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Medicine - Radiology | Fluorescence Spectroscopy and Microscopy - Methods and Protocols

Fluorescence Spectroscopy and Microscopy

Methods and Protocols

Series: Methods in Molecular Biology, Vol. 1076

Engelborghs, Yves, Visser, Antonie J.W.G. (Eds.)

2014, XXI, 816 p. 244 illus., 171 illus. in color.

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  • Explores the methods and applications in the expanding fluorescence field 
  • Provides step-by-step detail essential for reproducible results
  • Contains key notes and implementation advice from the experts

Reflecting the expanding field’s need for reliable protocols, Fluorescence Spectroscopy and Microscopy: Methods and Protocols offers techniques from a worldwide team of experts on this versatile and vital subject.  The topics covered fall into four broad categories: steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, fluorescent probe development, and the various sub-categories of fluorescence microscopy, such as fluorescence recovery after photobleaching (FRAP), live cell FRET imaging (FRETim), fluorescence lifetime imaging (FLIM), fluorescence fluctuation spectroscopy (FFS), and single-molecule fluorescence spectroscopy (smFS).  Written as a part of the popular Methods in Molecular Biology series, chapters include the kind of unambiguous detail and key implementation advice that proves essential for successful results.


Comprehensive and practical, Fluorescence Spectroscopy and Microscopy: Methods and Protocols aims to guide both ‘novice’ and established scientists toward furthering their research with these invaluable techniques.

Content Level » Professional/practitioner

Keywords » Cuvette-based studies - Fluorescence Fluctuation Spectroscopy - Fluorescence Lifetime Imaging - Fluorescence Recovery After Photobleaching - Fluorescent Probe Development - Förster Resonance Energy Transfer Imaging - Green fluorescent protein (GFP) - Single-Molecule Fluorescence Spectroscopy - Steady-State Fluorescence Spectroscopy - Time-Resolved Fluorescence Spectroscopy

Related subjects » Analytical Chemistry - Life Sciences - Radiology

Table of contents 

Part I: Steady-State Fluorescence Spectroscopy (SSFS)


1. How to Collect National Institute of Standards and Technology (NIST) Traceable Fluorescence Excitation and Emission Spectra

            Adam Matthew Gilmore


2. Steady State Fluorescence Polarization/Anisotropy for the Study of Protein Interactions

            Nicholas G. James and David M. Jameson


3. Quantitative Fluorescence Spectral Analysis of Protein Denaturation

            Ivo H.M. van Stokkum and Sergey P. Laptenok


4. High-Pressure Fluorescence Applications

            Mariano Dellarole and Catherine A. Royer


Part II: Time-Resolved Fluorescence Spectroscopy (TRFS)


5. Frequency Domain Fluorometry: Theory and Application

            Carissa M. Vetromile and David M. Jameson


6. Polar Plot Representation of Time-Resolved Fluorescence

            John Paul Eichorts, Kai wen Teng, and Robert M. Clegg


7. Ensemble and Single Molecule Detected Time Resolved FRET Methods in Studies of Protein Conformations and Dynamics

            Tomer Orevi, Eitan Lerner, Gil Rahamim, Dan Amir, and Elisha Haas


8. MD + QM Correlations with Tryptophan Fluorescence Spectral Shifts and Lifetimes

            Patrik R. Callis and Jose R. Tusell


9. Analysis of Time-Dependent Red Shifts in Fluorescence Emission from Tryptophan Residues in Proteins

            Dmitri Toptygin


10. Global Analysis of Time-Resolved Fluorescence Data

            Anatoli V. Digris, Eugene G. Novikov, Victor V. Skakun, and Vladimir V. Apanasovich


11. Nanometrology

            David J.S. Birch and Philip Yip


12. Upconversion Spectrophotofluorometry

            Arianna Biesso, Jianhua Xu, and Jay R. Knutson


13. Subpicosecond Kerr-Gate Spectrofluorometry

            Sergey P. Laptenok, Patrick Nürnberger, Andras Lukacs, and Marten H. Vos


14. Photo-Induced Electron Transfer Modeling to Simulate Flavoprotein Fluorescence Decay

            Nadtanet Nunthaboot, Kiattisak Lugsanangarm, Arthit Nueangaudom, Somsak Pianwanit, Sirirat Kokpol, and Fumio Tanaka


Part III: Fluorescent Probe Development (FPD)


15. Biosynthetic Incorporation of Trp Analogs in Proteins

            Jaap Broos


16. Optimization of Fluorescent Proteins

            Daphne S. Bindels, Joachim Goedhart, Mark A. Hink, Laura van Weeren, Linda Joosen, and Theodorus W.J. Gadella, Jr.


17. Monitoring Membrane Properties and Apoptosis Using Membrane Probes of the 3-Hydroxyflavone Family

            Zeinab Darwich, Andrey S. Klymchenko, and Yves Mély


Part IV: Fluorescence Microscopy: Fluorescence Recovery After Photobleaching (FRAP)


18. Rectangle FRAP for Measuring Diffusion with a Laser Scanning Microscope

            Ranhua Xiong, Hendrik Deschout, Jo Demeester, Stefaan C. De Smedt, and Kevin Braeckmans


Part V: Fluorescence Microscopy: Förster Resonance Energy Transfer Imaging (FRETim)


19. A Quantitative Protocol for Intensity-Based Live Cell FRET Imaging

            Clemens F. Kaminski, Eric. J. Rees, and Gabriele S. Kaminski Schierle


Part VI: Fluorescence Microscopy: Fluorescence Lifetime Imaging (FLIM)


20. Widefield Fluorescence Lifetime Imaging with Multi-Anode Detectors

            Roland Hartig, Yury Prokazov, Evgeny Turbin, and Werner Zuschratter


21. Global Analysis of FRET- FLIM Data in Live Plant Cells

            Sergey P. Laptenok, Joris J. Snellenburg, Christoph A. Bücherl, Kai R. Konrad, and Jan Willem Borst


22. Time-Resolved Fluorescence Anisotropy Imaging

            Klaus Suhling, James Levitt, and Pei-Hua Chung


23. Multi-Modal Fluorescence Imaging Spectroscopy

            Martijn H.W. Stopel, Christian Blum, and Vinod Subramaniam


Part VII: Fluorescence Microscopy: Fluorescence Fluctuation Spectroscopy (FFS)


24. Application of Fluorescence Correlation Spectroscopy (FCS) to Measure the Dynamics of Fluorescent Proteins in Living Cells

            Thomas Weidemann


25. Fluorescence Cross-Correlation Spectroscopy (FCCS) in Living Cells

            Xiaoxiao Ma, Yong Hwee Foo, and Thorsten Wohland


26. Quantifying Lipid-Protein Interaction by Fluorescence Correlation Spectroscopy (FCS)

            Ana M. Melo, Manuel Prieto, and Ana Coutinho


27. PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching

            Markus Sauer and Hannes Neuweiler


28. Z-Scan Fluorescence Correlation Spectroscopy as a Tool for Diffusion Measurements in Planar Lipid Membranes

            Tomáš Steinberger, Radek Macháň, and Martin Hof


29. Scanning Fluorescence Correlation Spectroscopy (SFCS) with a Scan Path Perpendicular to the Membrane Plane

            Paul Müller, Petra Schwille, and Thomas Weidemann


30. Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS

            Jelle Hendrix and Don C. Lamb


31. Quantitative Study of Protein-Protein Interactions in Live Cell by Dual Color Fluorescence Lifetime Cross-Correlation Spectroscopy

            Sergi Padilla-Parra, Nicolas Audugé, Maïté Coppey-Moisan, and Marc Tramier


32. Brightness Experiments

            Patrick J. Macdonald, Jolene Johnson, Yan Chen, and Joachim D. Mueller


33. Global Analysis of Autocorrelation Functions and Photon Counting Distributions in Fluorescence Fluctuation Spectroscopy

            Victor V. Skakun, Anatoli V. Digris, and Vladimir V. Apanasovich


34. Simulation of Autocorrelation Function and Photon Counting Distribution in Fluorescence Fluctuation Spectroscopy

            Igor P. Shingaryov, Victor V. Skakun, and Vladimir V. Apanasovich


Part VIII: Fluorescence Microscopy: Single-Molecule Fluorescence Spectroscopy (smFS)


35. Single-Molecule Fluorescence of Nucleic Acids

            Kaley McCluskey, Euan Shaw, Daniel A. Lafontaine, and J. Carlos Penedo


36. Photoswitchable Fluorescent Proteins for Superresolution Fluorescence Microscopy Above the Diffraction Limit of Light

            Susana Rocha, Herlinde De Keersmaecker, Hiroshi Uji-i, Johan Hofkens, and Hideaki Mizuno

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