An Introduction to Fluorescence Correlation Spectroscopy represents a comprehensive introduction to fluorescence correlation spectroscopy (FCS), a biophysical experimental technique increasingly used to study and quantify molecular mobility, concentrations and interactions in vitro, as well as in living cells and multicellular organisms. Students and researchers who are new to FCS can use the book as the first introduction to the technique, while those who are already using FCS regularly in their research may find it useful to deepen their understanding of the technique, its possibilities, limitations, and potential pitfalls as well as ways to avoid them.
This book introduces the reader to all aspects of FCS needed for practical usage of the technique in their research. In the beginning the concept of fluorescence intensity fluctuations and their auto- and cross-correlation functions are explained to give readers an understanding of the underlying principles. This is followed by an overview of instrumental FCS setups and various ways of data collection and processing, the derivations of theoretical models relating the experimentally obtained correlation functions to the underlying molecular processes, and the description of the fitting of experimental data with those models. Mathematically more involved portions are separated from the rest of the text and can be easily skipped by readers more interested in the conceptual and practical aspects of FCS. The book contains interactive graphics and is accompanied by an interactive computable document file allowing the reader to test the dependence of FCS results on a variety of experimental parameters, and to gain practical insights into FCS data fitting.
Key Features
- Introduces the concepts of FCS in an accessible way, supported by animations and graphics in the ebook.
- Includes a supplementary interactive computable document file that allows the reader to experiment with various FCS setup and fit parameters, allowing readers to test their understanding and simulate experimental outcomes.
- Provides rigorous mathematical derivations of fundamental FCS equations and models.
- Pedagogical features include questions, short reviews and critical discussions of literature relevant to the particular chapter that include applications and fundamental developments in the field of FCS.
Author(s): Thorsten Wohland, Sudipta Maiti, Radek Machan
Series: Biophysical Society–IOP Series
Publisher: IOP Publishing
Year: 2021
Language: English
Pages: 367
City: Bristol
PRELIMS.pdf
Preface
Acknowledgements
About the authors
Thorsten Wohland
Sudipta Maiti
Radek Macháň
CH001.pdf
Chapter 1 Introduction
1.1 What is fluorescence correlation spectroscopy all about?
1.2 What do ‘fluorescence’, ‘correlation’ and ‘spectroscopy’ have to do with measuring change?
1.3 What can FCS do for you?
1.4 What does an FCS measurement involve?
1.5 A brief history of FCS
1.5.1 Early work
1.5.2 The year of the invention
1.5.3 The initial progress
1.6 Critical technical steps of the revolution
1.6.1 Fluorescence: towards single molecule sensitivity
1.6.2 Microscopic volume
1.6.3 The confocal technique
1.6.4 Modern detectors
1.6.5 The data processors
1.7 Where is FCS now?
References
CH002.pdf
Chapter 2 Correlation functions
2.1 Introduction
2.2 Fluctuations
2.3 Correlations
2.4 From correlation coefficient to correlation function
2.5 The autocorrelation function (ACF) and its properties
2.6 The cross-correlation function (CCF) and its properties
2.7 Fluctuations and correlations
2.8 Synopsis
2.9 Exercises
References
CH003.pdf
Chapter 3 Fluorescence excitation and detection
3.1 The probe volume in FCS
3.1.1 Introduction
3.1.2 The significance of the size of the probe volume
3.1.3 A brief introduction to the generation of the fluorescence signal
3.1.4 Optical designs applied to obtain an appropriate probe volume for FCS
3.2 Photon detection
3.2.1 Photon counting
3.2.2 Array detectors
3.2.3 Photomultiplier tubes
3.3 Exercises
References
CH004.pdf
Chapter 4 Data structure, correlation and processing
4.1 Software correlators
4.1.1 Binned intensity trace—linear correlator
4.1.2 Binned intensity trace—multiple-tau correlator
4.1.3 Time-tagged intensity trace
4.1.4 Cross-correlation calculation and correlation function amplitude
4.1.5 Correlation function calculation via Fourier transform
4.2 Hardware correlators and their comparison with software correlators
4.3 Temporal resolution of correlation functions
4.4 Statistical filtering in correlation function calculation
4.4.1 Fluorescence lifetime and its integration into FCS datasets
4.4.2 Generation of statistical filters in fluorescence lifetime correlation spectroscopy (FLCS)
4.4.3 Generalisation of the FLCS principle -fluorescence spectral correlation spectroscopy (FSCS) and filtered FCS (fFCS)
4.5 Synopsis
4.6 Exercises
References
CH005.pdf
Chapter 5 Theoretical FCS models
5.1 The autocorrelation function for diffusion
5.2 General characteristics of the ACF for diffusion
5.3 Including multiple particles
5.4 Anomalous diffusion
5.5 Flow
5.6 Including multiple processes
5.7 Spatial and spatiotemporal correlation techniques
5.7.1 Two-focus FCS
5.7.2 Scanning FCS
5.7.3 Circular scanning fluorescence correlation spectroscopy
5.7.4 Image correlation spectroscopy (ICS)
5.7.5 Spatiotemporal image correlation spectroscopy (STICS)
5.7.6 Raster image correlation spectroscopy (RICS)
5.7.7 Imaging fluorescence correlation spectroscopy (Imaging FCS)
5.7.8 The FCS diffusion law
5.8 Other FCS modalities
5.9 Synopsis
5.10 Exercises
References
CH006.pdf
Chapter 6 Theoretical fluorescence cross-correlation spectroscopy (FCCS) models
6.1 Introduction
6.2 Dual-colour FCCS (DC-FCCS)
6.2.1 Cross-correlation amount
6.2.2 Unequal and non-aligned observation volumes
6.2.3 Spectral crosstalk
6.2.4 Non-correlated background
6.2.5 Non-fluorescent binding partners and free fluorophores
6.2.6 Fluorescence quenching and Förster resonance energy transfer (FRET) between fluorophores a and b
6.2.7 Complex stoichiometry
6.3 FCCS modalities derived from DC-FCCS
6.3.1 Single-wavelength FCCS (sw-FCCS)
6.3.2 FCCS with alternating laser excitation
6.4 Statistical filtering in FCCS
6.4.1 Statistical filtering and sources of DC-FCCS artefacts
6.4.2 Statistical filtering and negative CCF amplitudes
6.4.3 Quasi pulsed interleaved excitation FCCS (PIE-FCCS)
6.4.4 Reaction kinetics studied by FCCS
6.5 Synopsis
6.6 Exercises
References
CH007.pdf
Chapter 7 Artefacts in FCS
7.1 Background
7.2 Rare events
7.3 Bleaching
7.4 Sample movement
7.5 Detector-related artefacts: after-pulsing and dead time
7.6 Detector saturation
7.7 Fluorophore saturation
7.8 Scattering
7.9 Autofluorescence
7.10 Sample topology
7.11 Immobile particles
7.12 Refractive index mismatch
7.13 Exercises
References
CH008.pdf
Chapter 8 Data fitting
8.1 Introduction
8.2 What do we minimize?
8.3 The data structure and bias in FCS
8.3.1 The data structure in FCS
8.3.2 The bias of correlation functions
8.4 The standard deviation in FCS
8.4.1 Koppel’s standard deviation and its modifications
8.4.2 Standard deviations from multiple measurements
8.4.3 Standard deviation derived from the intensity trace
8.4.4 Standard deviation and bias within the ACF
8.4.5 The problem of correlations within the ACF
8.5 Non-linear least squares fit
8.5.1 Least squares and the χ2 function
8.5.2 The Levenberg–Marquardt fitting algorithm
8.6 Generalized least squares fit
8.6.1 The covariance matrix for the ACF
8.6.2 Regularization
8.7 Global fit
8.8 Maximum entropy method
8.9 Pairwise model selection using the F-test
8.10 Bayes model selection
8.11 Practical aspects
8.12 Synopsis
8.13 Exercises
References
CH009.pdf
Chapter 9 FCS and FCCS measurement strategies
9.1 Measuring concentrations by FCS
9.1.1 Concentration range accessible by FCS
9.1.2 Effective observation volume calibration
9.1.3 Molecular brightness determined by FCS
9.2 Characterising molecular diffusion by FCS
9.2.1 Confocal observation volume radius calibration
9.2.2 Calibration-free FCS modalities
9.2.3 Imaging FCS and its calibration
9.2.4 Separation of multiple processes contributing to the ACF temporal decay
9.2.5 Diffusion in two-dimensional systems studied by FCS
9.3 Molecular interactions studies by FCS
9.3.1 Using changes in the ACF amplitude
9.3.2 Using changes in the ACF temporal decay
9.4 Molecular interactions studies by FCCS
9.4.1 The importance of relating the CCF amplitude to ACF amplitudes
9.4.2 Determination of the FCCS experiment dynamic range
9.5 Synopsis
9.6 Exercises
References
CH010.pdf
Chapter 10 Where to go from here?
10.1 Introduction
10.2 What FCS can and cannot do
10.3 Data acquisition
10.4 Data analysis
10.5 Related techniques
10.6 Some final remarks
References
APP1.pdf
Chapter
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Chapter 6
Chapter 7
Chapter 8
Chapter 9
