Spatial Image Correlation Spectroscopy Research Articles

Light-Induced Condensates Show Accumulation-Prone and Less Dynamic Properties in the Nucleus Compared to the Cytoplasm

Biomolecular condensates, including membraneless organelles, are ubiquitously observed in subcellular compartments. However, the accumulation and dynamic properties of arbitrarily induced condensates remain elusive. Here, we show the size, amount, and dynamic properties of subcellular condensates using various fluorescence spectroscopic imaging analyses. Spatial image correlation spectroscopy showed that the size of blue-light-induced condensates of cryptochrome 2-derived oligomerization tag (CRY2olig) tagged with a red fluorescent protein in the nucleus was not different from that in the cytoplasm. Fluorescence intensity measurements showed that the condensates in the nucleus were more prone to accumulation than those in the cytoplasm. Single-particle tracking analysis showed that the condensates in the nucleus are predisposed to have stationary dynamics compared to those in the cytoplasm. Therefore, the subcellular compartment may, in part, affect the characteristics of self-recruitment of biomolecules in the condensates and their movement property.

Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images.

Size determination of subcellular structures such as inclusion bodies (IBs) and granules from fluorescent images is important for identification and structural characterization. However, it is often time-consuming just for the comparison of the average size of the structures. Here, we introduce a high-throughput procedure to represent the average size of structures in fluorescent images using Spatial Image Correlation Spectroscopy (SICS). This procedure provides an easier comparison of bodies and granular structures such as inclusion bodies (IBs) including misfolded protein aggregation, granules containing RNA (e.g., stress granules and processing bodies).

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy.

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy

Optineurin (OPTN) plays an important role in membrane trafficking processes such as exocytosis and autophagy. The sizes and rate of formation of accumulated structures comprising OPTN, such as foci or inclusion bodies (IBs), are often disrupted by amyotrophic lateral sclerosis (ALS) and glaucoma-associated mutants of OPTN. Therefore, methods for the quantitative measurement of the size of the accumulated structure are necessary. Here, we show that, using spatial image correlation spectroscopy (ICS), the average diameter of accumulated structures of the wild-type and disease-associated mutants in living cells may be easily determined. Although OPTN was found to frequently form foci in the cytoplasm, regardless of ALS- and glaucoma-associated mutation, the diameter of OPTN foci decreased in an ALS-associated mutant and increased in a glaucoma-associated mutant. However, a portion of cells carried IBs of the ALS-associated mutant that were larger than micrometre and ellipse-like shape, suggesting that this mutant accumulates non-uniformly in the IBs. The findings suggest that changes in their accumulation, determined via quantitative comparison of the OPTN foci and IBs in the cells, are involved in pathological features of ALS. In addition, this method enables rapid comparison of the average sizes of various other intracellular structures such as granules.

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.

Accuracy and Dynamic Range of Spatial Image Correlation and Cross-Correlation Spectroscopy

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static subdiffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables and the particle population densities and interactions and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented map regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR) to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 μm −2 within a 121 × 98 pixel subregion of a single cell.