Handbook Of Fluorescence Spectroscopy And Imaging - From Single Molecules To Ensembles

Providing much-needed information on fluorescence spectroscopy and microscopy, this ready reference covers detection techniques, data registration, and the use of spectroscopic tools, as well as new techniques for improving the resolution of optical microscopy below the resolution gap.
Starting with the basic principles, the book goes on to treat fluorophores and labeling, single-molecule fluorescence spectroscopy and enzymatics, as well as excited state energy transfer, and super-resolution fluorescence imaging.
Examples show how each technique can help in obtaining detailed and refined information from individual molecular systems.

Markus Sauer studied chemistry in Karlsruhe, Saarbrucken, and Heidelberg, and finished his PhD in Physical Chemistry at the University of Heidelberg under the guidance of Prof. Jurgen Wolfrum. After a short-term visit at LBNL, Berkeley in the group of Prof. Shimon Weiss, he was decorated with the BioFuture award to perform independent research on single-molecule handling, detection, and identification. He finished his habilitation in Heidelberg at the Institute of Physical Chemistry and is now Professor for Applied Laser Physics and Laser Spectroscopy at the University in Bielefeld.

Johan Hofkens received his MSc. (1988) and Ph.D. degree (1993) in Chemistry from the University of Leuven (K.U.Leuven). After postdoctoral research with Prof. Masuhara at Osaka University and Prof. Barbara at the University of Minneapolis, he rejoined the K.U.Leuven supervising the Single Molecule Unit in the group of Prof. De Schryver. In 2005 he was appointed Research Professor at the K.U.Leuven and recently he was promoted to full professor. His research interests are fast spectroscopy, (single molecule) fluorescence microscopy and nanoscopy and the application of these techniques in material science and biosciences.

Jörg Enderleins studied physics at the Mechnikov University in Odessa (Ukraine) from 1981 until 1986, and defended his PhD from Humboldt University in Berlin (Germany) in 1991. Thereafter he worked at PicoQuant until 1996, when he joined the group under Richard A. Keller in Los Alamos, USA as a visiting scientist for one year before becoming an assistant professor at the University of Regensburg, Germany. Since 2001 he had been a Heisenberg Fellow of the German Research Council (DFG) and established his research group at the Insitute for Biological Information processing 1 at the Forschungszentrum Julich. After an appointment as Professor for Biophysical Chemistry at Eberhard-Karls-University Tubingen, he became professor for biophysics at the Georg-August-University in Gottingen in 2007.

Professor Enderlein's research focuses on the development of new single-molecule spectroscopic and imaging techniques for biophysics applications.

Preface.

1 Basic Principles of Fluorescence Spectroscopy.

1.1 Absorption and Emission of Light.

1.2 Spectroscopic Transition Strengths.

1.3 Lambert–Beer Law and Absorption Spectroscopy.

1.4 Fluorophore Dimerization and Isosbestic Points.

1.5 Franck–Condon Principle.

1.6 Temperature Effects on Absorption and Emission Spectra.

1.7 Fluorescence and Competing Processes.

1.8 Stokes Shift, Solvent Relaxation, and Solvatochroism.

1.9 Fluorescence Quantum Yield and Lifetime.

1.10 Fluorescence Anisotropy.

References.

2 Fluorophores and Fluorescent Labels.

2.1 Natural Fluorophores.

2.2 Organic Fluorophores.

2.3 Different Fluorophore Classes.

2.4 Multichromophoric Labels.

2.5 Nanocrystals.

References.

3 Fluorophore Labeling for Single-Molecule Fluorescence Spectroscopy (SMFS).

3.1 In Vitro Fluorescence Labeling.

3.2 Fluorescence Labeling in Living Cells.

References.

4 Fluorophore Selection for Single-Molecule Fluorescence Spectroscopy (SMFS) and Photobleaching Pathways.

References.

5 Fluorescence Correlation Spectroscopy.

5.1 Introduction.

5.2 Optical Set-Up.

5.3 Data Acquisition and Evaluation.

5.4 Milliseconds to Seconds: Diffusion and Concentration.

5.5 Nanoseconds to Microseconds: Photophysics, Conformational Fluctuations, Binding Dynamics.

5.6 Picoseconds to Nanoseconds: Rotational Diffusion and Fluorescence Antibunching.

5.7 Fluorescence Lifetime Correlation Spectroscopy.

5.8 Conclusion.

References.

6 Excited State Energy Transfer.

6.1 Introduction.

6.2 Theory of (Förster) Energy Transfer.

6.3 Experimental Approach for Single-Pair FRET-Experiments.

6.4 Examples and Applications of FRET.

7 Photoinduced Electron Transfer (PET) Reactions.

7.1 Fluorescence Quenching by PET.

7.2 Single-Molecule Fluorescence Spectroscopy to Study PET.

7.3 Single-Molecule Sensitive Fluorescence Sensors Based on PET.

7.4 PET Reporter System.

7.5 Monitoring Conformational Dynamics and Protein Folding by PET.

7.6 Biological and Diagnostic Applications.

References.

8 Super-Resolution Fluorescence Imaging.

8.1 Diffraction Barrier of Optical Microscopy.

8.2 Multi-Photon and Structured Illumination Microscopy.

8.3 Stimulated Emission Depletion.

8.4 Single-Molecule Based Photoswitching Microscopy.

8.5 Background and Principles of Single-Molecule Based Photoswitching Microscopy Methods.

8.6 Temporal Resolution of Super-Resolution Imaging Methods.

References.

9 Single-Molecule Enzymatics.

9.1 Introduction: Why Study Enzymes on a Single-Molecule Level?

9.2 Biochemical Principles of Enzymatic Activity: the Michaelis–Menten Model.

9.3 ‘‘Looking’’ at Individual Enzymes.

9.4 Data Analysis of Fluorescence Intensity Time Traces of Single-Turnover Experiments. 

9.5 Conclusions.

References.

Index.