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ScienceDirect Applications of imaging fluorescence correlation spectroscopy Anand P Singh1,2 and Thorsten Wohland1,2 Imaging fluorescence correlation spectroscopy (imaging FCS), or the acquisition of fluorescence correlation functions at contiguous points in an imaging format, is a recent addition to quantitative bioimaging. Imaging FCS has been implemented in various modalities. These techniques provide excellent time resolution, have single molecules sensitivity and can be combined with super-resolution techniques, thus combining high spatial and temporal resolution. Although still at its beginning it has been applied in different forms to biological problems. This review looks at applications of imaging FCS in the last two years with the aim to give the reader an overview of the capabilities of these new techniques. Addresses 1 Department of Biological Sciencesand Centre for Bio-Imaging Sciences, National University of Singapore, Singapore, 117557, Singapore 2 Department of Chemistry and Centre for Bio-Imaging Sciences, National University of Singapore, Singapore, 117557, Singapore Corresponding author: Wohland, Thorsten ([email protected]
Current Opinion in Chemical Biology 2014, 20:29–35 This review comes from a themed issue on Molecular imaging Edited by Christian Eggeling and Mike Heilemann
http://dx.doi.org/10.1016/j.cbpa.2014.04.006 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction Fluorescence correlation spectroscopy (FCS) has been widely applied in cells, tissues and organisms to study biological processes at the molecular level [1–3]. Its almost exclusive implementation in confocal microscopes, however, imposed a range of limitations. Mainly single points were collected, restricting the available statistics per sample; the resolution was limited to the optical diffraction limit (200 nm); and the illumination in confocal optics extends over a much wider region than the actual observation volume, resulting in unnecessary photobleaching of the sample and thus restricting the number of measurement that could reliably be taken. A number of FCS modalities, which differ in illumination and detection schemes, were introduced to address these restrictions (Figures 1 and 2) [4,5]. The recording of multiple points in FCS in a confocal mode was introduced by using multiple confocal volumes created by microlens arrays, spatial light modulators, line-confocal scanning detection www.sciencedirect.com
or using a spinning disk confocal microscope [6–9]. But they suffered from the fact that confocal elements will cross-talk and thus need to be placed at a sufficiently large distance from each other, preventing it from being used as a true imaging technique. However, the confocal techniques have better time resolution compared to camera-based FCS and can provide some spatial information. Raster image correlation spectroscopy (RICS) circumvents the cross-talk problems by scanning the confocal volume through the sample. It uses the inherent spatial and temporal information in the scanned confocal images to calculate spatio-temporal correlations over multiple areas within the image thus being able to provide maps of diffusion coefficients, concentrations and interactions. But due to the scanning process RICS modalities do not collect data at all points simultaneously, have anisotropic time resolution, and still illuminate a much larger region than what is actually observed. With the illumination of selected planes in a sample — either as total internal reflection (TIR) for surfaces or as single plane illumination microscopy (SPIM) for 3D samples — and the advent of fast sensitive cameras, these restrictions were overcome [10,11]. This was first used in spatio-temporal image correlation spectroscopy (STICS), which uses confocal or TIR illumination to record spatio-temporal correlations, and was then implemented in imaging TIR-FCS (ITIRFCS) and SPIM-FCS to calculate temporal correlations at each pixel or spatio-temporal correlations between any groups of pixels in an image (Figure 3) [12,13]. ITIRFCS and SPIM-FCS collect correlation functions at thousands of contiguous points in an imaging mode in which only those areas are illuminated which are also observed, resulting in better statistics, lower photodamage per measurement and more measurements per sample over longer periods of time (Figure 1). In the following we review the applications of these different imaging FCS modalities to biological problems with an emphasis on their capabilities to gain new biological information.
Membrane dynamics and organization Biological cell membranes are dynamic, complex structures consisting of a large variety of lipids, carbohydrates and proteins. They not only create a barrier between the inside and outside of the cell but regulate molecular transport across the membrane and are important in cell adhesion, communication and signaling. While cell membrane compositions have been determined, the structure and dynamics still pose a challenge. Imaging of the organization at the nanometer scale with a time resolution in the millisecond range, which are thought to be relevant in biological processes, is possible with few instruments [14,15]. Different approaches to obtain quantitative FCS Current Opinion in Chemical Biology 2014, 20:29–35
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Illustration of illumination and observation region: (Blue color for illumination path and green color for the observed region): (a) Raster laser scanning approach to create point by point sequential images. (b) Spinning disk to create multiple observation volumes (ML — microlens disk, PH — pinhole disk). Planar illumination and parallel imaging to array detectors: (c) An objective-type total internal reflection microscope creates a homogeneous illumination plane (0.1–0.2 mm thick) to access the bottom cell membrane or structures close to the coverslip. (d) SPIM imaging by a light sheet created by a cylindrical lens. SPIM creates a thin section in the sample. The sample is translated through the light sheet to create 3D images. As indicated by the gray areas confocal approaches illuminate and induce photobleaching in the whole sample, while light-sheet based methods illuminate only the observed region.
images have been able to meet at least some of these challenges. RICS, developed by Digman et al. [16,17], was used for mapping the diffusion coefficient of the membrane protein myelin oligodendrocyte glycoprotein (MOG) in OLN-93 cells . Hedde et al. combined stimulated emission depletion (STED) microscopy with RICS  (see Figure 2). This was the first attempt to combine any super-resolution approach to an image correlation method providing simultaneous temporal resolution at the ms range and spatial resolution of 40–80 nm. Information on membrane organization is not only accessible by direct imaging but can also be inferred from the dependence of the membrane dynamics with observation area and temperature. Freely diffusing particles have a diffusion coefficient, which does not depend on the observation area. But if the particle experiences hindrance to diffusion at different length scales the diffusion coefficient will change when the observation area crosses from one length scale range to another. This was formulated as the so-called ‘FCS diffusion law’ [20,21]. In addition, the temperature dependence of the diffusion coefficient contains information about the diffusion activation energy, which can be related to lipid packing and phases . In order to understand the membrane organization, Di Rienzo et al. and Bag et al. used the ‘FCS diffusion law’ and the temperature dependence of the diffusion coefficient to understand the spatial-temporal dynamic organization of biological membranes [23,24]. This was shown on the examples of the transferrin receptor (TfR), which is restricted in its movement by the actin cytoskeleton, and glycosylphosphatidylinositol (GPI), which is assumed to interact with cholesterol and sphingolipid dependent domains or rafts. TfR clearly showed a different spatial Current Opinion in Chemical Biology 2014, 20:29–35
organization in presence and absence of the actin cytoskeleton, while GPI showed a bimodal distribution for raft trapped and free diffusion. Bag et al. used the imaging FCS diffusion law and Arrhenius plots obtained from temperature dependence of diffusion for measuring cell membrane compositional and/or organizational heterogeneity in live cells . They showed that the membrane diffusion of different cell types correlates with their respective protein content and assumed raft fraction. These approaches, although their spatial resolution is diffraction limited, can provide important information on membrane structures even below the diffraction limit.
Protein dynamics in cytosol and nucleus Protein dynamics in the cytosol and nucleus are much faster than in membranes and thus FCS modalities with a time resolution below about 50 ms are necessary . This can be reached only with few cameras and novel techniques are required [10,11]. Heuvelman et al. introduced camera based line-confocal microscopy to characterize chromatin-remodeling complexes in living cells . Oh and colleagues developed time-integrated multipoint moment analysis (TIMMA), an imaging method capable of resolving spatial variation in concentration, brightness and diffusion dynamics at the time scale down to 20 ms . TIMMA analyses the mean and variance of the fluorescence signal in each pixel as a function of the camera exposure time. This analysis contains the same information as can be obtained by FCS but is now limited only by the exposure time of the camera, not by the readout time and thus provides faster time resolution. They used TIMMA to study the internal gradient of GTPase Ran protein distribution and dynamics in live HeLa cells. Although implemented on a spinning disk microscope www.sciencedirect.com
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Schematics of detection schemes and the principles of correlation analysis: (a) In pulsed interleaved excitation (PIE) different colored labels are excited with alternating pico-second pulses. Thus photons can be collected for either the red or green label only, resulting in cross-talk free data. Collection of data at fast time scales provides information on the fluorophore lifetimes, while correlations at long time scales provides information on fluorophore dynamics [47,50]. (b) The observation volume for RICS can be significantly decreased using stimulated emission depletion (STED) illumination . (c) Time image series that can be either used for imaging FCS (20 20 pixels region of interest, recording at 2000 fps) or STICS analysis (full frame, recording at 10–30 fps). (d) Raster scan image (50–100 frames, pixel dwell time 6–20 ms and pixel size 40–60 nm). (e) kICS analysis: a 2D fast Fourier transform (2D-FFT) of each time frame image is performed to calculate a k-space time stack series which can then be analyzed [27,51].
with a stationary disk to produce multiple confocal volume elements, the same principle could be applied to light-sheet based methods for the investigation of spatial dynamics and gradients in live cells with excellent time resolution.
Protein–protein and ligand–receptor interaction Several techniques were used to measure molecular interactions although truly spatial resolved interaction maps have not yet been provided. Branda˜o et al. studied the binding kinetics of cholera toxin B (CTxB) to its glycolipid receptor (GM1) on live cells by using reciprocal k-space ICS (kICS) www.sciencedirect.com
 (see Figure 2e). kICS analysis, similar in the data recording to STICS, provides a simple way to separate binding from spatio-temporal dynamics. RICS allows distinguishing diffusion and binding events although often the signal to noise ratio is not good enough for live cell measurements . However, Hof and colleagues applied the method for studying biomacromolecular dynamics and distinguished diffusion and transient binding events in nanofiber scaffolds used in tissue engineering . Choi et al. implemented dual-color cross-correlation RICS for detecting protein complexes in live cells, and found that binding between paxillin and focal adhesion kinase (FAK) is strongly dependent on the phosphorylation state of paxillin Current Opinion in Chemical Biology 2014, 20:29–35
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nucleation and monitors the time dependent growth of hIAPP/lipid domains in time lapse imaging FCS movies on live cells. These applications demonstrate the strength of imaging FCS approaches, which can monitor local fast dynamics with millisecond time resolution over an area of 100 mm2 over many minutes to hours.
Intracellular transport and cellular uptake (a)
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Principle of imaging FCS (ITIR/SPIM-FCS): (a) Acquisition of a time image series with millisecond resolution or better. Typically 10 000–50 000 frames are acquired of which a single frame is shown here. The colored line depicts the trace of a diffusing particle. (b) Temporal fluorescence fluctuation of a single pixel (inset image), which consists of the crossing of many single particles, and its auto correlation function (ACF in blue color and fit in gray color). (c) and (d) Diffusion and concentration maps obtained from the autocorrelation functions for each pixel.
Cell migration and the complex transport of various cytoskeleton proteins in living organism are tightly regulated in both space and time, and contribute to tissue repair, regeneration and cell polarization . Toplak et al. employed dual color spatio-temporal image cross-correlation spectroscopy (STICCS), which estimates and allows distinguishing various transport and co-transport modes of binding adhesion proteins labeled with two spectrally distinct fluorescent proteins in live cells . The bivariate correlation fits follow the time evolution of isotropic and anisotropic transport. Often parameters determined by STICS/ STICCS require filtering to avoid the contribution of the dominant immobile fraction. The extension of ICS to the time-frequency domain (n-nu; nICS) provided a solution to this problem and the method was applied to quantify the retrograde flow of integrin (aLb2) and paxillin in CHO.B2 cells . Recently, lipid-complexes, nanoparticles and single wall carbon nanotubes (SWNTs) were used to study spatial cellular uptake pathways and the temporal dynamics in specific region of live cells by using STICS and RICS [37– 41]. These noninvasive spatio-temporal image correlation approaches provide a robust quantitative tool for spatial visualization and temporal intracellular dynamics of bioactive materials in living system.
Measuring dynamics in tissues and organisms (positions Y31 and Y118), which plays an important role in regulating nascent adhesion complex formation . These and the recently developed SPIM-FCCS have the potential to provide interaction maps for a sample even in a 3D live sample .
Aggregation of proteins and peptides Protein and peptide aggregation play an important role in cell signaling but also in human diseases and the distinction between the actions of monomers and oligomers is important in understanding the underlying mechanisms. Imaging FCS has been used in a number of ways to help elucidate protein and peptide aggregation. Vetri et al. studied the aggregation of Concanavalin A (ConA) in live cells by combining RICS and number-brightness analysis . The results from this study suggest that the cell membrane encourages ConA protein aggregation at higher local concentration and causes cell toxicity. In another membrane associated cyto-toxicity study it was found that monomeric human islet amyloid polypeptide (hIAPP) significantly damages the cell membrane at concentrations smaller than 1 mM [32,33]. The method allows localizing the region of Current Opinion in Chemical Biology 2014, 20:29–35
Imaging FCS approaches are feasible even in tissues. Recently, Brewer et al. used cross-correlation RICS (ccRICS) to examine the transdermal penetration of liposomes through different depths of human skin . However, the discrepancy between the illuminated region and the actually detected region increases with the size of the specimen and the advantages of light sheet-based microscopy become increasingly important as they reduce photodmage to the sample (Figure 1) [43–45]. SPIM-FCS was, for example, used to measure blood flow in live zebrafish embryos  and green fluorescent protein (GFP) fused with a nuclear localization signals (NLS) in isolated wing imaginal discs of Drosophila melanogaster larvae .
Summary and outlook Imaging FCS is still at its beginning regarding technical implementations but especially also in its applications. However, as is evident these methods already provide new information on membrane organization, molecular transport and cellular trafficking pathways on both spatial and temporal scales. Disadvantages of imaging FCS exist either on the temporal scale (camera based approaches) or on the problem of specimen exposure and isotropic time and www.sciencedirect.com
Applications of imaging FCS Singh and Wohland 33
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Single color and dual color SPIM-FCS measurements on live cells: (a) Diffusion and concentration maps of monomeric enhanced green fluorescent protein (EGFP) in RBL-2H3 cells. (b) c-Fos-EGFP expressed in HeLa cells (we thank Jo¨rg Langowski for the kind gift of the c-Fos-EGFP plasmid and Antonija Burcˇul for the cell preparation). (c) Simultaneous diffusion and concentration measurement of membrane proteins which possess two different labels. Here we used a plasma membrane targeting (PMT) sequence labeled with a monomeric red fluorescent protein (mRFP) or enhanced green fluorescent protein (EGFP). (d) Simultaneous recording of membrane localized mRFP-PMT and cytosolic EGFP in CHO cells, clearly showing the distinct diffusion dynamics of membrane and cytosol.
spatial resolution (scanning based approaches); and both approaches are typically limited in spatial resolution. Recently, several new modalities address some of these issues. STED-RICS is the first combination of a superresolution technique with imaging FCS significantly improving spatial resolution . The use of pulsed interleaved excitation (PIE) with RICS reduces cross-talk and combines lifetime information with imaging FCS (see Figures 1 and 2) . And camera based FCS could soon reach nanosecond time resolution with the development of single photon avalanche diode (SPAD) arrays [48,49]. The existing imaging FCS technologies have not yet reached the same temporal resolution as the already existing single point technologies. However, their multiplexing advantage and their combination of high spatial and temporal resolution make them excellent quantitative bioimaging techniques, which provide new information not previously accessible www.sciencedirect.com
(Figure 4). Although we are at an early stage, Imaging FCS techniques develop fast and provide new opportunities for the investigation of complex spatio-temporal processes in live cells and organisms.
Acknowledgements APS is the recipient of a National University of Singapore graduate scholarship and TW gratefully acknowledges funding by a Singapore Ministry of Education grant, R-154-000-534-112.
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