Spatial fluorescence cross‐correlation spectroscopy by means of a spatial light modulator

Blancquaert, Yoann; Gao, Jie; Derouard, Jacques; Delon, Antoine

doi:10.1002/jbio.200810007

Cited by 8 publications

(8 citation statements)

References 38 publications

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“…(For an instructive explanation of the pair correlation method, see Digman and Gratton (64).) Earlier implementation of the pair correlation concept involved cross correlation of fluorescence from two laser foci separated by a fixed distance (e.g., (65)(66)(67)). An advantage of the approach described above is that position pairs are sampled over the entire scan range rather than only at the two foci of the split laser beam (64).…”

Section: Single-molecule Aspects Of Fcsmentioning

confidence: 99%

Fluorescence Correlation Spectroscopy: Past, Present, Future

Elson

2011

Biophysical Journal

379

326

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In recent years fluorescence correlation spectroscopy (FCS) has become a routine method for determining diffusion coefficients, chemical rate constants, molecular concentrations, fluorescence brightness, triplet state lifetimes, and other molecular parameters. FCS measures the spatial and temporal correlation of individual molecules with themselves and so provides a bridge between classical ensemble and contemporary single-molecule measurements. It also provides information on concentration and molecular number fluctuations for nonlinear reaction systems that complement single-molecule measurements. Typically implemented on a fluorescence microscope, FCS samples femtoliter volumes and so is especially useful for characterizing small dynamic systems such as biological cells. In addition to its practical utility, however, FCS provides a window on mesoscopic systems in which fluctuations from steady states not only provide the basis for the measurement but also can have important consequences for the behavior and evolution of the system. For example, a new and potentially interesting field for FCS studies could be the study of nonequilibrium steady states, especially in living cells.

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Section: Single-molecule Aspects Of Fcsmentioning

confidence: 99%

Fluorescence Correlation Spectroscopy: Past, Present, Future

Elson

2011

Biophysical Journal

379

326

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“…Generally images acquired using Macintosh operating systems are stored as "big endian" and those from Windows are stored as "little endian". Any stack acquired in "big endian" can be converted to "little endian" by open source image processing software like ImageJ [2].…”

Section: Parsing the Tiff File Structurementioning

confidence: 99%

ImFCS: A software for Imaging FCS data analysis and visualization

Sankaran¹,

Shi²,

Ho³

et al. 2010

Opt. Express

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The multiplexing of fluorescence correlation spectroscopy (FCS), especially in imaging FCS using fast, sensitive array detectors, requires the handling of large amounts of data. One can easily collect in excess of 100,000 FCS curves a day, too many to be treated manually. Therefore, ImFCS, an open-source software which relies on standard image files was developed and provides a wide range of options for the calculation of spatial and temporal auto- and cross-correlations, as well as differences in Cross-Correlation Functions (ΔCCF). ImFCS permits fitting of standard models to correlation functions and provides optimized histograms of fitted parameters. Applications include the measurement of diffusion and flow with Imaging Total Internal Reflection FCS (ITIR-FCS) and Single Plane Illumination Microscopy FCS (SPIM-FCS) in biologically relevant samples. As a compromise between ITIR-FCS and SPIM-FCS, we extend the applications to Imaging Variable Angle-FCS (IVA-FCS) where sub-critical oblique illumination provides sample sectioning close to the cover slide.

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“…Flow velocity and flow directions have been determined in vivo and demonstrated in zebrafish blood vessels (24)(25)(26). Recently, spatial CCS was carried out with the use of a spatial light modulator (27).…”

Section: Introductionmentioning

confidence: 99%

Diffusion, Transport, and Cell Membrane Organization Investigated by Imaging Fluorescence Cross-Correlation Spectroscopy

Sankaran

Manna

Guo

et al. 2009

Biophysical Journal

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Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Abeta, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.

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Spatial fluorescence cross‐correlation spectroscopy by means of a spatial light modulator

Cited by 8 publications

References 38 publications

Fluorescence Correlation Spectroscopy: Past, Present, Future

Fluorescence Correlation Spectroscopy: Past, Present, Future

ImFCS: A software for Imaging FCS data analysis and visualization

Diffusion, Transport, and Cell Membrane Organization Investigated by Imaging Fluorescence Cross-Correlation Spectroscopy

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