Comparison between time-correlated single photon counting and fluorescence correlation spectroscopy in single molecule identification

AbstractSingle-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson’s disease, with an improved sensitivity of >100,000-fold over bulk measurements.

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Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy

Journal of Biomedical Optics ◽

10.1117/1.2187425 ◽

2006 ◽

Vol 11 (2) ◽

pp. 024012 ◽

Cited By ~ 26

Author(s):

Steffen Rüttinger ◽

Rainer Macdonald ◽

Benedikt Krämer ◽

Felix Koberling ◽

Martin Roos ◽

...

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Single Photon ◽

Photon Counting ◽

Correlation Spectroscopy ◽

Single Pair ◽

Single Photon Counting ◽

Fluorescence Correlation ◽

Excitation Time ◽

Resonant Energy Transfer ◽

Pulsed Interleaved Excitation

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FoCuS-point: software for STED fluorescence correlation and time-gated single photon counting

Bioinformatics ◽

10.1093/bioinformatics/btv687 ◽

2015 ◽

Vol 32 (6) ◽

pp. 958-960 ◽

Cited By ~ 33

Author(s):

Dominic Waithe ◽

Mathias P. Clausen ◽

Erdinc Sezgin ◽

Christian Eggeling

Keyword(s):

Single Photon ◽

Photon Counting ◽

Super Resolution ◽

Correlation Spectroscopy ◽

Single Photon Counting ◽

Fluorescence Correlation ◽

Point Data ◽

User Friendly ◽

Kinetics Of ◽

Focus Point

Abstract Motivation: Fluorescence Correlation Spectroscopy (FCS) is a popular tool for measuring molecular mobility and how mobility relates to molecular interaction dynamics and bioactivity in living cells. The FCS technique has been significantly advanced by its combination with super-resolution STED microscopy (STED-FCS). Specifically, the use of gated detection has shown great potential for enhancing STED-FCS, but has also created a demand for software which is efficient and also implements the latest algorithms. Prior to this study, no open software has been available which would allow practical time-gating and correlation of point data derived from STED-FCS experiments. Results: The product of this study is a piece of stand-alone software called FoCuS-point. FoCuS-point utilizes advanced time-correlated single-photon counting (TCSPC) correlation algorithms along with time-gated filtering and innovative data visualization. The software has been designed to be highly user-friendly and is tailored to handle batches of data with tools designed to process files in bulk. FoCuS-point also includes advanced fitting algorithms which allow the parameters of the correlation curves and thus the kinetics of diffusion to be established quickly and efficiently. Availability and implementation: FoCuS-point is written in python and is available through the github repository: https://github.com/dwaithe/FCS_point_correlator. Furthermore, compiled versions of the code are available as executables which can be run directly in Linux, Windows and Mac OSX operating systems. Contact: [email protected]

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Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy

European Biophysics Journal ◽

10.1007/s00249-002-0212-8 ◽

2002 ◽

Vol 31 (3) ◽

pp. 241-241 ◽

Cited By ~ 1

Author(s):

Lutz Jermutus ◽

Reto Kolly ◽

Zeno Földes-Papp ◽

Jozef Hanes ◽

Rudolf Rigler ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Fluorescence Correlation Spectroscopy ◽

Correlation Spectroscopy ◽

Single Molecule Level ◽

Fluorescence Correlation

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Microphotonic control of single molecule fluorescence correlation spectroscopy using planar optofluidics

Optics Express ◽

10.1364/oe.15.007290 ◽

2007 ◽

Vol 15 (12) ◽

pp. 7290 ◽

Cited By ~ 31

Author(s):

D. Yin ◽

E. J. Lunt ◽

A. Barman ◽

A. R. Hawkins ◽

H. Schmidt

Keyword(s):

Single Molecule ◽

Fluorescence Correlation Spectroscopy ◽

Correlation Spectroscopy ◽

Single Molecule Fluorescence ◽

Fluorescence Correlation

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Fluorescence correlation spectroscopy: The technique and its applications in soft matter

Physical Sciences Reviews ◽

10.1515/psr-2017-0104 ◽

2018 ◽

Vol 4 (4) ◽

Author(s):

Anjali Gupta ◽

Jagadish Sankaran ◽

Thorsten Wohland

Keyword(s):

Single Molecule ◽

Fluorescence Correlation Spectroscopy ◽

Soft Matter ◽

Photophysical Properties ◽

Spatiotemporal Analysis ◽

Correlation Spectroscopy ◽

Basic Principles ◽

Chemical Reaction Kinetics ◽

Fluorescence Correlation ◽

Wide Range

Abstract Fluorescence correlation spectroscopy (FCS) is a well-established single-molecule method used for the quantitative spatiotemporal analysis of dynamic processes in a wide range of samples. It possesses single-molecule sensitivity but provides ensemble averaged molecular parameters such as mobility, concentration, chemical reaction kinetics, photophysical properties and interaction properties. These parameters have been utilized to characterize a variety of soft matter systems. This review provides an overview of the basic principles of various FCS modalities, their instrumentation, data analysis, and the applications of FCS to soft matter systems.

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