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An introduction to fluorescence correlation spectroscopy /

"Version: 20201101"--Title page verso.Includes bibliographical references.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 -- 1.6. Critical technical steps of the revolution -- 1.7. Where is FCS now?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. Exercises3. Fluorescence excitation and detection -- 3.1. The probe volume in FCS -- 3.2. Photon detection -- 3.3. Exercises4. Data structure, correlation and processing -- 4.1. Software correlators -- 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 -- 4.5. Synopsis -- 4.6. Exercises5. 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 -- 5.8. Other FCS modalities -- 5.9. Synopsis -- 5.10. Exercises6. Theoretical fluorescence cross-correlation spectroscopy (FCCS) models -- 6.1. Introduction -- 6.2. Dual-colour FCCS (DC-FCCS) -- 6.3. FCCS modalities derived from DC-FCCS -- 6.4. Statistical filtering in FCCS -- 6.5. Synopsis -- 6.6. Exercises7. 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. Exercises8. Data fitting -- 8.1. Introduction -- 8.2. What do we minimize? -- 8.3. The data structure and bias in FCS -- 8.4. The standard deviation in FCS -- 8.5. Non-linear least squares fit -- 8.6. Generalized least squares fit -- 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. Exercises9. FCS and FCCS measurement strategies -- 9.1. Measuring concentrations by FCS -- 9.2. Characterising molecular diffusion by FCS -- 9.3. Molecular interactions studies by FCS -- 9.4. Molecular interactions studies by FCCS -- 9.5. Synopsis -- 9.6. Exercises10. 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.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, potential pitfalls, and 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. Part of Biophysical Society-IOP series.Students (undergraduate and graduate) and researchers of biophysics, in particular in bioimaging and fluorescence spectroscopy; facility managers in light microscopy facilities; biologists with interest in quantitative measurements using fluorescence spectroscopy.Also available in print.Mode of access: World Wide Web.System requirements: Adobe Acrobat Reader, EPUB reader, or Kindle reader.Thorsten Wohland is a Professor in the Departments of Biological Sciences and Chemistry at the National University of Singapore (NUS). He was awarded an Alexander von Humboldt research fellowship and the Outstanding Scientist Award of the faculty of science at NUS. His interests lie in the development of fluorescence techniques and their application to biological systems. He continues to develop new FCS modalities and is passionate about teaching FCS which motivated the writing of this book. Sudipta Maiti is a Professor of Chemical Sciences at the Tata Institute of Fundamental Research (Mumbai, India). His research focuses on protein aggregation, neurotransmission and development of microscopy techniques. He organizes the long-running annual FCS Workshop in India. He is the co-founder and President of the Fluorescence Society (India) and President of the International Society on Optics Within Life Sciences ('OWLS', Germany). He is a Fellow of the Indian Academy of Sciences. Radek Mach?a?en has worked as imaging scientist in microscopy core facilities since 2016, first at Charles University in Prague and then at the Singapore Centre for Environmental Life Sciences Engineering located at Nanyang Technological University in Singapore. Throughout his work at the microscopy core facilities, Radek has continued to promote FCS and related techniques by assisting facility users with FCS experiments and by teaching FCS at courses and practical workshops.Title from PDF title page (viewed on December 4, 2020).

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Call Number
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Publisher
: .,
Collation
1 online resource (various pagings) :illustrations (some color).
Language
English
ISBN/ISSN
9780750320801
Classification
543/.08584
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Media Type
-
Carrier Type
-
Edition
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Subject(s)
Biophysics.
SCIENCE / Life Sciences / Biophysics.
Fluorescence spectroscopy.
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