Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform

Burkhardt, Markus; Heinze, Katrin G.; Schwille, Petra

doi:10.1364/ol.30.002266

Cited by 39 publications

(27 citation statements)

References 9 publications

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“…By choosing dyes with an appropriate Stokes shift, three colors can also be monitored with a single wavelength with one photon excitation [85], circumventing the problems of imperfect overlap of the different excitation volumes when using multiple wavelengths. A more flexible filterless multicolor separation using a grating or prism based detection system can resolve up to four distinct colors [86,87].…”

Section: Multiplexing Fcsmentioning

confidence: 99%

Fluorescence correlation spectroscopy in vivo

Mütze

Ohrt

Schwille

2010

Laser & Photonics Reviews

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Fluorescence Correlation Spectroscopy (FCS) is an optical technique used to accurately probe the dynamics, local concentration, and photophysics of single molecules both in vitro and in vivo. It is based on the analysis of intensity fluctuations of fluorescently labeled molecules diffusing through a focused laser beam. Fluorescence Cross-correlation Spectroscopy (FCCS) is a powerful extension to FCS which allows quantitative analysis of molecular interactions between differently labeled molecules. This review will focus on the application and potential of FCS and FCCS in vivo. Practical issues and sources of artifacts when performing measurements in living cells are discussed. Finally, several extensions to conventional FCS, such as multiphoton excitation, scanning FCS, Fluorescence Lifetime Correlation Spectroscopy, multiplexing FCS and recent approaches to reach smaller excitation volumes are reviewed.

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Section: Multiplexing Fcsmentioning

confidence: 99%

Fluorescence correlation spectroscopy in vivo

Mütze

Ohrt

Schwille

2010

Laser & Photonics Reviews

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“…On the other hand, the only reported FCS systems that used dispersive elements were conceived for filtering the scattered excitation light and Raman scattering of water using a prism monochromator for rotational FCS experiments [12]; and a grating-based detection setup consisting of a fiber array coupled to individual APDs. The gratingbased setup was developed for simultaneously measuring autocorrelations of four distinct quantum dots [78].…”

Section: Dispersive Opticsmentioning

confidence: 99%

“…Other benefits of quantum dots include long-term photostability, high quantum yield, multiple labeling with several colors, and single wavelength excitation for all colors. Due to its long Stokes shift, multicolor FCS experiments [78] have been performed to detect heterogeneities in lipid bilayer membranes [93], combined with submicrometer fluidic channels for isolation and detection [94] and to measure the binding constants of quantum dot-labeled streptavidin-biotin with SW-FCCS [43] and two-photon excitation FCCS [95]. Although quantum dots have been used in fluorescence imaging of live cells and even whole organisms, singlemolecule experiments with quantum dots have been limited.…”

Section: Quantum Dotsmentioning

confidence: 99%

Recent Advances in Fluorescence Cross-correlation Spectroscopy

Hwang

Wohland

2007

Cell Biochem Biophys

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Fluorescence cross-correlation spectroscopy (FCCS) is a method that measures the temporal fluorescence fluctuations coming from two differently labeled molecules diffusing through a small sample volume. Cross-correlation analysis of the fluorescence signals from separate detection channels extracts information of the dynamics of the dual-labeled molecules. FCCS has become an essential tool for the characterization of diffusion coefficients, binding constants, kinetic rates of binding, and determining molecular interactions in solutions and cells. By cross-correlating between two focal spots, flow properties could also be measured. Recent developments in FCCS have been targeted at using different experimental schemes to improve on the sensitivity and address their limitations such as cross-talk and alignment issues. This review presents an overview of the different excitation and detection methodologies used in FCCS and their biological applications. This is followed by a description of the fluorescent probes currently available for the different methods. This will introduce biological readers to FCCS and its related techniques and provide a starting point to selecting which experimental scheme is suitable for their type of biological study.

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“…[6][7][8]16 In addition, dichroics and barrier filters are carefully chosen to maximize signal and minimize cross talk to alleviate the degradation of FCCS signals. 5 Thus, there exists a lower limit for detecting reaction complexes based on multicolor cross-correlation analysis and other analysis approaches such as photon counting histogram and moment analysis.…”

mentioning

confidence: 99%

“…Multicolor FCS with up to four different channels was recently described, but is precluded by species with overlapping spectra, and simultaneous photon counting for all spectral channels was not possible. 16 Global analysis of FCS data has been reported to be an effective tool to accurately recover concentrations and diffusion times of species in solution. 26,27 The combination of global analysis of FCS data and FRET (2CG-FCS) has been reported to accurately recover the diffusion properties, molecular concentration, and biomolecular interactions for two color fluorescent mixtures.…”

mentioning

confidence: 99%

Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

et al. 2008

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Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto-and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.Fluorescence correlation spectroscopy (FCS) was developed to study the temporal correlations of thermodynamic concentration fluctuations in a reactive chemical system and was described in a series of papers by Magde, Webb, and Elson. 1,2 FCS monitors temporal variations of the fluorescence signal in an optically defined focal volume. These intensity fluctuations originate from important dynamic properties, such as diffusion, aggregation, chemical and enzyme kinetics, flow, and active transport. [1][2][3] The amplitude of these fluctuations is proportional to the square root of the number of molecules in the volume. 3,4

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Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform

Cited by 39 publications

References 9 publications

Fluorescence correlation spectroscopy in vivo

Fluorescence correlation spectroscopy in vivo

Recent Advances in Fluorescence Cross-correlation Spectroscopy

Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

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