FRET - Förster Resonance Energy Transfer -  - E-Book

FRET - Förster Resonance Energy Transfer E-Book

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Meeting the need for an up-to-date and detailed primer on all aspects of the topic, this ready reference reflects the incredible expansion in the application of FRET and its derivative techniques over the past decade, especially in the biological sciences. This wide diversity is equally mirrored in the range of expert contributors. The book itself is clearly subdivided into four major sections. The first provides some background, theory, and key concepts, while the second section focuses on some common FRET techniques and applications, such as in vitro sensing and diagnostics, the determination of protein, peptide and other biological structures, as well as cellular biosensing with genetically encoded fluorescent indicators. The third section looks at recent developments, beginning with the use of fluorescent proteins, followed by a review of FRET usage with semiconductor quantum dots, along with an overview of multistep FRET. The text concludes with a detailed and greatly updated series of supporting tables on FRET pairs and Förster distances, together with some outlook and perspectives on FRET. Written for both the FRET novice and for the seasoned user, this is a must-have resource for office and laboratory shelves.

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Contents

Cover

Related Titles

Title Page

Copyright

Preface

List of Contributors

Part One: Background, Theory, and Concepts

Chapter 1: How I Remember Theodor Förster

Chapter 2: Remembering Robert Clegg and Elizabeth Jares-Erijman and Their Contributions to FRET

2.1 Biographical Sketch of Bob Clegg

2.2 Biographical Sketch of Eli Jares-Erijman

2.3 The Pervasive Influence of Gregorio Weber

2.4 Contributions by Bob Clegg to FRET

2.5 Contributions by Eli Jares-Erijman to FRET

2.6 A Final Thought

References

Chapter 3: Förster Theory

3.1 Introduction

3.2 Pre-Förster

3.3 Bottom Line

3.4 9000-Form, 9-Form, and Practical Expressions of the R60 Equation

3.5 Overlap Integral

3.6 Zones

3.7 Transfer Mechanisms

3.8 Kappa-Squared Basics

3.9 Ideal Dipole Approximation

3.10 Resonance as an All-or-Nothing Effect

3.11 Details About the All-or-Nothing Approximation of Resonance

3.12 Classical Theory Completed

3.13 Oscillator Strength–Emission Spectrum Relation for the Donor

3.14 Oscillator Strength–Absorption Spectrum Relation for the Acceptor

3.15 Quantum Mechanical Theory

3.16 Transfer in a Random System

3.17 Details for Transfer in a Random System

3.18 Concentration Depolarization

3.19 FRET Theory 1965–2012

Acknowledgments

References

Chapter 4: Optimizing the Orientation Factor Kappa-Squared for More Accurate FRET Measurements

4.1 Two-Thirds or Not Two-Thirds?

4.2 Relevant Questions

4.3 How to Visualize Kappa-Squared?

4.4 Kappa-Squared Can Be Measured in At Least One Case

4.5 Averaging Regimes

4.6 Dynamic Averaging Regime

4.7 What Is the Most Probable Value for Kappa-Squared in the Dynamic Regime?

4.8 Optimistic, Conservative, and Practical Approaches

4.9 Comparison with Experimental Results

4.10 Smart Simulations Are Superior

4.11 Static Kappa-Squared

4.12 Beyond Regimes

4.13 Conclusions

Acknowledgments

References

Chapter 5: How to Apply FRET: From Experimental Design to Data Analysis

5.1 Introduction: FRET– More Than a Four-Letter Word!

5.2 FRET: Let's get started!

5.3 FRET: The Basic Concept

5.4 FRET: Inevitable Mathematics

5.5 FRET: The Experiment

5.6 FRET beyond Förster

5.7 Summary and Outlook

Acknowledgment

References

Chapter 6: Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

6.1 Introduction

6.2 Bioconjugation

6.3 Organic Materials

6.4 Biological Materials

6.5 Inorganic Materials

6.6 Multi-FRET Systems

6.7 Summary and Outlook

Acknowledgments

References

Part Two: Common FRET Techniques/Applications

Chapter 7: In Vitro FRET Sensing, Diagnostics, and Personalized Medicine

7.1 Introduction

7.2 Small Organic Molecules and Synthetic Organic Polymers

7.3 Carbohydrate–Lipid

7.4 The Biotin–Avidin Interaction

7.5 Proteins and Peptides

7.6 Antibodies

7.7 Nucleic Acid (DNA/RNA)

7.8 Aptamers

7.9 High-Throughput and Point-of-Care Devices

7.10 Conclusions

References

Chapter 8: Single-Molecule Applications

8.1 Introduction

8.2 Single-Molecule FRET of Immobilized Molecules

8.3 Single-Molecule FRET of Freely Diffusing Molecules

8.4 Single-Molecule FRET Studies Involving Multiple FRET Partners

8.5 Conclusions and Perspectives

References

Chapter 9: Implementation of FRET Technologies for Studying the Folding and Conformational Changes in Biological Structures

9.1 Introduction to Using FRET in Biological Systems

9.2 Förster Formalism in the Determination of Biological Structures

9.3 FRET Experiments in Complex Biological Systems

9.4 Biological Model System 1: The Ribosome

9.5 Biological System 2: Nascent Polypeptide Structure

9.6 Biological System 3: Chaperone-Mediated Protein Folding

9.7 Biological System 4: Mature Protein Folding Intermediates

9.8 Biological System 5: Intersubunit Distance in Multimeric Protein Complexes

9.9 Biological System 6: Protein–Protein Interactions in the Assembly of Protein Polymers

9.10 Biological System 7: FRET in Nucleic Acid Systems

References

Chapter 10: FRET-Based Cellular Sensing with Genetically Encoded Fluorescent Indicators

10.1 Introduction

10.2 Enzymes

10.3 Metabolites

10.4 Second Messengers

10.5 Conclusions

Acknowledgments

References

Part Three: FRET with Recently Developed Materials

Chapter 11: FRET with Fluorescent Proteins

11.1 Introduction to FPs

11.2 Using FPs for FRET Imaging

11.3 Conclusions

Acknowledgments

References

Chapter 12: Semiconductor Quantum Dots and FRET

12.1 Introduction

12.2 A Quick Review of FRET

12.3 Quantum Dots

12.4 Quantum Dots and FRET

12.5 Quantum Dots as Donors in Biological Applications

12.6 Quantum Dots as Acceptors in Biological Applications

12.7 Energy Transfer between Quantum Dots and Other Nanomaterials

12.8 Nonbiological Applications of Quantum Dots and FRET

12.9 Summary

References

Chapter 13: Multistep FRET and Nanotechnology

13.1 Introduction

13.2 Fundamentals of Multistep FRET

13.3 Energy Transfer in Photosynthesis

13.4 Photonic Wires and Multistep FRET in Nanotechnology

13.5 Summary

13.6 Note Added in Proof

References

Part Four: Supporting Information and Conclusions

Chapter 14: Data

14.1 Tables before 1987

14.2 Introduction to the Table of Traditional Chromophores

14.3 Förster Distances and Other FRET Data before 1994

14.4 Förster Distances for Traditional Probes More Recent Than 1993

14.5 FRET Data on Fluorescent Proteins

14.6 FRET Data on Quantum Dots

14.7 Donor–Acceptor Pairs with a Förster Distance in a Given Range

14.8 Table–Reference Directory

References

Chapter 15: Outlook on FRET: The Future of Resonance Energy Transfer

15.1 A Rosy Crystal Ball View of FRET

15.2 Do Not Ask What FRET Can Do for You, Ask What You Can Do for FRET

15.3 FRET: Future Research with an Exciting Technology

15.4 Future of FRET

15.5 Outlook on Single-Molecule FRET

15.6 Outlook on FRET with Fluorescent Proteins

15.7 Luminescent Nanoparticles: Scaffolds for Assembling “Smarter” FRET Probes

References

Index

Related Titles

(also available in digital formats)

Balzani, V., Ceroni, P., Juris, A.

Photochemistry and Photophysics

Concepts, Research Topics, Applications

2014

ISBN: 978-3-527-33479-7

Kubitscheck, U. (ed.)

Fluorescence Microscopy

From Principles to Biological Applications

2013

ISBN: 978-3-527-32922-9

Valeur, B., Berberan-Santos, M. N.

Molecular Fluorescence

Principles and Applications Second edition

2012

ISBN (Hardcover): 978-3-527-32837-6

ISBN (Softcover): 978-3-527-32846-8

Sauer, M., Hofkens, J., Enderlein, J.

Handbook of Fluorescence Spectroscopy and Imaging

From Single Molecules to Ensembles

2011

ISBN: 978-3-527-31669-4

Bräuchle, C., Lamb, D. C., Michaelis, J. (eds.)

Single Particle Tracking and Single Molecule Energy Transfer

2010

ISBN: 978-3-527-32296-1

Yanagida, T., Ishii, Y. (eds.)

Single Molecule Dynamics in Life Science

2008

ISBN: 978-3-527-31288-7

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Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.

© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978-3-527-32816-1

ePDF ISBN: 978-3-527-65605-9

ePub ISBN: 978-3-527-65604-2

Mobi ISBN: 978-3-527-65603-5

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Cover Design Adam-Design, Weinheim, Germany

Typesetting Thomson Digital, Noida, India

Preface

Several factors over the last few years have come together to contribute to the origin and development of this book. First and foremost is the incredible expansion in the application of FRET and its derivative techniques, especially in the biological sciences. Web of Science (www.thomsonreuters.com) provides more than 12,000 items using the search term FRET as a unique topic, while PubMed (www.ncbi.nlm.nih.gov/pubmed) provides almost 6000 items. The latter site also allows tracking of these items over time with 0 hits shown for 1979 and >1000 predicted for 2013 – an impressive and eye-opening increase. This stands in stark contrast to the years 1970–1990 that have less than 20 publications in total. This is not to say that nothing significant happened during this time period, but rather it reflects how specialized the field was and reminding us also of how poor the performance of journal citation and referencing tools were before the 1990s. In terms of just citations alone, Förster's original 1948 paper in Annalen der Physik has to its credit a remarkable ∼5000 citations, although it is safe to say that only a minority of those who cite this article have read it (especially in the original German). Indeed, some scientists consider this to be one of the most cited papers that has never been actually read.

Development of and access to a wide range of versatile fluorescent materials in conjunction with improved, easy-to-use and yet incredibly sophisticated microscopes and fluorometers have coincided with, helped drive, and also increased FRET usage. Fluorophores that are utilized in FRET now commonly encompass organic dyes, fluorescent proteins, semiconductor quantum dots, metal chelates, various noble metal and other nanoparticles, intrinsically fluorescent amino acids, biological cofactors, and polymers, to name but a few members of this growing library. Hand in hand with materials development is the growing availability of numerous reactive and bioorthogonal chemistries to site–specifically attach such fluorophores to all types of biological molecules ranging from proteins to DNA. This, in conjunction with FRETs unique ability to consistently provide nanoscale inter- and intramolecular separation distances, has meant that its utility is also rapidly growing in structural studies of biomolecules and biological complexes. We have also seen the implementation of intracellularly expressed fluorescent protein-based FRET sensors expand so rapidly over the past 15 years that it is now not uncommon to encounter students who do not know where this technology originated from (Roger Tsien, University of California San Diego). Perhaps this is the ultimate form of a compliment in science – when something becomes so commonplace and widely accepted that people forget who invented it. Concomitant with this rapid development of materials, commercial microscope systems combined with sophisticated analytical software are now widely available providing direct access to many different FRET techniques and their derivatives. Another proof that FRET techniques have come of age is the recent meeting entitled “Förster Resonance Energy Transfer in the Life Sciences” held at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, in March 2011. This relaxed and wonderfully stimulating discussion meeting organized by Donna Arndt-Jovin, Stefan Hell, Thomas Jovin, Claus Seidel, and Jürgen Troe focused on all the different aspects of FRET from analytical techniques and microscopy to new materials. That FRET could be the stand-alone subject of an international scientific meeting speaks volumes to its growing utility. In addition, not many people realize that the genomics revolution of the past 15–20 years owes a large debt to the use of FRET. Richard Mathies and Alex Glazer at the University of California Berkeley were among the first to realize that use of a dye-based FRET system could drastically simplify the instrumental requirements for DNA sequencing. By linking a single donor dye with four different acceptors (representing the four DNA bases), they were able to provide a set of four common DNA primers. These constructs used FRET to create four spectrally well-separated windows that could be excited by a single laser wavelength in any electrophoretic system instead of requiring two or three separate lasers. This strategy quickly became the workhorse of DNA analysis and, as is typical for any successful and proprietary technology, this also became the subject of considerable litigation. Similar FRET systems form the basis for numerous genotyping tests such as the Taqman assay that have also contributed quite considerably to genomics. A variety of other FRET assay formats for monitoring enzymatic activity and the like have also become quite commonplace in biosensing, biological research, and drug discovery.

Niko and I both come from laboratories that are very interested in understanding how newer materials such as quantum dots and/or long-lifetime rare-earth chelates engage in FRET and other forms of energy transfer. These materials provide for fascinating energy transfer configurations that were not described or even considered in Förster's seminal treatise. For example, can FRET occur when the acceptor is as well excited as a donor while manifesting a much longer excited state lifetime as in the case of pairing an organic dye donor such as fluorescein with a red-emitting quantum dot. With questions such as this in mind, one of the main FRET resources that is almost invariably consulted first in the pursuit of appropriate background is Lakowicz's excellent primer –Principles of Fluorescence Spectroscopy (Springer). Although still one of the most readable textbooks ever published, this resource provides only a limited amount of data and discussion on the intricacies of FRET. Van Der Meer's Resonance Energy Transfer Theory and Data (Wiley-VCH Verlag GmbH) is far more detailed about FRET mechanics and is another well-cited reference in this area; however, this has unfortunately fallen out of print and is quite hard to find. Thus, it was that we both found ourselves lamenting the lack of an up-to-date and detailed resource/primer on all aspects of FRET when Wiley-VCH graciously approached us about undertaking a book on an important scientific area of our choice. It did not take us long to decide the subject that we were going to propose.

The authors who contributed the individual chapters in this book reflect not only some of the principal experts in the field but also the wide diversity of FRET application itself. We very much appreciate the support of leaders in this field, including Robert E. Campbell, Bo Albinsson, and Jonas Hannestad. We were also overjoyed when Wieb van Der Meer agreed to make several major contributions to this project. The book is divided into four major parts. Part One provides some background, theory, and key concepts. This part begins with a personal remembrance of Theodor Förster by one of his former students and colleagues Herbert Dreeskamp (Chapter 1). Thomas Jovin then notes the recent passing of Robert Clegg and Elizabeth Jares-Erijman by describing their important contributions to FRET (Chapter 2). Wieb van der Meer then takes us in detail through the Förster theory and tackles the always important yet continually vexing issue of kappa-squared (Chapters 3 and 4). Niko Hildebrandt then provides a detailed primer on how to apply FRET – from experimental design to data analysis (Chapter 5). Finally, Kim Sapsford updates a previous 2006 paper that describes the ever-growing FRET toolbox of diverse fluorophores (Chapter 6). Part Two of the book focuses on some common FRET techniques and applications. Kim Sapsford and her colleagues from the U.S. Food and Drug Administration again contribute with a discussion of FRET application for in vitro sensing and diagnostics (Chapter 7). Thomas Pons then reviews single-molecule FRET applications, which represent another rapidly growing and important area (Chapter 8). Cheryl Woolhead provides a description of FRET utility in the determination of protein, peptide, and other biological structures (Chapter 9). This part ends with a contribution from Jonathan Claussen on FRET-based cellular biosensing with genetically encoded fluorescent indicators (Chapter 10). Part Three looks at recent developments starting with the use of fluorescent proteins from Robert Campbell (Chapter 11). This is followed by a review of FRET usage with semiconductor quantum dots from W. Russ Algar and colleagues (Chapter 12) along with an overview of the growing area of multistep FRET from Bo Albinsson and Jonas Hannestad (Chapter 13). The concluding Part Four includes a detailed and vastly updated series of supporting tables on FRET pairs and Förster distances collected and collated by Wieb van der Meer (Chapter 14). These tables were so useful in his previous book that we could not let this opportunity to update them go unused. Finally, some of the authors provide their own outlook on and perspectives of FRET (Chapter 15).

We want to thank all of the authors for not only their time and contributions but also for their incredible patience with us as this book slowly came together. The same is true for our coworkers at Wiley, including Eva-Stina Müller and Heike Nöthe. We have tried to focus on the important aspects that will both help the FRET novice and reinforce the understanding of a seasoned FRET user. Given the details and growth of this technology, we realize that we could not include everything we wanted and our apologies are further extended for any and all omissions along with any errors. We admit that we were rather naïve and a little overly hopeful in some of our initial ideas and desires for this book. A detailed historical accounting of Förster's life and work is still missing from the literature and although we tried repeatedly, we could not bring this to fruition for the current book. We also wanted a dedicated Web site to accompany this book where FRET data, especially on newer donor–acceptor pairs, could be continuously updated along with providing a forum for discussion and solicitation of experimental advice. Alas, such a home is not to be found as of yet. Finally, although he tried quite valiantly, Niko could not convince any television producers or comic book publishers to introduce a new superhero for children (and scientists), namely, Captain FRET who solves complicated situations with the application of resonance energy transfer while carefully explaining the subsequent photophysical analysis for the layman. If there is ever an update to this book, we will redouble our efforts to bring these additional ideas to reality.

In the preface to Resonance Energy Transfer Theory and Data, Wieb van Der Meer outlines how most scientists with interest in energy transfer can be subdivided into two groups: those interested in homotransfer, the homotransferites (primarily biochemists), and those interested in heterotransfer, the heterotransferites (primarily physical chemists). In the same manner as him, this book is written for all and not just one group. However, in adhering to the culture of our times and rather than differentiating between groups, we would like to add a new all-inclusive description to the FRET user anthology; if you are able to utilize FRET successfully (in any form), then you should be considered and referred to as a “FRET jock” and this should be a moniker of distinction and pride among your scientific colleagues. It is our fervent hope that well-worn copies of this book find their way onto your office and laboratory shelves.

Niko Hildebrandt

Igor L. Medintz

List of Contributors

Bo Albinsson

Chalmers University of Technology

Department of Chemistry and Biotechnology/Physical Chemistry

41296 Gothenburg

Sweden

W. Russ Algar

University of British Columbia

Department of Chemistry

2036 Main Mall

Vancouver

British Columbia

V6T 1Z1

Canada

Alice G. Byrne

Western Kentucky University

Department of Physics and Astronomy

1906 College Heights Blvd

Bowling Green

KY 42101

USA

Matthew M. Byrne

Western Kentucky University

Department of Physics and Astronomy

1906 College Heights Blvd

Bowling Green

KY 42101

USA

Robert E. Campbell

University of Alberta

Department of Chemistry

11227 Saskatchewan Drive

Edmonton

Alberta

T6G 2G2

Canada

Jonathan C. Claussen

U.S. Naval Research Laboratory

Center for Bio/Molecular Science and Engineering

Code 6900

4555 Overlook Avenue, SW

Washington

DC 20375

USA

and

George Mason University

College of Science

Fairfax

VA 22030

USA

George Coker, III

Western Kentucky University

Department of Physics and Astronomy

1906 College Heights Blvd

Bowling Green

KY 42101

USA

Yidan Ding

University of Alberta

Department of Chemistry

11227 Saskatchewan Drive

Edmonton

Alberta

T6G 2G2

Canada

Herbert Dreeskamp

Technische Universität Braunschweig

Germany

Kelly B. Gemmill

U.S. Naval Research Laboratory

Center for Bio/Molecular Science and Engineering

Code 6900

4555 Overlook Avenue, SW

Washington

DC 20375

USA

Jessica Granek

U.S. Food and Drug Administration

CDRH/OSEL/DB

WO64 RM3028 HFZ-110

10903 New Hampshire Avenue

Silver Spring

MD 20993

USA

Jonas K. Hannestad

Chalmers University of Technology

Department of Chemistry and Biotechnology/Physical Chemistry

41296 Gothenburg

Sweden

Niko Hildebrandt

Université Paris-Sud

Institut d'Electronique Fondamentale

NanoBioPhotonics

Btiment 220

91405 Orsay Cedex

France

Hiofan Hoi

University of Alberta

Department of Chemistry

11227 Saskatchewan Drive

Edmonton

Alberta

T6G 2G2

Canada

Thomas M. Jovin

Max Planck Institute for Biophysical Chemistry

Laboratory of Cellular Dynamics

37077 Göttingen

Germany

Ulrich J. Krull

University of Toronto Mississauga

Department of Chemical and Physical Sciences

3359 Mississauga Rd. North

Mississauga

Ontario

L5L 1C6

Canada

Angela Mariani

U.S. Food and Drug Administration

CDRH/OSEL/DB

WO64 RM3028 HFZ-110

10903 New Hampshire Avenue

Silver Spring

MD 20993

USA

Melissa Massey

University of Toronto Mississauga

Department of Chemical and Physical Sciences

3359 Mississauga Rd. North

Mississauga

Ontario

L5L 1C6

Canada

Igor Medintz

U.S. Naval Research Laboratory

Center for Bio/Molecular Science and Engineering

Code 6900

4555 Overlook Avenue, SW

Washington

DC 20375

USA

Thomas Pons

ESPCI–CNRS–UPMC (UMR8213)

Laboratoire de Physique et d'Étude des Matériaux

10, rue Vauquelin

75005 Paris

France

Philip J. Robinson

University of Glasgow

College of Medical, Veterinary and Life Sciences

Institute of Molecular, Cell and Systems Biology

Glasgow

Lanarkshire G12 8QQ

Great Britain

Kim E. Sapsford

U.S. Food and Drug Administration

CDRH/OSEL/DB

WO64 RM3028 HFZ-110

10903 New Hampshire Avenue

Silver Spring

MD 20993

USA

Seth L. Sloan

Western Kentucky University

Department of Physics and Astronomy

1906 College Heights Blvd

Bowling Green

KY 42101

USA

Christopher Spillmann

U.S. Naval Research Laboratory

Center for Bio/Molecular Science and Engineering

Code 6900

4555 Overlook Avenue, SW

Washington

DC 20375

USA

Samantha Spindel

U.S. Food and Drug Administration

CDRH/OSEL/DB

WO64 RM3028 HFZ-110

10903 New Hampshire Avenue

Silver Spring

MD 20993

USA

B. Wieb van der Meer

Western Kentucky University

Department of Physics and Astronomy

1906 College Heights Blvd

Bowling Green

KY 42101

USA

Daniel M. van der Meer

TelaPoint

9500 Ormsby Station Road, Suite 402

Louisville

KY 40223

USA

Steven S. Vogel

National Institutes of Health

National Institute on Alcohol Abuse and Alcoholism

Laboratory of Molecular Physiology

5625 Fishers Lane

Bethesda

MD 20892

USA

Bridget Wildt

U.S. Food and Drug Administration

CDRH/OSEL/DB

WO64 RM3028 HFZ-110

10903 New Hampshire Avenue

Silver Spring

MD 20993

USA

Cheryl A. Woolhead

University of Glasgow

College of Medical, Veterinary and Life Sciences

Institute of Molecular, Cell and Systems Biology

Glasgow

Lanarkshire G12 8QQ

Great Britain

Andrew B. Yeatts

U.S. Food and Drug Administration

CDRH/OSEL/DB

WO64 RM3028 HFZ-110

10903 New Hampshire Avenue

Silver Spring

MD 20993

USA

Part One

Background, Theory, and Concepts

1

How I Remember Theodor Förster*)

Herbert Dreeskamp

I first met Theodor Förster in 1959, after my postdoctoral years in the United States, at a conference on fast reactions organized by Manfred Eigen in the Harz Mountains. Very prominent scientists attended, such as Eyring, Noyes, and the three men seen in Figure 1.1 (from left to right) George Porter, Theodor Förster, and Albert Weller.

Figure 1.1

Half a century ago “fast reaction” meant flash photolysis in the microsecond range by Norrish and Porter or relaxation methods by Eigen. As I remember, it was Förster who pointed out clearly that the term “fast” characterized our technical facilities at that time rather than the scientific problem at hand. The time range of fast chemical reactions may better be characterized by the rearrangement of electrons in the 10−16 s range, the vibration of nuclei in the 10−12 s range, or the deactivation of electronically excited states in the 10−9 s range. Thus, if there are reactions of electronically excited states – and after all, molecules do have characteristically different properties in their different electronic states – you will be able to investigate these reactions in the nanosecond range by just studying fluorescence, which is emitted in competition to these photochemical reactions. And since Förster had, at the beginning of his career, studied the absorption spectra of organic compounds, that is, the electronic structure of their ground and excited states, he was able to find the proteolytic reactions of aromatic compounds as the classical example of using fluorescence to investigate fast chemical reactions. To me this example shows clearly, in a nutshell, Förster's approach to the scientific problem and why he was so extremely successful in opening new avenues in photochemistry.

Sometimes it was said that he was gifted by a remarkable intuition. I am sure his intuition was the result of strict devotion to science, very hard work, his enormous knowledge of the literature, and his insistence to reach a complete understanding of the problem at hand.

Theodor Förster was a son of Frankfurt am Main, like Otto Hahn (as shown in Figure 1.2, second from left, and Förster is the first from right). He got a training there as a theoretical physicist when both he and quantum mechanics were quite young (Figure 1.3). As an assistant to Karl Friedrich Bonhoeffer in Leipzig, he came under the influence of such eminent men – besides Bonhoeffer of course – as Heisenberg, Kautsky, and, I think particularly, Peter Debye. Since those Leipzig days there is the most remarkable and efficient interplay between theory and experiment in the work of Theodor Förster.

Figure 1.2

Figure 1.3

His aim was to search for the most appropriate solution of the scientific problem, or in his favorite words: “Die richtige Deutung einer Beobachtung” (The correct interpretation of an observation). This included taking carefully all information into account, separating the important from the trivial, designing a simple experiment, and arriving at the correct interpretation, if possible, without any too elaborate computer analysis. For me, this picture (Figure 1.4) from the Posen or Göttingen years – the 1940s – may illustrate what I tried to say: Brains seem to be more important than fancy equipment or powerful computers.

Figure 1.4

Very often it was both a relief and a delight for all of us who were present, when after a somewhat incomprehensible seminar talk he would stand up and quite politely say, “If I understand you correctly, you meant to say this and that…” and he would give in a few words a lucid interpretation of the topic at hand.

I once asked Förster how to grade a thesis paper, and he advised me to be not too strict. But for him, Theodor Förster, his scientific work had to meet the highest standards. Things had to be correct, of course, but equally important: it had to include the most concise analysis of the problem, a perfect logic of the solution, and a clear statement on the significance of the results. He did not publish much, but the things he did publish can be a source of inspiration still today.

You may know that the phenomenon of fluorescence depolarization was an important step that ultimately led to an understanding of electronic excitation energy transfer between molecules. After a large amount of empirical material had been accumulated by others, Förster, in a lucidly written review article, gave a brilliant analysis of this effect and brought a long discussion ultimately to an end. Thus, may I advice you, once in a while, to take your time off from the lab and go to the library and study a paper of his, or better still his most admirable monograph “Fluoreszenz organischer Verbindungen.” You may be rewarded by getting a hint on how problems may be solved by putting them in the right perspective, a strategy in which Theodor Förster was a superb master.

My picture of Förster would be incomplete without remembering how much he enjoyed the company of colleagues, or of his students, for example, at a Christmas party in the lab (Figure 1.5).

Figure 1.5

Very often prominent colleagues from abroad came to Stuttgart, gave a talk, and certainly were invited by him and his wife Martha (Figure 1.6) to their home. Regularly, younger members of the department were also invited to these evenings.

Figure 1.6

For me, certainly the most memorable of these meetings was when James Franck visited Stuttgart, I think in 1964 (Figure 1.6). You all will know the fundamental work in atomic physics done in Berlin and Göttingen by Franck and Hertz in the 1920s, or the direct proof of a radiationless energy transfer between atomic systems by Franck and Cario. But I think we should also remember that James Franck was the initiator of the Franck Report of 1945.

In the last picture (Figure 1.7), you see James Franck and Theodor Förster many years after the war, evidently discussing at a scientific conference. Also the topic of their discussion was – I am pretty sure, – the phenomenon of light harvesting, energy transfer, and photosynthesis, questions that fascinated both these men for many years. Franck gave the first experimental proof that the electronic energy may be exchanged radiationlessly among atoms, and Förster, some 25 years later, on the basis of his deep understanding of quantum mechanics, gave us the theory of the nontrivial transfer of electronic energy in molecular systems, the “Förster resonance energy transfer” (FRET), which gave us a formula that has become extremely important in biological sciences.

Figure 1.7

The contributions of Theodor Förster to modern photochemistry are most impressive, but equally fascinating to me is the way in which he elaborated these things. If you have a look at his strategy, I am certain you will have a good chance to profit also from this aspect of the work of Theodor Förster.

Note

* This chapter is based on a talk given by the author at the International Bunsen Discussion Meeting on “Light Harvesting and Solar Energy Conversion,” March 29, 2010, Stuttgart-Hohenheim, commemorating the 100th birthday of Theodor Förster (1910–1974). The author studied physics in Bonn and Paris, spent decisive years 1960–1970 with Förster in Stuttgart, and was professor of physical chemistry at the Technische Universität Braunschweig. He thanks Dr. Eberhard Förster, son of Theodor Förster, for the pictures used in this chapter.

2

Remembering Robert Clegg and Elizabeth Jares-Erijman and Their Contributions to FRET

Thomas M. Jovin

This is a rather personal account, yet not biographical, of a scientific “family” bound by circumstances and a common pervasive scientific theme. It is perhaps the essential nature of Förster resonance energy transfer (FRET) – a near-field resonance phenomenon – that engenders “resonating” interactions between individuals. For Robert (“Bob”) Clegg and Elizabeth (“Eli”) Jares-Erijman, as well as for the redactor of this account, Thomas (“Tom”) Jovin, there were distinct circles of scientific and personal influences that dictated how FRET entered their lives and careers. As in the case of most scientists, the initial event was exposure to key literature. In the emerging FRET field after World War II, a number of highly cited original papers and reviews stimulated innumerable scientists to incorporate FRET into their conceptual and experimental strategies. The reason lay with their authors, leading protagonists and innovators, who in historical order included Theodor Förster [1], Gregorio Weber [2], Izchak Steinberg [3], Lubert Stryer [4], and Ludwig (Lenny) Brand [5]. There are other equally valuable sources; I cite these because they predominated in my case and were also highly influential for Bob and Eli, ultimately leading to their own valuable contributions.

2.1 Biographical Sketch of Bob Clegg

Robert MacDonald Clegg succumbed to cancer on October 15, 2012. His tragic death represents an immense loss not only to the members of his immediate family (wife Margitta, and sons Benjamin, Niels, and Robert), but also to his very extensive “scientific family” of colleagues and friends.

Bob received his PhD in physical chemistry from Cornell University in 1974, having worked with Elliot Elson on the theory and practice of rapid chemical kinetics, specifically chemical relaxation following pressure perturbation. Elliot was a product of the lab of Buzz Baldwin in the Stanford Biochemistry Department and his influence on Bob's view of science and research cannot be overemphasized. As a postdoc (with me) in the Department of Molecular Biology at the Max Planck Institute for Biophysical Chemistry, Bob quickly demonstrated his depth of knowledge and unique leadership and innovative skills. He assumed the position of Senior Staff Research Associate with an independent group in 1976. Over the next two decades, he pursued numerous lines of research, devising and applying quantitative thermodynamic, kinetic, and spectroscopic techniques, particularly fluorescence – which he had not used previously – to studies of macromolecular systems such as RNA polymerase. He became one of the best expounders worldwide of the theory and practice of FRET (energy transfer) and was involved in pioneering implementations and applications of fluorescence lifetime imaging microscopy (FLIM). Bob is well remembered in Göttingen for being “big and broad” in both science and physique, but also for his invariably cheerful and gentle disposition. He was ever ready to offer help in the form of advice or action. He was everybody's friend.

During a sabbatical leave in 1996 at the University of Illinois (UIC) in Champaign, Bob established a close working relationship with fluorescence pioneer Gregorio Weber. Weber's distinguished disciple Enrico Gratton induced Bob's repatriation to the United States (UIC) in 1998, with an appointment as professor in the Departments of Physics and Bioengineering and as a member of the faculty of the Biophysics program (at the time of his death, Bob was its director and an affiliate of the Institute for Genomic Biology). In this academic environment, Bob quickly established himself as a leading researcher in numerous biophysical disciplines as well as an extraordinarily dedicated and capable teacher.

Bob was both an excellent experimentalist and theoretician, and consistently sought a fundamental understanding of the phenomena under investigation. In so doing, he displayed a unique capacity for deciphering structure–function relationships in complex systems involving transitions in molecular conformation and association of proteins and nucleic acids. His contributions to the expansion of optical microscopy into new fields of biology and biotechnology were also numerous and profound.

The breadth of Bob's interests and associations was reflected in his membership in the Biophysical Society, the American Physical Society, FASEB, the Optical Society of America, and the American Chemical Society. In 2009, the Biophysical Society recognized his contributions to fluorescence with the Weber Award for Excellence in Theory and Experiments in Fluorescence, Fluorescence Subgroup. That same year, he received from the Society for Experimental Biology and Medicine the Alan MacDiarmid Best Paper Award in the interdisciplinary research category. The paper in question was “Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors,” a typical (for Bob) synthesis of biology, chemistry, and physics.

2.2 Biographical Sketch of Eli Jares-Erijman

Elizabeth Andreas Jares-Erijman, professor in the Department of Organic Chemistry at the University of Buenos Aires, Argentina, died of cancer on September 29, 2011. She was 50 years old. As in the case of Bob Clegg, we, the scientific community, lost an excellent, innovative scientist, a stimulating teacher, and a wonderful friend.

A chemist by training, Elizabeth Eli received her PhD from the University of Buenos Aires in Argentina in 1989. After a postdoctoral period in the Department of Chemistry at UIC, she was transferred to my lab in Göttingen in 1993, accompanied by her husband Leonardo (Leo) – also a postdoc in the department – and her daughter Paula (see more details later). Three years later, Eli returned to Argentina and rejoined the Department of Organic Chemistry in the Faculty of Exact and Natural Sciences. She advanced through the academic hierarchy and occupied a pivotal role in the teaching and research activities of the department. Eli had her second child, Florencia, in 1998.

In 2004, the Max Planck Institute for Biophysical Chemistry recognized her seminal contributions and involvement with the research program of the institute and proposed her for appointment as Head of a Max Planck Partner Group of the institute. This came to pass after an evaluation by an outside commission. Hers was the first partner group to be established in Argentina, in fact, the first in all of Latin America.

Eli was one of the few individuals in Latin American science who crossed rigid departmental lines in order to establish a comprehensive and systematic research effort in what is currently designated as chemical and supramolecular biology. She established a Laboratory of Nanotools and Bioimaging to promote the design and use of novel organic probes and multifunctional nanoparticles as biosensors and “nanoactuators.” New implementations of FRET, for example, exploiting the phenomenon of photochromism, were an important feature in many of these systems. However, the biological applications were always at the research focus, as illustrated in recent publications devoted to α-synuclein, the “amyloid” protein in Parkinson's disease (PD).

Eli was an excellent citizen of her scientific community, serving in many commissions, both at the local and at the national levels. By all indicators, she was an inspired and very competent teacher. A significant indicator of her persuasive and inspiring leadership is the quality and success of the people who worked with her. She received numerous awards for her scientific achievements, which included the prize Eduardo G. Gros and the prize Bernado A. Houssay (an Argentine Nobel laureate), and was nominated in 2011 for the prestigious UNESCO-L'Oréal Award for Women in Science. In 2012, the Argentine Foundation of Nanotechnology (FAN) established a prize for Scientific Quality “Doctora Elizabeth Jares-Erijman,” and the newly established CONICET Institute of Nanosciences bears her name.

2.3 The Pervasive Influence of Gregorio Weber

As has already been stated, Gregorio Weber plays a central role in this account, first of all due to the preeminent position he occupied in the scientific world. He is recognized as the person responsible for much of the theoretical and experimental developments in/of modern fluorescence spectroscopy. In particular, he pioneered the application of this technique in the biological sciences. In so doing and by virtue of his extraordinary human qualities, he served as an inspirational teacher to generations of spectroscopists and biophysicists working in basic science, biomedicine, and on industrial implementations of the numerous instrumentation and techniques developed in his lab. His list of achievements is extensive and unique: synthesis and application of small-molecule probes of hydrodynamic properties, polarity, and microviscosity; theory of fluorescence polarization and FRET; intrinsic fluorescence of the amino acids and of complexes of FAD and NADH; development of frequency domain fluorimetry; and studies of protein structure under pressure.

In our work in Göttingen, we were of course guided by the publications of this illustrious “father of biological fluorescence,” and a more personal association started in the 1970s. Gregorio and I shared an Argentine origin and thus it was not by accident that in 1993, during a visit to UIC, he introduced me to the Argentine husband–wife scientist pair Leo and Eli. Leo was Gregorio's (last) postdoc and Gregorio recommended that both he and Eli extend their postdoctoral experience with a stint in our institute in Göttingen, Germany. It was in this manner that Leo came to work with Bob Clegg and Eli came to work with me. In the three ensuing years, as well as thereafter, we kept “growing up with FRET” together, sharing our admiration of Gregorio Weber with other celebrated postdoctoral “FRETists” (now professors), such as Dorus Gadella Jr., Gerard Marriott, and Philippe Bastiaens. We also benefited from Gregorio's insight and advice offered during occasional visits to the institute. Bob's (and my) sentiments regarding Gregorio Weber are well expressed in Figure 2.1, taken from one of his many lectures on fluorescence and FRET, years later. Gregorio passed away, also of cancer, in 1997.

Figure 2.1 Bob Clegg's expression of indebtedness to Gregorio Weber.

2.4 Contributions by Bob Clegg to FRET

It is perhaps appropriate to invoke at this stage a particular formulation of the scientific method: (i) a scientific hypothesis can never be shown to be absolutely true, and must potentially be disprovable; (ii) the hypothesis must be useful until it is disproved; and (iii) the simplest hypothesis must be favored, unless it can be shown to be false (Ockham's razor). Bob was meticulous about the application of these precepts and his contributions accordingly took two forms: direct (offering new insights) and indirect (providing inspiration to others). In his work he applied a keen innate physical intuition and was highly social in the sense of exhibiting an invariably pleasant, cheerful, and helpful disposition.

Bob's research interests can be summarized as follows: (i) development and applications of fluorescence lifetime-resolved imaging microscopy (FLI and FLIM) and the development of unique dedicated software for analysis of such data; (ii) development of instruments for rapid relaxation kinetics (T- and P-jump), and microsecond rapid mixing; and (iii) applications of the advanced instrumentation to an exceedingly wide range of biological systems, animal and vegetal. The underlying molecular mechanisms investigated included nucleic acid conformational equilibria and kinetics, and multisubunit functional proteins and photosynthetic systems. During his 21 years in Germany (1977–1998), Bob published 68 papers and contributions, most of which dealt with the structure of nucleic acids. Nine were devoted to the theory and practice of FRET and another nine were devoted to FLIM. Two FRET reviews had (and still have) a great impact on the field. The first [6] was a detailed blueprint for the FRET practitioner. The underlying theory was thoroughly presented as well as numerous techniques for the evaluation of population distributions and determinations of the FRET efficiency E: sensitized emission, donor quenching, decrease in donor lifetime, and changes in donor and acceptor anisotropy. In conclusion, Bob stated, “The measurements cannot be better than the molecules that are being measured, so extreme care must be taken to ensure that the samples are well defined and pure. FRET with specifically labeled nucleic acids will surely become more popular in the near future; the method has the potential to distinguish many structural features and symmetries ranging over molecular distances less than 100 A.” How true! The second FRET review already dealt with FRET microscopy [7]. Bob contrasted in detail and unique clarity a quantum mechanical and two classical derivations of the FRET phenomenon, an issue he returned to repeatedly in later reviews and historical accounts (see later).

A great deal of Bob's research in Germany (and later, back in the United States) was devoted to the study of DNA helices, junctions, bulges, and kinks, much of it in close collaboration with David Lilley. FRET was an essential ingredient of this research, as is well illustrated in three publications that received wide attention [8–10]. One of these [9] featured the modulation of FRET according to the relative geometric disposition of FRET donor and acceptor positioned around a DNA helix (Figure 2.2).

Figure 2.2 FRET strategy (geometrical model) for determination of DNA helical parameters: (a) the D–A vector R “swings around” the helix as a function of separation N in the sequence and axial displacement L of the fluorophores relative to the bases to which they are attached, (b) FRET efficiency as a function of N (in base pair units). Inset: (ratio)A, a widely adopted FRET measure introduced by Bob; (ratio)A = (acceptor emission sensitized by the donor and excited directly at the donor absorption band)/acceptor emission directly excited at its absorption band. Adapted from Ref. [9].

The development of FLI instrumentation was a key and long-range element of Bob's research program, during both the German and subsequent US phases of his career. The emphasis was always on maximal speed and multiparametric acquisition, a pioneering example being the PhD thesis work of Peter Schneider [11]. Our extensive departmental involvement with FRET (flow cytometry and then imaging) and FLIM in the years leading up to the fall of the Berlin Wall was greatly facilitated by the participation of a large number of excellent colleagues from the Institute of Biophysics in Debrecen, Hungary. This circumstance was brought about by the farsighted efforts and perseverance of its director Sandor Damjanovich. Some of these individuals are featured in the group photograph taken at the symposium in honor of Theodor Förster (“Förster resonance energy transfer in life sciences”) in Göttingen, March 2011, where some of us saw Bob for the last time (Figure 2.3). It was on this occasion that Bob managed to track down the house where Förster originated his theory and wrote his famous book Fluoreszenz organischer Verbindungen.

Figure 2.3 Bob Clegg (far left) and some former members of his group and other alumni of the Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, who attended the symposium honoring Theodor Förster in March 2011, Göttingen.

Back in the United States (as of 1998) and established in an academic environment, Bob developed his capabilities to the full, including those as a gifted “gadgeteer.” He shared responsibility for the celebrated nationally funded research resource center, the LFD (Laboratory for Fluorescence Dynamics), established by Enrico Gratton in 1986 at UIC and relocated in 2006 to the University of California, Irvine. The LFD provided an excellent environment for cutting-edge technology development. Thus, in the decade of 2000–2010, a real-time field FLIM instrument, fully compatible with confocal optical configurations and with high contrast and sensitivity, was devised, allowing the high-speed acquisition of three-dimensional imaging and including spectral resolution. This instrumentation was applied to process prostate biopsies in an attempt to facilitate diagnosis of prostate cancer. In addition, the redistribution of a phototherapeutic/diagnostic compound (PpIX) in live tumor cells was investigated, just one activity establishing FLIM as an important technique in dermatology research [12]. A FRET redox biosensor was developed to measure the oxidation–reduction potentials in fluids and cells [13]. In parallel, a pressure-jump instrument was devised for studying photosynthetic plant systems, including living organisms such as algae [14]. The same method was applicable to kinetic studies of RNA/DNA conformational changes and binding of ligands. In fact, numerous post-2000 publications, most using FRET but not cited here, were dedicated to nucleic acid studies: four-way junctions (largely collaborations with Taekjip Ha and David Lilley), hammerhead ribozymes, protein–DNA interactions, ribosomal intersubunit dynamics, and probe–DNA interactions; the latter studies were conducted in the course of a very long-term collaboration with a close friend and colleague, Frank Loontiens.

Data analysis was always a central issue for Bob in his work. As one example, the FLIM polar plot analysis of frequency domain FLIM image data was established [15] and extended by incorporating FRET-relevant spectral resolution [16] and newly created image analysis algorithms for selecting important image locations via their morphology using “wavelets” [17]. In addition, novel ways of “denoising” images depending on the type of noise (Poissonian or Gaussian) were devised [18], dramatically increasing the accuracy of the FLI. The potential of lifetime-resolved imaging in small organisms as well as live biological mammalian cells and photosynthesis (algae as well as higher plants) was to be enhanced for 3-D imaging using sample excitation single plane illumination microscopy (SPIM). Unfortunately, this work did not proceed beyond the planning stage due to Bob's sickness.

Bob did not neglect his dedication to the promotion of FRET history awareness and the applications of the technique, almost invariably coupled with FLIM. Accordingly, numerous reviews appeared (FRET [19–21] and FLIM [22–26]), which complemented and extended the earlier publications. Bob's service to the scientific community was also evident in his many years as a member of the faculty of the long-standing Annual Workshop on FRET Microscopy, organized by Ammasi Periasamy, another prolific contributor to the FRET field and its literature.

2.5 Contributions by Eli Jares-Erijman to FRET

Eli arrived in our lab in 1993 after a postdoc at UIC working in the lab of Ken Rinehart on the synthesis, isolation, and characterization of very complex natural products. She was an accomplished organic chemist but had had little exposure to fluorescence techniques, biophysical methods, and biomolecules. This situation changed in a very short time, such that 3 years later, Eli would return to Argentina as an accomplished biophysicist and an expert in fluorescence technology and probes. In fact, the latter served as primary objectives and motivators of this development, inasmuch as Eli displayed a keen ability to recognize the potential of new structural motifs, scaffolds, and mechanisms in creating innovative probes of molecular states, transitions, and localization. One of the first applications was in a study of noncanonical DNA such as Z-DNA and so-called parallel-stranded DNA (psDNA), using the FRET-based approach pioneered by Bob Clegg, while at the same time extending it conceptually and experimentally (new ratio functions). The left-handed character of Z-DNA was confirmed [27], but the helical sense of psDNA containing AA and GG base pairs and also presumed to be left-handed was not published because the extensive data posed (and still pose) problems of interpretation.

Eli's chemical acumen became very evident in the next FRET-based studies of photochromic compounds (diarylethenes) that provided a switchable acceptor function by virtue of dual (“open” and “closed”) states interconvertible by cycles of near-UV and visible light [28]. The distinctive absorption spectra of the closed and open forms differ in the degree of superposition with the emission of an appropriately selected donor, thereby leading to a change in the overlap integral J, one of the factors defining the Förster transfer distance Ro. The mechanism of photochromic FRET (pcFRET) is depicted in Figure 2.4; it was explored by systematic structural modifications and careful thermodynamic and kinetic studies after Eli returned to Argentina [28–30].

Figure 2.4 Photochromic FRET (pcFRET). Exposure of the diarylethene (open form) to near-UV light (250–320 nm) induces photocyclization to the closed form. The latter has an absorption band in the visible region overlapping with the emission band of a (the) donor, thus enabling FRET. Visible light leads to cycloreversion. The bistable diarylethenes exhibit little fatigue such that multiple cycles are feasible.

At this juncture, Eli was operating as a Partner Group of our Max Planck Institute and was PI and co-PI on a number of nationally and internationally funded programs. Her major focus was on the development and application of smart sensors and devices combining luminescent, photochromic, and other small molecules with nanostructures such as quantum dots (QDs). In about 1998, QDs became commercially available through the auspices of Quantum Dot Corp. (QDC). Eli was probably the first person to conduct FRET experiments utilizing QDC QDs as donors and was instrumental in one of the first in-depth characterization of these new materials [31]. She readily perceived that emerging technologies based on a combination of chemistry, physics, and molecular biology were creating demand for smart materials serving as reporters and sensors in micro- and nanosystems [32]. The pioneering study of epidermal growth factor receptor (EGFR) activation and dynamics by microscopy of living cells using QD-EGF as ligands [33] was one of the first responses to this challenge, and stimulated numerous applications to other systems, including the insulin receptor [34,35]. Meanwhile, pcFRET was shown to operate at the level of a single particle [36] and to offer a new means for conducting isothermal relaxation kinetic measurements [37]. The pcFRET principle was extended to systems of core–shell QDs wrapped with an amphiphilic polymer containing photochromic groups and, in some constructs, a second fluorophore [38–40]. Such nanoparticles are water dispersible and can be reversibly modulated in fluorescence (quenched) by exercising the photochromic cycle. Other “J -engineered” QDs for sensing pH were based on FRET from the QD core to a shell of indicator molecules with pH-sensitive absorption spectra [41].

A quite different endeavor featured the use of “nanodevices” to sense and control the aggregation of amyloid proteins (specifically α-synuclein) in vitro and in the cellular context, thereby contributing to a better understanding of the molecular processes underlying the etiology of PD. This line of research led to the discovery of novel supramolecular intermediates in the aggregation pathway of α-synuclein preceding the formation of amyloid fibrils [42]. A prominent example is the “acuna” (amyloid “cradle”), a submicrometer structure that may be (at least partially) responsible for the toxicity and functional loss of dopaminergic neurons underlying PD [42]. The effort required a very interdisciplinary approach, combining numerous technologies such as organic synthesis, surface chemistry, physical and biophysical analysis, and quantitative microscopy to develop the sensors and apply them in context of cellular biology. Fluorogenic bisarsenical ligands [43], ratiometric [44] and/or solvatochromic probes [45], and NIR cyanines [46] also provided potential and actual novel FRET strategies for microscopy-based investigations of amyloid proteins in vitro and in living cells [47–51]. Current efforts in Eli's as yet functional research group are also being directed at the design and synthesis of optimal photoswitchable probes for the emerging superresolution microscopies.

As in the case of Bob Clegg, Eli published reviews on FRET imaging that have had a wide acceptance [52–54]. They are somewhat unusual in presenting novel views of photophysical phenomenon, such as the concept of a fluorophore as a photonic “enzyme” [52], and in offering an open-ended classification scheme for FRET methods. The latter include the donor and acceptor photobleaching techniques that originated from our FRET community in Göttingen, largely inspired by the unique publications, for example [55], of Tomás Hirschfeld, another illustrious member of the pantheon of spectroscopists. In publications [50,53,54], and in fact already in Ref. [27], it was proposed that in many FRET situations, calculations based on the ratio may be preferable to the classical , such that one can “bid farewell” to E and . FRET is a moving target.

2.6 A Final Thought

People survive in our memories if we keep them there by willfully recalling their personal qualities as well as their achievements. In Figure 2.5, we can appreciate that Bob and Eli, despite their distinctive ways and views, were two of a kind. We miss them both very much.

Figure 2.5 Eli and Bob on the grounds of the riverside campus of the University of Buenos Aires in 2010.

I am greatly indebted to Bob Clegg's family and other colleagues for material that made the writing of this chapter possible. Errors of commission and omission are mine alone.

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