of video photobleaching measurements. Principles and optimization.

The major use of the fluorescence recovery after photobleaching (FRAP) technique is to measure the translational motion of the molecular components in various condensed media. In a conventional laser spot photobleaching experiment, a photomultiplier is used to measure the total brightness levels of the bleached region in the sample, so no spatial information can be directly obtained. In video-FRAP, a series of images after photobleaching is acquired, allowing the spatial character of the recovery to be determined; this permits direct detection of both anisotropic diffusion and flow. To utilize all of the available image data to determine the transport coefficients, a two-dimensional spatial Fourier transform analysis of the images after photobleaching was employed. The change in the transform between two time points reflects the action of diffusion during the interim. An important advantage of this method, which involves taking the ratio of image transforms at different time points, is that it does not require a specific initial condition to be created by laser photobleaching. The ability of the analysis to extract transport coefficients from computer-simulated diffusional recovery is assessed in the presence of increasing amounts of noise. Experimental data analysis from the diffusion of proteins in viscous solutions and from the diffusion of protein receptors on cell surfaces demonstrate the feasibility of the Fourier analysis to obtain transport coefficients from the video FRAP measurement.