Introduction to Fluorescence Correlation Spectroscopy

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