Energy transfer in the red fluorescent protein DsRed in confined optical fields

Energieüberträge in dem rot fluoreszierenden Protein DsRed in definiert begrenzten optischen Feldern

Frank Schleifenbaum


Energieüberträge in dem rot fluoreszierenden Protein DsRed in definiert begrenzten optischen Feldern
ISBN: 978-3-938807-44-6
Veröffentlicht: August 2008, 1. Auflage, Einband: Broschur, Seiten 150, Format DIN A5, Gewicht 0.22 kg
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Energy transfer in the red fluorescent protein DsRed in confined optical fields

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Frank Schleifenbaum

Energy transfer in the red fluorescent protein DsRed in confined optical fields

(Energieüberträge in dem rot fluoreszierenden Protein DsRed in definiert begrenzten optischen Feldern)

Dissertation an der Fakultät für Chemie und Pharmazie der Eberhard-Karls-Universität Tübingen

Band 3 der Schriftenreihe LIFE SCIENCES. 150 Seiten. Zahlreiche Abbildungen, 21 davon in Farbe. Broschur. Preis: 29,50 Euro. ISBN 978-3-938807-44-6. Rhombos-Verlag, Berlin 2008

Institut für Physikalische und Theoretische Chemie:


Fluorescence resonance energy transfer (FRET) is one of the most fascinating topics in many fields of science, ranging from nano-engineering to biology. Here novel approaches to study and control FRET by confinement of the optical field are presented. Starting from high-resolution multiparameter single molecule fluorescence and Raman spectroscopy to characterise the red fluorescent protein DsRed as a prominent FRET system, it is shown how the rigidly coupled chromophores can be isolated by a subwavelength microresonator. Further on it is shown how the energy transfer can be precisely controlled and adjusted using microresonator structures. The volume concludes with an outlook to applications showing up possibilities to determine fluorescence quantum yields in sensor systems based on optical microresonator systems.



Scope of this thesis

Spectroscopic methods have gained more and more importance over the last four decades in all fields of life sciences. Due to the rapid development of both, electronics and computational systems spectroscopy has established as a standard method in laboratories over the world. This exceptional success of the technique is based on the direct insight into molecular properties which can be gained from the interaction between electromagnetic radiation and a target molecule. Hence, X-ray investigations give insight into the crystal structure of solids, ultraviolet and visible light elucidate electronic transitions within molecules whereas infrared radiation uncovers inter-atomic vibrations, revealing an individual chemical fingerprint of the investigated substances. Finally, radio frequencies allow for the exploration of the complete chemical structure of a molecule or a solid. This capacity constitutes spectroscopic techniques as powerful tools towards the understanding of molecular processes.
One key-role within this variety of spectroscopic methods is held by the field of fluorescence spectroscopy in the visible regime of electromagnetic radiation (over 100.000 publications referring to fluorescence techniques over the last decade). This is due to the fact that it combines ultra high sensitivity providing excellent image contrasts in microscopic applications with structural information. Especially biological sciences benefit from fluorescence spectroscopy since it is possible to exclusively label distinct parts of e. g. a living cell with a fluorescent marker, thus gaining an in-vivo insight into the processes in a living organism (see for example references). Advanced techniques such as the transfer of excitation energy between two fluorescent molecules can be used to study processes on a length-scale with sub-nanometer spatial resolution, thus allowing for the investigation of e. g. protein folding or transport phenomena inside a cell.
In spite of these advantages the structural information which can be gained from fluorescence studies is often very limited. In this thesis fluorescence studies in confined optical fields are presented. Changing the electromagnetic nano-environment of a molecule directly influences its fluorescence response.
Hence, a precise control of the electromagnetic field impinging on a fluorescent molecule should allow for tailoring the emission properties of the molecule, for rendering a customized fluorescence emission possible or for opening the field for a deeper understanding of photophysical processes.      

Due to the high sensitivity of fluorescence-based methods it is possible to investigate the emission properties of even a single isolated emitter, thus circumventing averaging effects inevitable when an ensemble of molecules is investigated at a time. In this work it is shown that by applying different spectroscopic methods such as fluorescence spectroscopy, fluorescence lifetime measurements or Raman spectroscopy on the same individual molecule a deeper insight in basic photophysical processes occurring within a molecule can be gained.

The measurements presented in this thesis have been accomplished using autofluorescent proteins as a fascinating family of target molecules.
Within the past 15 years the green fluorescent protein GFP, which had been fairly unnoticed up to this point, developed to be a prevalently used tool in the fields of molecular biology, medicine and cell biology. This is mainly due to the fact that by planting the GFP gene inside a cell the GFP is produced by the organism itself and the green fluorescent chromophore is generated by an autocatalytic reaction without any further cofactors. Thus autofluorescent proteins provide the potential to monitor biological processes inside a living cell without any external fluorescent labeling, which often turns out to be cell-toxic. A major drawback of the GFP is that the fluorescence emission of its chromophore showing a maximum at ~505 nm strongly overlaps with the autofluorescence of living cells, a fact which hampers its applicability for sensitive fluorescence imaging techniques. Due to this overlap, the image contrast is so strongly reduced that it often is not possible to distinguish between fluorescent marked areas in a cell and the autofluorescent background.
A milestone to overcome this severe drawback seemed to be found with the discovery of a red fluorescent protein from discosoma red reef coral, DsRed, in the late 1990s exhibiting a strong red fluorescence with an emission maximum at 580 nm which is fairly remote from the green cell autofluorescence.
However, for an accurate interpretation of the achieved data a deep understanding of the underlying photophysical processes is crucial (see for example). Whereas this requirement can be complied by classical methods in case of GFP, the situation is considerably more complex for the DsRed protein. This is mainly due to the fact that DsRed is known to form obligate tetramers even at nanomolar concentrations. These proteins are composed of monomeric subunits containing either a green or red emitting chromophore. It has been shown by different approaches that these chromophores do not act independently from each other but are energy coupled via a Förster energy transfer mechanism. Hence, upon excitation of the proteins in the blue spectral region the observed red emission is not due to a direct excitation of the red chromophore but the result of a transfer of the excitation energy from the green to the red emitting fluorophore.
For a deeper understanding of the underlying processes inside a DsRed tetramer it is necessary to investigate the isolated chromophores without impact of energy transfer processes.
However, it has been shown that it is not possible to separate the tetramers into functional monomers containing only one type of chromophore by chemical or physical means.

This thesis presents three potent approaches to overcome this restriction. First, the results of multiparameter spectroscopy on the single molecule level are presented, allowing for the first time to quantitatively derive a complete set of spectroscopic parameters to describe Förster energy transfer processes in the DsRed system without any further assumptions. Moreover, a novel technique is presented which allows for imaging and spectroscopy of both the fluorescence and Raman signal emitted from the same individual protein molecule. Assigning distinct Raman bands to fluorescent features gives insight into photophysical and chemical properties of the individual investigated proteins.

The second part of this thesis presents a novel technique which allows for the disentanglement of the coupled chromophoric units by changing the photonic mode density of the electromagnetic field using a sub-wavelength Fabry-Perot based microresonator. Moreover, it is shown that the presented metal-clad microresonator allows for the precise control of energy transfer processes of embedded molecules, making it a strong tool to enhance the efficiency of optical energy transport in a wide-spread field of applications.

A novel approach to directly determine fluorescence quantum yields for ultra-small amounts of dye-molecules in solution by time domain spectroscopy is introduced in the third section of this work. It is shown that this microresonator-based method can serve as a powerful tool to investigate influences of the local chemical environment on the photophysical properties of fluorescent dye molecules. The knowledge of these influences is of great interest in fields related to molecular biology, where distinct proteins are fluorescence labeled to be monitored inside a living cell. If the impact of the conjugated protein-structure on the fluorescence marker is unknown, the obtained results involve a high degree of uncertainties making a quantitative analysis impossible.


Outline of this thesis

All investigations presented in this thesis have been carried out using confocal optical microscopy and spectroscopy. The principles of this technique as well as the fundamental knowledge for the studies described in this work are provided in chapter 1. An introduction into the principles of fluorescence emission and Raman scattering is given. The experimental setup is described in detail, followed by a discussion on the optical properties of the microresonator system. The successive sections can be studied almost independently, based on the introductory chapter 1. As the design of the microresonators had to be individually adapted to the distinct experiments carried out and presented in this work, each chapter on microresonator-controlled emission measurements starts with a detailed description of the respective microcavity design utilized.
Each section starts with a brief introduction of the main objective followed by the discussion of the experimental realization. In the end of each chapter a discussion and interpretation of the measurements is provided.
The thesis is organized as follows:

In chapter the experimental confocal setup is described. At this juncture the techniques of spectral and time domain spectroscopy are shortly discussed. Additionally a short introduction of the fluorescence and Raman processes and Förster resonance energy transfer (FRET) is given.
Chapters 0 and 2.2.3 refer to the fabrication and the optical principles of the presented microresonator design. Herein the preparation of the used silver mirrors is presented and followed by a description of the experimental determination and characterization of the optical properties of the microresonator.
In Chapter 2.3 the preparation of the dye matrices for bulk and single-molecule measurements is presented.
Chapter 2.4 provides a brief introduction into autofluorescent proteins in general and the red fluorescent protein DsRed in particular.

In section 1 the investigation of isolated DsRed tetramers in free space by multiparameter single molecule spectroscopy is reported. In detail, it is shown that by means of combining spectrally and time resolved spectroscopy
it is possible for the first time to achieve a complete set of experimental spectroscopic data of the isolated chromophores within the DsRed tetramer. Thus one is able to describe and calculate the Förster energy transfer processes accurately, purely based on experimental data.

In section 4 a new approach for combined fluorescence and Raman spectroscopy on single protein-molecules is presented. This new technique gives a direct proof that Raman spectroscopy on single emitters is possible, a fact which has recently been subject to vivid discussions. The use of thin silver island-films to enhance the Raman signal allows for highly sensitive Raman imaging, thus opening this field to microscopic applications.

Section 1 refers to the microresonator controlled fluorescence emission of embedded bichromophoric energy transfer coupled dye-systems. For the FRET system DsRed proof is given that the microresonator allows for the disentanglement and the spectral and spatial isolation of the respective chromophores. Moreover the microresonator exceeds the energy transfer efficiency for randomly oriented dipole emitters by approximately one order of magnitude. The chapter concludes with a presentation of microresonator controlled spectroscopy of single DsRed energy transfer pairs. Here for the first time proof is given that it is possible to investigate the donor chromophore of a single Förster pair in presence of the acceptor by means of microresonator control.

In section 1 time-resolved spectroscopy of DsRed embedded in the microresonator is presented. Experimental results revealing that the microresonator allows for selective dequenching of the donor emission of rigidly coupled dye-pairs are presented. Moreover, it is shown that the magnitude of donor-dequenching can be precisely controlled and a theoretical description of the observed effects is given.

Section 1 refers to possible applications of the microresonator-system for the direct measurement of fluorescence quantum yields. Experimental data of various dye-systems embedded between the microresonator mirrors are shown, both in condensed and liquid phase matrices.
Additionally, precise ab-initio modeling is shown which allows for the direct determination of parameters like the fluorescence quantum yield and the fluorescence lifetime of the embedded molecules. The influence of rotational diffusion of embedded dye-molecules on the microresonator controlled fluorescence lifetime is presented and discussed.



In this thesis, novel approaches to investigate and control fluorescence resonant energy transfer (FRET) by optical field confinement are presented. As a model system, the red fluorescent protein DsRed was studied. DsRed is known to form rigid tetramers consisting of diverse fluorescent subunits in a random composition. This makes it an appropriate system for FRET-studies, since the rigidity of the structure circumvents chromophoric diffusion whereas the random composition of the distinct tetramers elucidates the potential of the presented techniques for studying complex energy transfer systems.
After an introduction into the topic and the experimental techniques is given in chapters 1 and 1, results of multiparameter spectroscopy of individual DsRed tetramers are presented in chapter 1. By combination of spectral and time resolved microscopy on the same single protein, for the first time it is possible to obtain a complete set of spectroscopic parameters to quantitatively describe the energy transfer process, purely based on experimental data. Here, the spectroscopic results reveal that the green emitting donor chromophore of DsRed exhibits an unexceptional long fluorescence lifetime when no acceptor chromophore is present. This knowledge provides new insights into the photophysics of DsRed since so far the green chromophore was believed to be a homologue to the chromophore of the green fluorescent protein GFP with a shorter fluorescence lifetime due to structural and chemical identity.
In chapter 4, fluorescence and Raman imaging as well as spectroscopy of the same individual protein-molecule and its photobleaching products is reported. Due to the combination of two spectroscopic modes it is proven that surface enhanced resonance Raman scattering (SERRS) is possible on single emitting species, a question which arose a vivid scientific discussion over the last decade. On basis of fluorescence spectroscopy prior to Raman measurements it was possible to assign distinct Raman bands to fluorescent features of the investigated individual proteins.
In this section, a novel approach for surface enhancement of the Raman signal is presented, using thin silver island-films as a SERS active system. Due to the homogeneous surface coating achieved by evaporating thin metal films on a surface, Raman-intensity profiles of the sample area can be recorded, opening the field of Raman imaging with high sensitivity and robustness to a broad variety of applications in life-sciences.
Moreover, it is shown that no chemical enhancement is required to achieve single molecule Raman sensitivity, which is in good agreement with theoretical treatments, yielding that the electromagnetic enhancement of the Raman process is several orders of magnitude higher than the relatively weak chemical enhancement effect.
Chapter 1 refers to the spectral control of Förster energy transfer of FRET-systems by varying the photonic mode of a sub-wavelength Fabry-Perot microresonator. As the non-radiative energy transfer is modified by the changed electromagnetic field, it is shown that the method enables the spectral isolation of chemically fixed FRET-pairs in the ensemble as well as on the single entity level. As the induced dipole moments of the embedded dyes are deformed by the introduced boundary conditions, the optical nearfield of the molecules is enhanced along the resonant longitudinal cavity mode, resulting in an increased critical Förster distance R0 for molecules embedded in the microresonator. It is shown that, even on the single entity level, the microresonator effectively controls the Förster energy transfer, thus allowing for the investigation of the unquenched donor emission in presence of an acceptor.
In chapter 1 it is shown that the microresonator geometry presented in chapter 1 allows for the disentanglement of a dipole-coupled dye-pair by modifying the absorption probability of the embedded dyes. The effect of donor-dequenching is shown as an increase of the donor-to-acceptor intensity ratio for both cw- and pulsed excitation.
Computational simulations are presented, supporting the experimental results. Moreover, the energy transfer efficiencies for various absorption probabilities are determined, revealing non-linear behavior for the Förster-process for externally confined electromagnetic fields. The photobleaching kinetics of the respective chromophores of the investigated DsRed FRET-system were studied, revealing dramatic changes for a variation of the mode density of the electromagnetic field.
Furthermore, fluorescence lifetime measurements are reported, revealing that donor-dequenching can be controlled by tuning the time delay of two succeeding light-pulses.
Chapter 1 refers to prospective applications using the presented microresonator design. A novel technique for direct determination of the fluorescence quantum yield in a native environment based on experimental investigations and computational modeling is presented. This new method is capable to outperform standard methods due to its independence of reference standards combined with a high cost-efficiency. Moreover, a modified microresonator geometry is introduced which allows for fluorescence studies in the liquid phase using exceptional small sample amounts. The impact of rotational diffusion on the microresonator controlled emission is discussed and shown by both experimental data and the results obtained from theoretical modeling. The ultra-low sample amounts required for microresonator studies combined with the ability to investigate liquid systems suggests the presented technique for applications as an analytical tool in integrated optical systems.


In dieser Arbeit werden neuartige Verfahren zur Untersuchung und zur Steuerung von Fluoresezenz Resonanz Energieüberträgen (FRET) mit Hilfe des optischen Feldes vorgestellt. Als Modellsystem wurde das rot fluoreszierende Protein DsRed untersucht. DsRed ist für die Ausbildung von stabilen Tetrameren bekannt, welche aus unterschiedlichen grün und rot fluorezierenden Untereinheiten in statistischer Zusammensetzung aufgebaut sind. Durch die Stabilität der Struktur ist zum einen eine feste räumliche Orientierung der chromophoren Einheiten zueinander sichergestellt, zum anderen stellt die statistische Verteilung von grün und rot fluoreszierenden Untereinheiten eine Herausforderung an die präsentierten Methoden dar.
Nach einer allgemeinen Einführung in das Themengebiet sowie der Vorstellung der verwendeten experimentellen Methoden in den Kapiteln 1 und 2, werden in Kapitel 3 Ergebnisse von multiparametrischer Spektroskopie an einzelnen DsRed-Tetrameren präsentiert. Durch die Kombination von spektral und zeitlich aufgelöster Mikroskopie gelingt zum ersten Mal die vollständige Zusammenstellung aller spektroskopischen Parameter zur quantitativen Beschreibung der Energietransferprozesse auf Basis rein experimenteller Daten. Die Befunde ergeben, dass der grün fluoreszierende Donor-Chromophor eine unerwartet lange Fluoreszenzlebensdauer aufweist, wenn kein Akzeptorchromophor als Fluoreszenzlöscher zugegen ist. Diese Erkenntnis gibt neuartige Einblicke in die Photophysik des DsRed, dessen grün fluoreszierender Chromophor bislang als Homologes des grün fluoresziereden Proteins GFP aufgrund der strukturellen und chemischen Gleichheit verstanden wurde.
In Kapitel 4 werden Ergebnisse von bildgebenden und spektral aufgelösten Fluoreszenz- und Ramanmessungen an demselben einzelnen fluoresziereden Proteinmolekül und seinen Photoprodukten vorgestellt. Durch die Kombination zweier spektroskopischer Methoden wird ein klarer Beweis geliefert, dass oberflächenverstärkte Resonanz-Ramanspektroskopie (SERRS) an einzelnen Emittern möglich ist, eine Frage, die während der letzten Dekade im Fokus lebhafter wissenschaftlicher Diskussionen stand. Durch die zuvor durchgeführten Fluoreszenzmessungen war es möglich, unterschiedlche Ramanbanden den Fluoreszenzcharakteristika der untersuchten einzelnen Proteine zuzuordnen.
Es konnte weiterhin gezeigt werden, dass keine chemische Verstärkung notwendig ist um Raman-Einzelmolekülsensitivität zu erhalten. Durch die Verwendung von Silberinselfilmen als Raman-aktives System steht ein Verfahren zur Verfügung, bei dem die Ramaninformation als hoch-sensitives und gleichzeitig robustes Signal für die bildgebende Mikroskopie verwendet werden kann.
Kapitel 5 beschäftigt sich mit der spektralen Kontrolle des Förster Energieübertrags in FRET-Systemen durch Variation der photonischen Mode in einem Lamda/2-Mikroresonator. Da der nicht-strahlende Energieübertrag durch das geänderte elektromagnetische Feld modifiziert wird, ist es möglich die einzelnen Chromophore in einem chemisch fixierten FRET-System sowohl im Ensemble als auch auf Einzelmolekülebene spektral zu isolieren. Da die induzierten Dipolmomente der eingebetteten Farbstoffmoleküle durch die durch den Mikroresonator eingeführten Grenzbedingungen deformiert werden, ist ihr optisches Nahfeld entlang der longitudinalen Resonatormode verstärkt. Hierdurch wird eine deutliche Zunahme des kritischen Förster-Abstandes R0 der eingebetten FRET-Systeme erreicht.
In Kapitel 6 wird gezeigt, dass die zuvor vorgestellte Mikroresonator-geometrie eine Entkopplung von Dipol-Dipol Wechselwirkungen in Farbstoffpaaren durch die Kontrolle der Absorptionswahrscheinlichkeit der eingebetteten Farbstoffe erlaubt. Die Unterbindung eines Energieübertrags von Donor- zu Akzeptorchromophor wird sowohl für kontinuierliche als auch für gepulste Anregung gezeigt. Darüber hinaus wurden die Energiebübertragseffizienzen für unterschiedliche Absorptions-wahrscheinlichkeiten bestimmt und ein nicht-lineares Verhalten des Förster-Pozesses in extern eingegrenzten optischen Feldern konnte sowohl experimentell als auch durch Computersimulationen gezeigt werden. Es wurde demonstriert, dass der Energieübertrag durch Einstellen einer definierten Verzögerungszeit zwischen zwei aufeinanderfolgenden Anregungspulszügen kontrolliert werden kann. Weiterhin konnte gezeigt werden, dass die Kinetik des Photobleichens der unterschiedlichen Chromophore eines FRET-Systems in einem Mikroresoantor deutlichen Änderungen für die jeweiligen Chromophore im Vergleich zum freien Raum mit elektromagnetischem Modenkontinuum unterliegen.
In Kapitel 7 werden mögliche Anwedungen des vorgestellten Mikroresonators diskutiert. Hierbei wird eine neuartige Methode zur direkten Bestimmung der Fluoreszenzquantenausbeute in nativer Umgebung vorgestellt. Das neue Verfahren ist konventionellen Techniken deutlich überlegen, da zum einen auf Referenzsubstanzen verzichtet werden kann und zum anderen eine hohe Kosteneffizienz durch die benötigten geringen Substanzmengen erreicht wird. Weiterhin wird in diesem Kapitel eine modifizierte Resonatorgeometerie vorgestellt, die Fluoreszenz-untersuchungen in flüssiger Phase zulässt. Der Einfluss von Rotationsdiffusion auf die Mikroresonator-kontrollierte Emission wird diskutiert und sowohl experimentell als auch durch Simulationsrechnungen gezeigt. Aufgrund der äußerst geringen erforderlichen Probenmengen bei gleichzeitig höchster Detektionssensitivität in Verbindung mit der Möglichkeit der Messung im Durchfluss wird das System als mögliches analytisches Detektionswerkzeug in integrierten optischen Systemen vorgeschlagen.


Table of Contents

    Table of Contents    VII
    List of Abbreviations    IX
1.    Introduction    1
1.1.    Scope of this thesis    1
1.2.    Outline of this thesis    4

2.    Theoretical background and instrumentation    7
2.1.    Principles of molecular emission    7
2.2.    Experimental methods and instrumentation    20
2.3.    Preparation of dye-matrices for bulk and single molecule studies    29
2.4.    Autofluorescent proteins    30

3.    Multiparameter spectroscopy on single DsRed proteins    37
3.1.    Introduction    38
3.2.    Experimental methods    39
3.3.    Spectral and time domain microscopy    40
3.4.    Calculations    46
3.5.    Conclusion    52

4.    Fluorescence spectroscopy and surface enhanced resonance Raman scattering (SERRS) on the same individual protein and its photoproducts    53
4.1.    Introduction    54
4.2.    Experimental    56
4.3.    Spectroscopic results    59
4.4.    Conclusion    68

5.    Spectral control of Förster resonant energy transfer (FRET) by the optical mode of a sub-wavelength microresonator    69
5.1.    Introduction    70
5.2.    Microresonator design    71
5.3.    Transmission properties of the microresonator    73
5.4.    Spatial resolution of chromophoric subpopulations within DsRed tetramers    74
5.5.    Microresonator controlled energy transfer of randomly dispersed donor-acceptor pairs    77
5.6.    Single molecule spectroscopy of DsRed in a microresonator    80
5.7.    Conclusion    83

6.    Controlled disentanglement of coupled dipole emitters by confinement of the electromagnetic field    85
6.1.    Introduction    86
6.2.    Microresonator design and characterisation    87
6.3.    Frequency-domain donor dequenching using continuous-wave irradiation    88
6.4.    Time-domain donor dequenching by pump-probe measurements    96
6.5.    Conclusion    102

7.    Determination of the fluorescence quantum efficiency by mode confinement of the electromagnetic field    103
7.1.    Introduction    104
7.2.    Experimental    105
7.3.    Modeling the fluorescence lifetime of molecules embedded in a microresonator        105
7.4.    Towards application: liquid-phase microresonators    107
7.5.    Conclusion    114

8.    References    115

9.    Appendix    127
9.1.    Acknowledgement    127
9.2.    Summary    130
9.3.    Zusammenfassung    132
9.4.    Curriculum Vitae    134
9.5.    Scientific Contributions    135
9.6.    Academic Teachers    138


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