Spatiotemporal Image Correlation Spectroscopy Research Articles

Quantifying superimposed protein flow dynamics in live cells using spatial filtering and spatiotemporal image correlation spectroscopy.

Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin-based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton.

Image correlation spectroscopy approaches to probe diffusion in cell.

Among the methods for studying molecular dynamics through fluorescence, fluorescence correlation spectroscopy (FCS) is one of the most effective in vivo experimental techniques. It returns information about the diffusion of fluorescent molecule pools in a single well-defined small volume by means of auto/cross-correlating fluorescence intensity fluctuation. Since probing a single point may not be representative of a complex landscape, we can use the spatio-temporal image correlation spectroscopy (stICS) method. Using a sequence of images taken on the same functional biological area, such a method computes the auto/cross-correlation over the entire region. Since the diffusive parameters are extracted from the autocorrelation map as an average over a region of interest, stICS can be susceptible to spatial inhomogeneities of the fluorescent moiety in the same area. However, vesicles, membranes, confinements are sometimes poorly detectable from the sole fluorescence intensity. If different diffusion pools are present, some of them may be missed from the analysis of the global autocorrelation curve on account of the signal-to-noise ratio and/or their dynamic characteristics. This work explores different independent approaches to reveal the spatial feature of the molecular dynamics inside the cell. These approaches required a sequence of images to perform FCS analysis pixel by pixel. Each single-pixel autocorrelation evaluation avoids mixing different contributions due to average dynamics pools over a large field of view. Among the developed algorithm, the most promising is related to an analysis based on Maximum Entropy, which, for each point, gives a spectrum of Brownian Diffusion coefficients. This information can help link morphological and dynamics feature, also through correlative analysis with other spatial techniques, enabling new imaging schemes. These approaches are applied to recover membrane characteristics in cell's membrane such as PDL1, a significant player in an immune-checkpoint also involved in cancer progression.

Förster Resonance Energy Transfer-Based Single-Cell Imaging Reveals Piezo1-Induced Ca2+ Flux Mediates Membrane Ruffling and Cell Survival.

A mechanosensitive ion channel, Piezo1 induces non-selective cation flux in response to various mechanical stresses. However, the biological interpretation and underlying mechanisms of cells resulting from Piezo1 activation remain elusive. This study elucidates Piezo1-mediated Ca2+ influx driven by channel activation and cellular behavior using novel Förster Resonance Energy Transfer (FRET)-based biosensors and single-cell imaging analysis. Results reveal that extracellular Ca2+ influx via Piezo1 requires intact caveolin, cholesterol, and cytoskeletal support. Increased cytoplasmic Ca2+ levels enhance PKA, ERK, Rac1, and ROCK activity, which have the potential to promote cancer cell survival and migration. Furthermore, we demonstrate that Piezo1-mediated Ca2+ influx upregulates membrane ruffling, a characteristic feature of cancer cell metastasis, using spatiotemporal image correlation spectroscopy. Thus, our findings provide new insights into the function of Piezo1, suggesting that Piezo1 plays a significant role in the behavior of cancer cells.

Stics Analysis Reveals the Role of Tau Phosphorylation in Regulating Lysosome Transport

Organelles move through a combination of active transport by motor proteins and diffusion. Tau, a protein that binds to mictrobule tracks, regulates the transport of organelles. To study how tau phosphorylation influences intracellular transport, we applied image correlation spectroscopy analysis to live-cell microscopy images of lysosomes in COS-7 cells expressing tau phosphomutants. Spatio-temporal image correlation spectroscopy (STICS) correlates fluorescence intensity fluctuations within an image through space and over time to measure velocities of mobile molecules and complexes. Compared to conventional tracking analysis, STICS does not rely on detecting and following individual fluorophores through space and time. Using total internal reflection fluorescence microscope, we performed live cell imaging on lysosomes labelled with lysotracker in COS-7 cells, and applied STICS to analyze the image time series. The STICS transport maps were further analyzed to generate histograms of the velocity magnitude and angle distributions of lysosome transport under different tau conditions. Using STICS analysis, we could resolve differences in the direction of lysosome transport under varying tau concentrations. Our results show that lysosome motility towards the cell nucleus increases, in presence of higher tau concentrations, whereas lower concentrations of tau result in vesicles moving both towards the periphery and the perinuclear region. Lysosomes at the central region of the cell are not inhibited by phosphomimetic tau. In contrast, lysosomes at the cell periphery are inhibited by wild-type and phosphomimetic tau. STICS analysis on lysosome motility shows that lysosomes exhibit different motility in the presence of tau according to their localization within the cell. Our results show that tau and its phosphorylation regulate cellular lysosome motility.

"Basal Cell Migration" in Regeneration of the Corneal Wound-Bed.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

Image Correlation Spectroscopy with Second Harmonic Generating Nanoparticles in Suspension and in Cells.

The absence of photobleaching, blinking, and saturation combined with a high contrast provides unique advantages of higher-harmonic generating nanoparticles over fluorescent probes, allowing for prolonged correlation spectroscopy studies. We apply the coherent intensity fluctuation model to study the mobility of second harmonic generating nanoparticles. A concise protocol is presented for quantifying the diffusion coefficient from a single spectroscopy measurement without the need for separate point-spread-function calibrations. The technique's applicability is illustrated on nominally 56 nm LiNbO3 nanoparticles. We perform label-free raster image correlation spectroscopy imaging in aqueous suspension and spatiotemporal image correlation spectroscopy in A549 human lung carcinoma cells. In good agreement with the expected theoretical result, the measured diffusion coefficient in water at room temperature is (7.5 ± 0.3) μm2/s. The diffusion coefficient in the cells is more than 103 times lower and heterogeneous, with an average of (3.7 ± 1.5) × 10-3 μm2/s.

Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series

Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells.

Spatio-temporal image correlation spectroscopy and super-resolution microscopy to quantify molecular dynamics in T cells.

Many cellular processes are regulated by the spatio-temporal organisation of signalling complexes, cytoskeletal components and membranes. One such example is at the T cell immunological synapse where the retrograde flow of cortical filamentous (F)-actin from the synapse periphery drives signalling protein microclusters towards the synapse centre. The density of this mesh however, makes visualisation and analysis of individual actin fibres difficult due to the resolution limit of conventional microscopy. Recently, super-resolution methods such as structured illumination microscopy (SIM) have surpassed this resolution limit. Here, we apply SIM to better visualise the dense cortical actin meshwork in T cell synapses formed against activating, antibody-coated surfaces and image under total-internal reflection fluorescence (TIRF) illumination. To analyse the observed molecular flows, and the relationship between them, we apply spatio-temporal image correlation spectroscopy (STICS) and its cross-correlation variant (STICCS). We show that the dynamic cortical actin mesh can be visualised with unprecedented detail and that STICS/STICCS can output accurate, quantitative maps of molecular flow velocity and directionality from such data. We find that the actin flow can be disrupted using small molecule inhibitors of actin polymerisation. This combination of imaging and quantitative analysis may provide an important new tool for researchers to investigate the molecular dynamics at cellular length scales. Here we demonstrate the retrograde flow of F-actin which may be important for the clustering and dynamics of key signalling proteins within the plasma membrane, a phenomenon which is vital to correct T cell activation and therefore the mounting of an effective immune response.

Spatiotemporal image correlation analysis of blood flow in branched vessel networks of zebrafish embryos.

Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images. Here, we develop and test advanced methods that allow mapping the flow fields in branched vessel networks at the resolution of 10 to 20 μm. The methods, based on the application of spatiotemporal image correlation spectroscopy and its extension to cross-correlation analysis, are applied here to the case of early stage embryos of zebrafish.

Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse

The cortical actin cytoskeleton has been shown to be critical for the reorganization and heterogeneity of plasma membrane components of many cells, including T cells. Building on previous studies at the T cell immunological synapse, we quantitatively assess the structure and dynamics of this meshwork using live-cell superresolution fluorescence microscopy and spatio-temporal image correlation spectroscopy. We show for the first time, to our knowledge, that not only does the dense actin cortex flow in a retrograde fashion toward the synapse center, but the plasma membrane itself shows similar behavior. Furthermore, using two-color, live-cell superresolution cross-correlation spectroscopy, we demonstrate that the two flows are correlated and, in addition, we show that coupling may extend to the outer leaflet of the plasma membrane by examining the flow of GPI-anchored proteins. Finally, we demonstrate that the actin flow is correlated with a third component, α-actinin, which upon CRISPR knockout led to reduced plasma membrane flow directionality despite increased actin flow velocity. We hypothesize that this apparent cytoskeletal-membrane coupling could provide a mechanism for driving the observed retrograde flow of signaling molecules such as the TCR, Lck, ZAP70, LAT, and SLP76.

Velocity Landscapes Resolve Multiple Dynamical Populations from Fluorescence Image Time Series

Molecular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With the advance of fluorescence microscopy modalities we can capture the movement of these objects at higher spatio-temporal resolutions and better sensitivity. Nevertheless, the underlying assumption of the existing approaches to quantify molecular flows, on the basis of Spatio-Temporal Image Correlation Spectroscopy (STICS), is that sampling windows contain one dominant dynamic population. In the complex cellular environment, two or more dynamic populations can sample the same spatio-temporal window. We propose here an approach, termed velocity landscape, to assess and extract from correlation of pixels‘ fluorescence fluctuations all of the components present in the window of analysis. First we demonstrate the robustness of this approach compared to the standard STICS mapping, through set of simulated image time series at varying signal-to-noise, molecular number densities and sampling window sizes. As a proof of concept biological study, we apply the velocity landscape to map the fluxes of the flow of STIM1 on microtubules towards the plasma membrane of primary human bronchial epithelial (HBE) cells.

Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization.

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area.

Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles

Fluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system’s symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed flow motions, when compared to SpatioTemporal Image Correlation Spectroscopy (STICS). Although we present a specific example (lipoplexes in living cells), the emphasis is in the discussion of the method, its basic assumptions and its validation on numeric simulations. Statement of SignificanceRecent advances in nanoparticle-based drug and gene delivery systems have pointed out the interactions at cellular and subcellular levels as key-factors for the efficiency of the adopted biomaterials. Such biochemical and biophysical interactions drive and affect the intracellular dynamics, that is commonly characterized by means of fluorescence microscopy and spectroscopy techniques. Here we present a novel Image Correlation Spectroscopy (ICS) method as a promising tool to capture the intracellular behavior of nanoparticles with high resolution and low background’s sensitivity. This study overcomes some of the approximations adopted so far, by decoupling the flow terms of the investigated dynamics and thus recovering ensemble’s information from specific single particle behaviors. Finally, relevant implications for nanoparticle-based drug delivery are shown.

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

By following single fluorescent molecules in a microscope, single-particle tracking (SPT) can measure diffusion and binding on the nanometer and millisecond scales. Still, although SPT can at its limits characterize the fastest biomolecules as they interact with subcellular environments, this measurement may require advanced illumination techniques such as stroboscopic illumination. Here, we address the challenge of measuring fast subcellular motion by instead analyzing single-molecule data with spatiotemporal image correlation spectroscopy (STICS) with a focus on measurements of confined motion. Our SPT and STICS analysis of simulations of the fast diffusion of confined molecules shows that image blur affects both STICS and SPT, and we find biased diffusion rate measurements for STICS analysis in the limits of fast diffusion and tight confinement due to fitting STICS correlation functions to a Gaussian approximation. However, we determine that with STICS, it is possible to correctly interpret the motion that blurs single-molecule images without advanced illumination techniques or fast cameras. In particular, we present a method to overcome the bias due to image blur by properly estimating the width of the correlation function by directly calculating the correlation function variance instead of using the typical Gaussian fitting procedure. Our simulation results are validated by applying the STICS method to experimental measurements of fast, confined motion: we measure the diffusion of cytosolic mMaple3 in living Escherichia coli cells at 25 frames/s under continuous illumination to illustrate the utility of STICS in an experimental parameter regime for which in-frame motion prevents SPT and tight confinement of fast diffusion precludes stroboscopic illumination. Overall, our application of STICS to freely diffusing cytosolic protein in small cells extends the utility of single-molecule experiments to the regime of fast confined diffusion without requiring advanced microscopy techniques.

Tracking and localization of calmodulin in live cells

The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of <100nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca2+. Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from <0.01μm2s−1 to ~5μm2 s−1, whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of >10μm2s−1. For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells.

Investigating the Dynamics of Vibrio Cholerae Virulence Initiation by Stics and Single Molecule Tracking

Single molecule imaging techniques have provided insight into a vast realm of new biological processes. However, many of these techniques rely on capturing a sufficiently high number of emitted photons to localize a molecule and can thus fail when the photon count is low. In a system where a large fraction of the fluorescent labels are very dim and/or moving quickly, localization methods can fail or be biased toward the slowest and brightest molecules in the system. Fortunately, correlation-based methods can provide insight into dynamics in these systems. Spatio-Temporal Image Correlation Spectroscopy (STICS) uses the full spatiotemporal correlation of a fluorescent image time series to provide information such as the diffusion constants and the densities of fluorescent molecules. STICS does not rely on the emitters positions and thus operates with higher precision in systems where sub-diffraction limited localization fails. We have previously developed STICS for imaging single molecules in living bacteria cells, and here we apply STICS to the specific case of the V. cholerae virulence signaling pathway. In particular, the membrane-bound TcpP, which controls downstream expression of the cholera enderotoxin, is a dynamic protein whose behavior is believed to be affected by various environmental signals including temperature and specific small molecule hormones. Here, we examine the fluorescent signals from single molecules of TcpP fused at the native locus to the photoactivatable fluorescent protein PAmCherry. We analyze these data by STICS and single molecule tracking to compare these two techniques in realistic imaging conditions. Because each technique operates with higher precision in a different imaging regime, we conclude that these approaches used together can provide highly complementary information about the dynamics of TcpP.

From Nanoscale to Mesoscale: Integrating Advanced Microscopy Techniques to Reveal the Ultrastructure and Coordinated Dynamics of Mechanosensory Podosomes

Podosomes are dynamic integrin- and actomyosin-based multi-molecular adhesion structures involved in cell protrusion, topography sensing and extracellular matrix degradation. Formed by a variety of cells, they have a dotted shape and undergo irregular cycles of growth and shrinkage driven by actin polymerization and myosin contractility. With an actine core of ∼0.5um, hundreds of podosomes organize in different mesoscale spatial arrangements ranging from large circular belts in bone-degrading osteoclasts to small rosettes in endothelial cells and well-defined large clusters in antigen-presenting cells. The molecular mechanisms regulating formation and maintenance of these mesoscale arrangements are still poorly defined. By integrating a variety of advanced microscopy techniques, we aim to unravel the nanoscale structural and dynamic complexity of individual podosomes as well as formation, architecture and function of mesoscale podosome clusters. We previously demonstrated that, at the nanoscale, single podosomes comprise a dense actin core with radiating actin filaments decorated by vinculin, and that integrins and talin densely cover the entire cluster area except the actin core regions. At present, by exploiting an extension of spatiotemporal image correlation spectroscopy (STICS), we measured the evolution of spatiotemporal molecular dynamics of podosome components, revealing self-organized dynamic spatial patterns of various cytoskeletal components throughout the clusters. Furthermore, by multi-color SIM and 3D STORM, we revealed the presence of actin substructures regulating the core nanoscale architecture that likely contribute to their protrusive behaviour and tensional integrity. Together, our findings propose podosomes as highly organized actin microarchitectures mediating tensional integrity at the nanoscale and providing a feedback mechanism for cellular mechanosensing at the mesoscale.

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

An Intermittent Model for Intracellular Motions of Gold Nanostars by k-Space Scattering Image Correlation

Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.