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

Kolin, David L.; Wiseman, Paul W.

doi:10.1007/s12013-007-9000-5

Cited by 258 publications

(236 citation statements)

References 120 publications

(187 reference statements)

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“…For such timedependent approaches, molecules must undergo diffusion or flow to be detected, and hence, the methods cannot be applied to fixed tissue samples. Image correlation spectroscopy (ICS), an extension of FCS to the spatial domain, can overcome some of those limitations (6). However, with ICS, samples with spatial fluorescence intensity discontinuities introduce artifacts in the spatial correlation functions that result in systematic errors in the measurements (10).…”

mentioning

confidence: 99%

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis

Godin¹,

Costantino²,

Lorenzo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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104

130

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Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA B receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 μm 2 ) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.homodimerization | quantitative immunocytochemistry | fluorescence fluctuations analysis | GABAB | EGF receptor

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mentioning

confidence: 99%

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis

Godin¹,

Costantino²,

Lorenzo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

130

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“…A recent review nicely summarizes the (expanding) variety of mathematical treatments to which these two different types of datasets can be submitted [124], including alternatives to the super-resolution imaging techniques by single-molecule localization mentioned previously [125,126]. As with FCS, image correlation spectroscopy techniques can also be used on single-molecule image series in order to get access to fast timescale information [127].…”

Section: (B) Single-molecule Detection and Analysis In Wide Field Geomentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

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Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

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“…Raster ICS overcomes this limitation by incorporating a time delay between pixels and rows of pixels in the image (Digman et al 2005). Other correlation microscopy methods include tools for analyzing single particles in dense environments, such as particle ICS (PICS) (Semrau and Schmidt 2007;Kolin and Wiseman 2007), total internal reflection FCS (TIR-FCS) which uses exponentially decaying EM waves to excite a thin layer of a specimen (Hassler et al 2005), and selective plane imaging microscopy FCS (SPIM-FCS), which enables measurement with high time resolution in a 3D image field (Wohland et al 2010).…”

Section: Dynamic Imagingmentioning

confidence: 99%

Imaging stress

Brielle

Gura

Kaganovich

2015

Cell Stress and Chaperones

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Recent innovations in cell biology and imaging approaches are changing the way we study cellular stress, protein misfolding, and aggregation. Studies have begun to show that stress responses are even more variegated and dynamic than previously thought, encompassing nano-scale reorganization of cytosolic machinery that occurs almost instantaneously, much faster than transcriptional responses. Moreover, protein and mRNA quality control is often organized into highly dynamic macromolecular assemblies, or dynamic droplets, which could easily be mistaken for dysfunctional Baggregates,^but which are, in fact, regulated functional compartments. The nano-scale architecture of stressresponse ranges from diffraction-limited structures like stress granules, P-bodies, and stress foci to slightly larger quality control inclusions like juxta nuclear quality control compartment (JUNQ) and insoluble protein deposit compartment (IPOD), as well as others. Examining the biochemical and physical properties of these dynamic structures necessitates live cell imaging at high spatial and temporal resolution, and techniques to make quantitative measurements with respect to movement, localization, and mobility. Hence, it is important to note some of the most recent observations, while casting an eye towards new imaging approaches that offer the possibility of collecting entirely new kinds of data from living cells. StressProtecting the cell from protein-associated damage is a matter of having the correct proteins at the right place at the right time: the cellular environment changes rapidly in different folding and stress conditions in order to avoid catastrophic consequences of too many misfolded polypeptides or not enough functional proteins. When a cell is exposed to stresses such as heat-shock, cold-shock, osmotic stress, starvation, or amino-acid analogues which cause rampant mutations, the cellular response propagates across the entire network of protein biogenesis.Although much research has focused on how these stresses affect protein synthesis, we are only now beginning to look closely at how stress effects protein localization and distribution. Whereas expression takes time, and is more difficult to accomplish under stress, protein localization is substantially more dynamic and therefore changes in protein distribution can be affected quickly, in time to deal with the stress. Hence, regulation of protein distribution deserves careful attention in the study of stress response. Indeed, recent work has shown that in the single cell eukaryote Saccharomyces cerevisiae more than half of the proteome radically changes its localization under different stress condition (Breker et al. 2013). By altering the spatial positioning of proteins, cells can change Electronic supplementary material The online version of this article

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

Cited by 258 publications

References 120 publications

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Imaging stress

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