Fluorescence Spectroscopy in Biology: Advanced Methods and their Applications to Membranes, Proteins, DNA, and Cells

314 Pages·2005·3.563 MB·English

by  M. Hof, V. Fidler, R. Hutterer (auth.), Professor M. Hof, Professor R. Hutterer, Professor V. Fidler (eds.)

3
Springer Series on Fluorescence
Methods and Applications
Series Editor:
O.S.Wolfbeis
Fluorescence Spectroscopy
in Biology
Advanced Methods and their Applications
to Membranes,Proteins,DNA,and Cells
Volume Editors: M.Hof · R.Hutterer · V.Fidler
3
About this series:
Fluorescence spectroscopy,fluorescence imaging and fluorescent probes are
indispensible tools in numerous fields of modern medicine and science,
including molecular biology,biophysics,biochemistry,clinical diagnosis and
analytical and environmental chemistry.Applications stretch from spectro-
scopy and sensor technology to microscopy and imaging,to single molecule
detection,to the development of novel fluorescent probes,and to proteomics
and genomics.The Springer Series on Fluorescence aims at publishing state-
of-the-art articles that can serve as invaluable tools for both practitioners
and researchers being active in this highly interdisciplinary field.The carefully
edited collection of papers in each volume will give continuous inspiration
for new research and will point to exciting new trends.
Library of Congress Control Number:2004114543
ISSN 1617-1306
ISBN3-540-22338-XSpringer Berlin Heidelberg New York
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Series Editor
Professor Dr.Otto S.Wolfbeis
University ofRegensburg
Institute ofAnalytical Chemistry,Chemo- and Biosensors
Universitätsstraße 31
93040 Regensburg
Germany
e-mail:[email protected]
Volume Editors
Professor M.Hof
J.HeyrovskýInstitute ofPhysical Chemistry
Academy ofSciences ofthe Czech Republic
and Centre for Complex Molecular Systems and Biomolecules
Dolejškova 3
18223 Prague 8
Czech Republic
e-mail:[email protected]
Professor R.Hutterer
Institute ofAnalytical Chemistry,Chemo- and Biosensors
Universitätsstraße 31
93040 Regensburg
Germany
e-mail:[email protected]
Professor V.Fidler
Faculty ofNuclear Sciences and Physical Engineering
Czech Technical University in Prague
Břehová 7
11519 Praha 1
Czech Republic
e-mail:[email protected]
Preface
Take any combination ofthe following features:supramolecular structures with
a specific fluorescent probe localized as you would like;nanoscale spatial resolu-
tion;tailor-made molecular and/or solid-state fluorescing nanostructures;user-
friendly and/or high- throughput fluorescence techniques;the ability to do what-
ever you wish with just one single (supra)molecule;utilization ofnon-linear optical
processes;and,last but not least,physical understanding ofthe processes result-
ing in a (biological) functionality at the single molecule level.What you will then
have is some recent progress in physics,chemistry,and the life sciences leading
to the development of a new tool for research and application.This was amply
demonstrated at the 8th Conference on Methods and Applications ofFluorescence:
Probes,Imaging,and Spectroscopy held in Prague,the Czech Republic on August
24th–28th, 2003. This formed a crossroad of ideas from a variety of natural
science and technical research fields and biomedical applications in particular.
This volume – the third book in the Springer-Verlag Series on Fluorescence –
reviews some of the most characteristic topics of the multidisciplinary area of
fluorescence applications in life sciences either presendted directly at th 8th MAF
Conference or considered to be a cruical development in the field.
In the initial contribution in Part 1 – Basics and Advanced Approaches,the ed-
itors explain the basics of fluorescence and illustrate the relationship between
some modern fluorescence techniques and classical approaches. The second
contrigution by B.Valeur,with his many years ofpersonal experience,helps the
fluorescence spectroscopist to answer teh perennial question ofwhether to use
pulse or phase modulation fluorescence detection.A technically demanding but
promising new approach for extracting distance information from fluorescence
kinetics data is presented by ist innovator L.Johansson in the third contribution.
The three subsequent contributions also have the pioneers ofeach new approach
among their authors:D.Birch – nanotomography,M.Hof – solvent relaxation
used micro-polarity and fluidity probing, and N. Thompson – total internal
reflection fluorescence microscopy.The last contribution in Part 1,written by
J. Enderlein, is devoted to single molecule spectroscopy using a quantitative
approach to data analysis in this important new experimental field.Part 2 – Fluo-
rescence in Biological Membranes – addresses a hot topic in membrane research,
i.e.,the formation ofmicrodomains.G.Duportail summarizes the recent results
in the study oflipid rafts using fluorescence quenching and L.Bagatolli demon-
strates the use offluorescence microscopy in the charcterization ofdomain for-
mation.
VIII Preface
Part 3 consisting ofcontributions ten and eleven deals with advanced fluores-
cene kinetics analysis in protein sciences.G.Krishnamoorthy’s chapter shows
what we can learn with time-resolved fluorescence about protein dynamics and
folding.Y.Mély combines time-resolved fluorescence with FCS to elucidate the
mechnaism ofinteraction ofthe HIV-1 nucleocapsid protein with hairpin loop
oligonucleotides.
The development ofefficient non-viral dug carriers is one ofthe most urgently
needed requirements in the biological sciences. It has become obvious that
modern fluorescence is capable ofhelping in the development ofsuch supramol-
ecular assemblies.Thus the two contributions (I.Blagbrough and M.Langner) in
Part 4 are devoted to this field.
The final part ofthis volume focuses on two new approaches in cell fluores-
cence microscopy. R. Brock shows how to characterize diffusion in cells by
fluorescence correlation spectroscopy.The last two contributions by S.Rosenthal
and O.Minet are devoted to photophysics and the use of quantum dots in cell
imaging.
Prague,October 2004 Martin Hof,Rudi Hutterer,and Vlastimil Fidler
Contents
Part 1
Fluorescence Spectroscopy:Basics and Advanced Approaches . . . 1
1 Basics ofFluorescence Spectroscopy in Biosciences . . . . . . . . 3
M.Hof,V.Fidler andR.Hutterer
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Fluorescence and its Measurement . . . . . . . . . . . . . . . . . 4
1.2.1 Molecular Electronic Relaxation . . . . . . . . . . . . . . . . . . 4
1.2.2 Detecting Fluorescence . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.3 Data Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3 Polarized Fluorescence . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1 Definition ofPolarization and Anisotropy . . . . . . . . . . . . . 7
1.3.2 Steady-State Fluorescence Anisotropy . . . . . . . . . . . . . . . 8
1.3.3 Time-Resolved Fluorescence Polarization . . . . . . . . . . . . . 9
1.3.3.1 Non-Spherical Particles in Homogenous Isotropic Medium . . . . 9
1.3.3.2 Segmental Mobility ofthe Chromophore . . . . . . . . . . . . . . 10
1.3.3.3 Hindered Rotors:Fluorescent Dyes in Biological Membranes . . . 10
1.4 Influence ofFluorescence Quenching . . . . . . . . . . . . . . . . 11
1.4.1 Fluorescence Quantum Yield and Lifetime . . . . . . . . . . . . . 11
1.4.2 Fluorescence Quenchers . . . . . . . . . . . . . . . . . . . . . . . 11
1.4.2.1 Solute Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.4.2.2 Solute Quenching in Protein Studies:an Application Example . . 13
1.4.2.3 Solvent Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.4.2.4 Self-Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.4.2.5 Trivial Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.5 Influence ofSolvent Relaxation on Solute Fluorescence . . . . . . 17
1.5.1 Basics ofSolvent Relaxation . . . . . . . . . . . . . . . . . . . . . 17
1.5.2 Influence ofSolvent Relaxation on Steady-State Spectra . . . . . . 18
1.5.2.1 Non-Viscous Solvents . . . . . . . . . . . . . . . . . . . . . . . . 18
1.5.2.2 Viscous and Vitrified Solutions . . . . . . . . . . . . . . . . . . . 18
1.5.3 Quantitative Characterization ofSolvent Relaxation
by Time-Resolved Spectroscopy . . . . . . . . . . . . . . . . . . . 19
1.6 Fluorescence Resonance Energy Transfer
as a Spectroscopic Ruler . . . . . . . . . . . . . . . . . . . . . . 20
1.6.1 Donor-Acceptor Pairs at Fixed Distances . . . . . . . . . . . . . 20
1.6.2 Donor-Acceptor Pairs at Variable Distances . . . . . . . . . . . 21
1.6.3 Some Applications ofFluorescence Resonance Energy Transfer 21
1.7 Irreversible Photobleaching . . . . . . . . . . . . . . . . . . . . 22
X Contents
1.8 Single Molecule Fluorescence . . . . . . . . . . . . . . . . . . . 23
1.9 Optical Sensors Based on Fluorescence . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2 Pulse and Phase Fluorometries:An Objective Comparison . . . . 30
B.Valeur
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2 General Principles ofTime-Resolved Fluorometry . . . . . . . . . 31
2.2.1 Pulse Fluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.2.2 Phase-Modulation Fluorometry . . . . . . . . . . . . . . . . . . . 32
2.2.3 Relation Between Harmonic Response andd-Pulse Response . . . 33
2.2.4 General Relations for Single Exponential
and Multiexponential Decays . . . . . . . . . . . . . . . . . . . . 34
2.3 Pulse Fluorometers . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4 Phase-Modulation Fluorometers . . . . . . . . . . . . . . . . . . 37
2.4.1 Phase Fluorometers Using a Continuous Light Source
and an Optical Modulator . . . . . . . . . . . . . . . . . . . . . . 38
2.4.2 Phase Fluorometers Using the Harmonic Content
ofa Pulsed Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.5 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.6 Specific Applications . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.6.1 Time-Resolved Spectra . . . . . . . . . . . . . . . . . . . . . . . . 42
2.6.2 Time-Resolved Emission Anisotropy . . . . . . . . . . . . . . . . 44
2.6.3 Lifetime-Based Decomposition ofSpectra . . . . . . . . . . . . . 45
2.6.4 Fluorescence Lifetime Imaging Microscopy (FLIM) . . . . . . . . 45
2.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . 47
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3 Non-Exponential Fluorescence ofElectronically Coupled
Donors Contains Distance Information . . . . . . . . . . . . . . 49
S.Kalinin,M.Isaksson andL.B.-Å.Johansson
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.4 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . 51
3.4.1 Synthetic Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.4.2 Experimental Data . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Contents XI
4 Fluorescence Nanotomography:Recent Progress,Constraints
and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . 56
O.J.Rolinski andD.J.S.Birch
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2 Fluorescence Resonance Energy Transfer . . . . . . . . . . . . . . 57
4.3 FRET Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.4 Fluorescence Nanotomography Theory . . . . . . . . . . . . . . . 60
4.4.1 An Inverse Problem . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.4.2 Separation ofVariables Approach . . . . . . . . . . . . . . . . . . 62
4.4.3 Numerical Simulations . . . . . . . . . . . . . . . . . . . . . . . 64
4.5 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.5.1 Bulk Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.5.2 Porous Polymer Nafion117 . . . . . . . . . . . . . . . . . . . . . 66
4.5.3 Phospholipid Bilayers . . . . . . . . . . . . . . . . . . . . . . . . 68
4.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5 Solvent Relaxation as a Tool for Probing Micro-Polarity
and -Fluidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
J.Sy´kora,R.Hutterer andM.Hof
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5.2 Basic Principles ofthe SR Method . . . . . . . . . . . . . . . . . 71
5.3 Applications ofthe SR Technique by Using Time-Correlated
Single Photon Counting . . . . . . . . . . . . . . . . . . . . . . . 73
5.3.1 SR in Phospholipid Bilayers . . . . . . . . . . . . . . . . . . . . . 73
5.3.2 SR in Reverse Micelles . . . . . . . . . . . . . . . . . . . . . . . . 75
5.3.3 SR in Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5.3.4 SR in Ionic Liquid . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5.3.5 SR in DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5.3.6 SR in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
6 Total Internal Reflection Fluorescence Microscopy:
Applications in Biophysics . . . . . . . . . . . . . . . . . . . . . 79
N.L.Thompson andJ.K.Pero
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.1.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.1.2 Optical Principles . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.1.3 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
6.1.4 Sample Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
6.2 Combination ofTIRFM with Other Methods . . . . . . . . . . . . 83
6.2.1 Fluorescence Recovery after Photobleaching . . . . . . . . . . . . 83
XII Contents
6.2.2 Evanescent Interference Patterns . . . . . . . . . . . . . . . . . . 85
6.2.3 Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . . 86
6.2.4 Fluorescence Resonance Energy Transfer . . . . . . . . . . . . . . 88
6.2.5 Variable Incidence Angles . . . . . . . . . . . . . . . . . . . . . . 89
6.2.6 Inverse Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
6.3 Advanced Topics . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.3.1 High Refractive Index Substrates . . . . . . . . . . . . . . . . . . 91
6.3.2 Thin Metal Films and Metallic Nanostructures . . . . . . . . . . . 92
6.3.3 Fluorescence Emission Near Planar Dielectric Interfaces . . . . . 92
6.3.4 Fluorescence Polarization . . . . . . . . . . . . . . . . . . . . . . 93
6.3.5 Fluorescence Lifetimes and Time-Resolved Anisotropies . . . . . 94
6.3.6 Two-Photon Excitation . . . . . . . . . . . . . . . . . . . . . . . 94
6.4 Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 95
6.4.1 Single Molecule Imaging . . . . . . . . . . . . . . . . . . . . . . 95
6.4.2 Imaging Cell–Substrate Contact Regions . . . . . . . . . . . . . . 96
6.4.3 Exocytosis and Secretion Vesicle Dynamics . . . . . . . . . . . . 97
6.4.4 Emerging Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
7 Single Molecule Spectroscopy:Basics and Applications . . . . . . 104
J.Enderlein
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
7.2 Photophysics,Probes and Markers . . . . . . . . . . . . . . . . . 105
7.3 Physical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . 109
7.3.1 Modified Flow Cytometry,Microchannels and Microdroplets . . . 109
7.3.2 Confocal Detection . . . . . . . . . . . . . . . . . . . . . . . . . 111
7.3.3 Wide-Field Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 113
7.4 Data Acquisition and Evaluation . . . . . . . . . . . . . . . . . . 116
7.4.1 Time-Tagged and Time-Correlated Photon Counting . . . . . . . 116
7.4.2 Fluorescence Correlation Spectroscop . . . . . . . . . . . . . . . 117
7.4.3 Fluorescence Intensity Distribution Analysis
and Related Techniques . . . . . . . . . . . . . . . . . . . . . . . 120
7.4.4 Molecule-by-Molecule Analysis . . . . . . . . . . . . . . . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Part 2
Application of Fluorescence Spectroscopy to Biological Membranes 131
8 Raft Microdomains in Model Membranes as Revealed
by Fluorescence Quenching . . . . . . . . . . . . . . . . . . . . . 133
G.Duportail
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8.2 Identification ofLipid Compositions Forming Rafts . . . . . . . . 134