Springer Series in chemical physics 96 Springer Series in chemical physics Series Editors: A W Castleman, Jr J P Toennies K Yamanouchi W Zinth The purpose of this series is to provide comprehensive up-to-date monographs in both well established disciplines and emerging research areas within the broad f ields of chemical physics and physical chemistry The books deal with both fundamental science and applications, and may have either a theoretical or an experimental emphasis They are aimed primarily at researchers and graduate students in chemical physics and related f ields Please view available titles in Springer Series in Chemical Physics on series homepage http://www.springer.com/series/676 Astrid Grăaslund Rudolf Rigler Jerker Widengren Editors Single Molecule Spectroscopy in Chemistry, Physics and Biology Nobel Symposium With 223 Figures 123 Editors Professor Astrid Grăaslund Professor Jerker Widengren Stockholm University Department of Biophysics 10691 Stockholm, Sweden E-Mail: astrid@dbb-su.se Royal Institute or Technology (KTH) Department of Biomolecular Physics 10691 Stockholm, Sweden E-Mail: jerker@biomolphysics.kth.se Professor Rudolf Rigler Swiss Federal Institute of Technology Lausanne (EPFL) 1015 Lausanne, Switzerland E-Mail: rudolf.rigler@epfl.ch Series Editors: Professor A.W Castleman, Jr Department of Chemistry, The Pennsylvania State University 152 Davey Laboratory, University Park, PA 16802, USA Professor J.P Toennies Max-Planck-Institut făur Străomungsforschung Bunsenstrasse 10, 37073 Găottingen, Germany Professor K Yamanouchi University of Tokyo, Department of Chemistry Hongo 7-3-1, 113-0033 Tokyo, Japan Professor W Zinth Universităat Măunchen, Institut făur Medizinische Optik ă Ottingerstr 67, 80538 Măunchen, Germany Springer Series in Chemical Physics ISSN 0172-6218 ISBN 978-3-642-02596-9 e-ISBN 978-3-642-02597-6 DOI 10.1007/978-3-642-02597-6 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009934497 © 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Media (www.springer.com) Nobel Symposium, June 2008, at the S anga Să aby Conference Foreword By selecting the first week of June 2008 for the Nobel Symposium “Single Molecular Spectroscopy in Chemistry, Physics and Biology”, Rudolf Rigler, Jerker Widengren and Astrid Gră aslund have once again won the top prize for Meeting Organizers, providing us with a Mediterranean climate on top of the warm hospitality that is unique to Sweden The S anga Să aby Conference Center was an ideal place to spend this wonderful week, and the comfort of this beautiful place blended perfectly with the high calibre of the scientific programme It was a special privilege for me to be able to actively participate in this meeting on a field that is in many important ways complementary to my own research I was impressed by the interdisciplinary ways in which single molecule spectroscopy has evolved and is currently pursued, with ingredients originating from physics, all branches of chemistry and a wide range of biological and biomedical research A beautiful concert by Semmy Stahlhammer and Johan Ull´en further extended the interdisciplinary character of the symposium I would like to combine thanks to Rudolf, Jerker and Astrid with a glance into a future of other opportunities to enjoy top-level science combined with warm hospitality in the Swedish tradition Ză urich, April 2009 Kurt Wă uthrich Participants of the Nobel-Symposium 138: First row: Sarah Unterkofler, Anders Liljas, Xiao-Dong Su, Birgitta Rigler, Carlos Bustamante, Toshio Yanagida, Steven Block, Xiaowei Zhuang, Sunney Xie Second row: Ivan Scheblykin, Lars Thelander, Petra Schwille, Watt W Webb, Rudolf Rigler, Jerker Widengren, Peter Lu, Shimon Weiss, William E Moerner, David Bensimon Third row: Anders Ehrenberg, Yu Ming, Fredrik Elinder, Kazuhiko Kinosita, Vladana Vukojevic, Masataka Kinjo, May D Wang, Yu Ohsugi, Shuming Nie, Andreas Engel, Peter G Wolynes, Michel Orrit, Hans Blom, Johan Hofkens Fourth row: Claus Seidel, Heike Hevekerl, Taekjip Ha, Evangelos Sisamakis, Per Ahlberg, Joseph Nordgren, Kurt Wthrich, Sune Svanberg, Bengt Nordn, Paul Alivisatos, Per Thyberg, Richard Keller, Andriy Chmyrov, Johan Elf, Per Rigler, Kai Hassler, Gustav Persson, Jă urgen Kă ohler, Eric Betzig, Thomas Schmidt, Christoph Bră auchle, Elliot Elson, Mans Ehrenberg, Dimitrios K Papadopoulos, Ingemar Lundstră om, Horst Vogel, Stefan Wennmalm, Hermann Gaub, H akan Wennerstră om, Yosif Klafter, Julio Fernandez Preface The development of Single Molecule Detection and Spectroscopy started in the late eighties The developments came from several areas Fluorescence-based single molecule spectroscopy developed in particular from (i) holeburning and zero phonon spectroscopy of organic molecules at cryo temperatures and (ii) confocal fluctuation spectroscopy of emitting molecules at elevated temperatures Of crucial importance for these approaches was the ability to suppress background radiation to the point where signals of single molecules could be detected Today, confocal single molecule analysis is the dominating approach, particularly in chemistry and in biosciences, but attempts to combine analysis at low and high temperatures are being pursued In parallel with this development, significant progress has been made in the field of single molecule force spectroscopy Approaches based on atomic force microscopy, optical trapping, microneedles or magnetic beads have made it possible to investigate mechanical properties, and not least, the interplay between mechanics and chemistry on a single molecule level In June 1999 the first Nobel Conference on Single Molecule Spectroscopy was organized in Să odergarn Mansion, Lidingă o (Sweden) and a comprehensive presentation of the results obtained in the first decade of single molecule analysis was given (Orrit, Rigler, Basche (eds.) 1999) Now after almost another decade, it was of interest to find out whether the developments and promises presented at the Săodergarn Conference were still valid or had even exceeded our expectations The contributions to this volume come from the pioneers of the early period of single molecule spectroscopy as well as from other laboratories which have made important contributions to demonstrate the importance of SM analysis in various applications in Chemistry, Physics and BioSciences The Nobel Symposium No 138 dedicated to Single Molecule Spectroscopy in Chemistry, Physics and Biosciences was held at the Mansion S anga-Să aby situated at the island of Ekeră o in Lake Măalaren outside Stockholm, from June 1–6, 2008 The Conference was blessed with pleasant weather and sunshine all the days Together with the wonderful surroundings this contributed to many X Preface stimulating opportunities for individual discussions, in parallel with outdoor excursions including swimming in the lake, jogging tours, walks in the forests and sauna The Symposium started with an evening session on molecules and dynamic processes by Kurt Wă uthrich and Martin Karplus The program of the next days included the presentation of the fields which initiated single molecule analysis in cryo temperatures (Moerner,Orrit) followed by confocal analysis of molecular fluctuations at room temperature (Keller, Rigler, Elson, Webb, Widengren, Schwille) Major topics in the following sessions included quantum dots (Alivasatos, Nie), the analysis of conformational dynamics (Weiss, Ha, Seidel), the motion of molecular motors (Yanagida, Kinosita) and replicating assemblies (Bustamante, Block) A special session was devoted to the analysis of forces operating on single molecules (Gaub, Fernandez) as well as to high resolution imaging of single molecules (Hell, Betzig, Zhuang, Engel) Stochastic single molecule events at the cellular level were another important topic (Xie, Schmidt, Vogel, Wolynes) as well as single molecule enzymology (Lu, Xie, Rigler, Hofkens, Klafter, Kă ohler), which together with atomic force microscopy formed the basis for intense discussions Several presentations brought the single molecule methodologies and perspectives to a sub-cellular and cellular context (Rigler, Schwille, Weiss, Bensimon, Axner, Hell, Betzig, Zhuang, Schmidt, Xie, Orwar, Bră auchle), which seems to form one of several exciting future directions of this field A special event was the evening concert with Semmy Stalhammer on the violin and Johan Ull´en on the piano The violin sonata of Cesar Franck and its masterly performance matched perfectly the level and tension of the scientific sessions As organizers we would like to thank all the invited speakers for their excellent contributions to this symposium, as well as all those who contributed with a chapter to this book We would also like to thank Margareta Klingberg and colleagues at the conference site of S anga-Să aby for the prerequisites and support of an excellent venue, and not the least the Nobel Foundation for supporting this Symposium Stockholm, July 2009 Astrid Gră aslund Rudolf Rigler Jerker Widengren Contents Part I Introductory Lecture: Molecular Dynamics of Single Molecules How Biomolecular Motors Work: Synergy Between Single Molecule Experiments and Single Molecule Simulations Martin Karplus and Jingzhi Pu Part II Detection of Single Molecules and Single Molecule Processes Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances William E Moerner 25 Single Molecules as Optical Probes for Structure and Dynamics Michel Orrit 61 FCS and Single Molecule Spectroscopy Rudolf Rigler 77 Part III Fluorescence-Correlation Spectroscopy Single-Molecule Spectroscopy Illuminating the Molecular Dynamics of Life Watt W Webb 107 Chemical Fluxes in Cellular Steady States Measured by Fluorescence-Correlation Spectroscopy Hong Qian and Elliot L Elson 119 558 P.G Wolynes Fig 28.4 The apparent “renormalized” law of mass action for a gene switch is shown in the outer panel The probability of the gene being off versus average population of transcription factor is shown The “Shea-Ackers” curve is the conventional macroscopic law of mass action Single-molecule physical chemistry reigns! Fig 28.5 The effective potential surfaces for a simple gene switch are shown – h gives the binding rate, f the unbinding rate, and g↑ and g↓ are the synthesis rates when the gene is on or off, respectively The different diagrams correspond with different sequences of binding/synthesis/unbinding events The upper plot shows the typical trajectory at high non-adiabaticity The lowest plot shows the adiabatic case A churning process gives an enhanced rate of protein number fluctuations in the intermediate “weakly non-adiabatic” case B simple gene switch can change its state Simple formulae have been found for the escape rates in the various regimes by Walczak, Onuchic, and Wolynes [18] In Fig 28.6, I show the typical escape rates from either attractor as a function of the ratio of the rate of single molecule binding to the growth/death rate parameters It is interesting that there is a “stochastic resonance” when these two input time scales are close to each other In other words, switches are most agile in the weakly non-adiabatic regime, corresponding to the case B, binding/growth/unbinding scenario It is 28 Single-Molecule Chemical Physics in a Natural Context 559 Fig 28.6 Comparison of the rate of spontaneous epigenetic switching for different values of the non-adiabaticity K The exact numerical results are shown as a solid curve Various approximations reflecting the mechanisms described in Fig 28.5 are also plotted A stochastic resonance occurs near the weakly non-adiabatic regime interesting that protein-DNA binding is indeed a very rapid process – one may argue unnaturally so Eigen [19] and Berg and von Hippel [20] have drawn attention to this fact very early and it is now clear that a variety of factors contribute to this rapidity – one-dimensional search via sliding, transfer between DNA chains, and flycasting dynamics of repressors assisted by electrostatics [21] An interesting question must, therefore, be – is this speed an adaptation to achieve the stochastic resonance? As the experimental study of gene regulation at the single-molecule level blossoms, I am sure we will soon have an answer to this question Acknowledgments This work was supported by the NSF grant to the Center for Theoretical Biological Physics The wonderful collaborations with Masaki Sasai, Aleksandra Walczak, Jos´e Onuchic, Daniel Schultz, and J.E Hornos are much appreciated References H Frauenfelder, S Sligar, P.G Wolynes, Science 254, 1598–1603 (1991) E.V Russel, N.E Israeloff, Nature 408, 695–698 (2000) R Zondervan et al., Proc Nat Acad Sci USA 104, 12628–12633 (2007) A.M Boiron, P Tamarat, B Loomis, R Brown, M Orrit, Chem Phys 247, 119–132 (1999) J Wang, P.G Wolynes, Phys Rev Lett 74, 4317–4320 (1995) H.P Lu, L.Y Xun, X.S Xie, Science 282, 1877–1882 (1998) 560 P.G Wolynes L Edman, R Rigler, Proc Natl Acad Sci USA 97, 8266–8271 (2000) B Schuler, W Eaton, Curr Opin Struct Biol 18, 16–26 (2008) W.J Greenleaf, M.T Woodside, S.M Block, Annu Rev Biophys Biomolec Struct 36, 171–190 (2007) 10 F Jacob, Mol Biol 3, 318 (1961) 11 M.L Delbră uck, J Chem Phys 8, 120 (1940) 12 M Sasai, P.G Wolynes, Proc Natl Acad Sci USA 100, 2374–2379 (2003) 13 D Chandler, P.G Wolynes, J Chem Phys 74, 4078–4095 (1981) 14 R.A Marcus, Angew Chem 32, 1111–1121 (1993) 15 N.E Buchler, U Gerland, T Hwa, Proc Natl Acad Sci USA 100, 5136– 5141 (2003) 16 J.E.M Hornos, D Schultz, G.C.P Innocentini, A.M Walczak, J Wang, J.N Onuchic, P.G Wolynes, Phys Rev E 72, 051907/1–5 (2005) 17 P.J Choi, L Cai, K Frieda, S Xie, Science 232, 442–446 (2008) 18 A.M Walczak, J.N Onuchic, P.G Wolynes, Proc Natl Acad Sci USA 102, 18926–18931 (2005) 19 P.H Richter, M Eigen, Biophys Chem 2, 255–263 (1974) 20 O.G Berg, R.G Winter, P.H von Hippel, Biochemistry 20, 6929–6948 (1981) 21 Y Levy, J.N Onuchic P.G Wolynes, J Am Chem Soc 3, 738–739 (2007) Index ϕ29, 239 ATP-γ-S, 275 a bottleneck state, 263 a neck linker, 225 a scanning probe, 221 a signal-to-background ratio, 80 a stretched exponential decay, ABEL Trap, 51 absorption, 62 accessibility of the channels, 540 acoustic resonances, 70 actin, 221 actin filaments, 226, 272 activation free energy, 125 activator, 402 activator-reporter pair, 402 active electric field trap, 96 active-site conformational motions, 481 actomyosin cortex, 143 adhesin, 338–340, 343, 350, 356, 357, 359 bond, 350, 351, 357 bond length, 350, 357 bond opening rate, 350 FimH, 338 PapG, 338, 357 adhesion, 337, 338, 356, 359 lifetime, 350, 357 mechanism, 338 organelle, 338, 339 properties, 343, 356, 359 system, 338, 339 ADP release, 274, 276, 277 adsorption time, 546 affinity, 278, 279 AFM, 208, 209, 291, 422–425, 428 Ago2, 148 alkaline phosphatase, 455 amphiphilic, 190 amphiphilic polymer, 451 amyloid aggregate formation, 111 Angstră om, 382 anomalous diffusion, 5, 146 anomalous sub-diffusion, 109 antenna complexes, 513 anthracene, 69 Anti-Brownian ELectrokinetic trap (ABEL trap), 52 antibodies, 405 antibunching, 32, 38, 79 anticorrelated fluctuation, 475, 476 APD imaging, 98 arginine finger, 275 argonaute proteins, 147 argonaute2, 148 Arrhenius, 320 artificial atom, 175, 179 artificial molecule, 185 ASCE, 238 ASCE family, 265 ASCE superfamily, 239 assembly, 296 astigmatism, 408 asymmetric hand-over-hand manner, 10 asymmetric incorporation, 150 562 Index atomic force microscope (AFM), 208, 418, 421 atomic Force Microscopy, 317 ATP, 292 ATP barrier, 294 ATP hydrolysis, 272, 274, 276 ATP synthase, 11, 271 ATP synthesis, 274, 275, 278, 280, 282 ATP-waiting dwell, 274, 275 attachment organelle, 338 attempt rate, 343, 344, 355, 356 attractor, 557 autocorrelation, 38 autocorrelation function (ACF), 156 autofluorescence, 489 autoinhibited, 290 avalanche photodiodes, 81, 141 axle, 281, 282 azide, 281 background-limited, 31 bacteria, 47 bacterial cell wall, 487 bacterial cells, 44 bacteriophages, 238, 239 bacteriorhodopsin, 422 barrier position histograms, 294 bead, 272 Bell’s equations, 346 bi-site, 276, 277 biased Brownian movement, 221 binding and unbinding, 474 binding cooperativity, 259–261 binding pocket, 292 bioimaging, 192 biological detection, 178 biological imaging, 175 biomolecular interactions, 492 bioreactor, 449 biotin, 300 biotinyated horseradish peroxidase, 89 blinking, 36, 43, 66, 178 bond adhesin, 350 head-to-tail, 342, 343, 349, 358 layer-to-layer, 342, 346, 350, 353, 355–358 length, 344–346, 353, 355, 357 opening length, 346, 349 opening rate, 343, 346, 347, 349–351, 353, 355 Boolean network, 121, 124 Brownian fluctuations, 247, 249 Brownian motion, 7, 51 buffer, 160 burst phase, 258 busy and lazy periods of horse radish peroxidase, 93 Cy3-ATP, 275, 276 caenorhabditis elegans embryos, 139, 143 caged ATP, 308 caged Ca2+ , 308 caged cAMP, 308 caged molecules, 306, 307, 309 caged mRNA, 311 calcium sensitive fluorophore, 161 cameleon, 44 cancer cells, 549 cancer chemotherapy, 547 Capturing Chromosome Conformation (3C), 205 carboxylic acids, 195 Carl Zeiss, 82 cascade reaction, 499 catalytic “groundstate”, 95 Caulobacter crescentus, 47 2π cavity, 79 CdTe, 188 cell divisions, 143 cell wall, 478 10G cells, 148 centroid, 386 Champagne, 242 channelrhodopsin 2, 309 chaperonin GroEL, 53 chemical network, chemical reaction, 328 2D chemical reactor, 463 chemical relaxation, 88, 159 chemotaxis, 230 Chinese hamster ovary (CHO) cell, 46 ChIP-chip, 204 ChIP-loop, 205 ChIPseq, 204 cholesterol, 46 cholesterol oxidase, 88 Index chromatin immunoprecipitation (ChIP), 204 chromophore FAD, chymotrypsin, 499 cis-trans, 385 clathrin-coated pit, 407 ClpX, 246 CMOS APD, 100 coil state, collapse, 331 colloidal stability, 192 color centers, 380 compact globule, competitive inhibitor, 273 complex network, 455 confined movement, 549 confocal, 369 confocal illumination, 80 confocal microscope, 496 confocal volume, 80 confocor 1, 2, 3, 82, 97 conformation, 289 conformation selection, 492 conformational fluctuation, 5, 438 conformational fluctuation of single DNA molecules, 84 conformational landscapes, 92 conformational memory effect, 472 conformational selection, 279 conformations of the transition state, 95 conjugated DNA, 463 connectivity of the domains, 540 constant force, 321 contour length, 294 contrast ratio, 412 controlled chemistry, 451 controlled release, 546 copolymeric nanocontainers, 97 core-shell, 196 correlation functions, 475 cortex, 146 cover neck bundle (CNB), 10 cover strand (CS), 10 cre-recombinase, 314 cross correlation, 135, 157 cross-correlation function, 132, 133, 475 cross-section, 62 cryogenic, 69 crystal structures, 281 563 CTAB (Cetyltrietylammoniumbromide), 542 cumulative distribution function, 506 Cy3–Cy5 dimer, 50 cyanine dye, 402 cysteine residues, 324 cystitis, 338 cytoplasm, 145 cytoplasmic reticulum, 99 cytostatica, 547 damping, 69 delay, 95 dephasing, 32 depot-effect, 546 detailed balance, 120 deterministic systems, 127 diagonal disorder, 516 dibenzoterrylene, 70 dichroic mirror, 86 γ-dictator mechanism, 274 dicyanomethylenedihydrofuran (DCDHF), 50 difference distribution function, 95 differential detection, 249 diffraction, 366, 373 diffraction limit, 400 diffusion, 454 diffusion and catch, 279 diffusion coefficient, 145, 150, 156, 484, 546 diffusion constant, 506 diffusion reaction equation, 86 diffusion tensor, 78 diffusion-to-capture, 47 diffusional transport, 450 dihedral space, 333 dihydro-rhodamine, 88 direct immunofluorescence, 409 direct writing ebeam lithography, 465 directed movement, 549 distance autocorrelation function, disulfide bond reduction, 324 DNA, 296, 463 DNA hybridization, 463 DNA packaging motor, 238 DNA sequencing, 445 564 Index DNA sequencing by single-molecule spectroscopy, 112 domain structure, 540 double-ChIP, 205 drifting distance, 484 driving force, 483 dronpa, 385 Drosophila chromosomes, 99 drug delivery systems, 546 drug screening, 97 DsRed, 47 dual color FCCS, 142, 150 dual-color cross-correlation, 140 dumbbells, 70 dwell phase, 258 dwell time, 274 dwell time analysis, 329 dwell time distributions, 253, 254 dynamic disorder, 471, 495 dynamic disorder-to-order transition, 10 dynamic nanoscale volumes, 457 dynamical heterogeneity, 72 dynamics, 471 EF1 , 277, 281 E coli uropathogenic, 338, 358 eigenvalues, 86 Einstein relationship, 485 electric field for concentrating very dilute samples, 97 electron transfer (ET), 5, 67, 83, 438 electroporation, 306, 307 electrostatic interaction, 483 endocytosis, 548 endoscope prototypes, 114 energetics, 277 energy barriers, 291 energy landscape, 289, 328, 343, 349, 483 energy potential surface, 484 ensembles of conformations, 334 entropic recoil, 331 entropy, 343, 349, 485 enzymatic function, 289 enzymatic reaction, 455 enzymatic reaction turnover trajectory, 478 enzymatic turnover cycle, 480 enzymatic turnover trajectories, 480 enzyme (busy phase), 93 enzyme active site, 471 enzyme catalysis, 328 enzyme conformational changes, 471 enzyme–product complex, 479 enzyme–substrate complex, 458 enzymes, 471 epi illumination, 82 epi-illumination microscope, 86 epi-TIRF FCS, 90 epidermal growth factor (EGF), 230, 549 equilibrium, 120 ER293, 149 ergodic theorem, 84 evanescent field excitation, 82 evanescent wave, 90 event echo, 95 excitation-energy transfer, 517 excited states lifetime, 79 excitonic interactions, 517 extended coiled-coil domain, 14 external force, 15 extinction, 557 extrinsic noise, 127 EYFP, 43 F1 -ATPase, 19, 271 F-PALM, 49 F1FO ATP synthase, 243 FAD isoalloxazine, far-field, 366, 371 far-field optical nanoscopy, 371 FCCS, 140 FCS Cross Correlation, 97 FCS in Confocal Volumes, 80 filtration, 192 first moment, 483 FITC, 88 flavin cofactor, 88 fluctuation analysis, 155 fluctuation–dissipation Theorem, 486 fluctuations, 119, 121, 126, 127, 132 fluorescamine, 195 fluorescein, 159, 456 fluorescein diphosphate, 455 fluorescence, 63, 190, 365 fluorescence brightness, 157, 158 Index fluorescence bursts, 81 fluorescence correlation spectroscopy (FCS), 41, 107, 132, 155–157, 160, 162, 163, 165, 380 fluorescence excitation, 31 fluorescence lifetime measurements, fluorescence microscopy, 399 fluorescence photoactivatable localization microscopy (FPALM), 386, 400 fluorescence polarization, 487 fluorescence recovery after photobleaching (FRAP), 109 fluorescence resonant energy transfer (FRET), 5, 44, 97, 463 fluorescence trajectories, 475 fluorescent protein, 403 fluorogenic substrate, 496 fluorophore, 370 flux, 120, 124, 125, 132 flycasting dynamics, 559 FM spectroscopy, 29 FM-Stark, 30 FM-US, 30 FMOT, 339 focal spot, 372 folding trajectories, 330 force constant of the potential surface, 485 force field, 483 force fluctuation, 486 force sensor, 296 force spectroscopy, 291 force-clamp, 317, 334 force-clamp spectroscopy, 320 force-extension, 319 force-quench, 330 forced shape transformation, 453 formation times, 480 forming an active enzyme–substrate complex, 481 Frauenfelder model, Fre/FAD protein complex, free energy, 277, 278, 280, 465 free energy landscape, 227 free energy surface, 3, Frenkel excitons, 518 frequency domain optical storage, 27 565 friction coefficient, 485 full-width at half maximum FWHM, 403 functionalized mesoporous nanoparticles, 538 functionalized mesoporous silica structures, 546 FXS, 97 10G, 149 galabiose, 338, 357 β-galactosidase, 91, 95, 439 Gaussian-like, 486 gene delivery system, 538, 547 gene expression, 440 gene expression profiling, 97 gene network, 557 gene regulation, 441, 553, 554, 559 generalized Langevian equation, 486 genetic switch, 444, 556 genetically encoded small-molecule labeling, 412 GFP-transcription factor, 99 giant liposome, 457 glasses, 72 “glassy” nature of the protein, globular base, 14 globular catalytic moiety (F1 ), 12 glutamate, 309 glycerol, 73 gold, 68 gold bead, 273 golgi stacks, 460 green fluorescent protein (GFP), 42, 110, 160 ground state depletion (GSD), 370 ground state depletion followed by single molecule return (GSDIM), 387 haloperoxidase, 501 Hanbury Brown and Twiss, 80 hand-over-hand mechanism, 223 harmonic oscillator, heavy water, 256 α-helices and β-strands, heterogeneity, 27, 33 high positioning, 544 high-resolution crystal structure, 14 566 Index higher order eigenvalues, 79 Hill coefficient, 243, 251, 258, 262 hinge-bending motion, 472 His-tag, 273 histidine tags, 271 homogeneous width, 27 horse radish peroxidase, 88, 89 host–guest interactions, 546 hydrodynamic, 189 hydrogen bonds, 322, 485 hydrolysis, 478 hydrolysis reaction, 16 hydrophobic surface energy, 463 hyperfine broadening, 40 hysteretic reorganization, 95 I5 M, 372 image of a single molecule, 33 imaging, 187 imaging in cells, 45 imaging speed, 411 immobilization, 544 immunofluorescence imaging, 405 inactivation, 499 incubation, 478 index of refraction, 65 indirect immunofluorescence, 409 induced conformational changes, 492 induced fit, 279 induced unfit, 279, 280 inhomogeneous broadening, 33 intensity minimum, 375 inter-subunit coordination, 258 interfacial enzymology, 508 interfacial tension, 465 interference filters, 31 intermittency, 67 internal force, 242 internal pressure, 242 internalization pathway, 549 intersystem crossing, 165 intrinsic noise, 127 ion channels, 232 ion concentration, 159 isomerization, 385 isoSTED, 379 joint probability distribution, 486 kinesin, 10, 223 kinesin and ATP synthase, 10 kinetics, 189 kinetics of the excited state and rotational correlations, 78 Krebs cycle, 459 lac operon, 441 lactose metabolism, 441 Langevin, 94 Langevin description, 94 Langevin simulations, 94 laser heating, 256 laser scanning confocal microscope (LSCM), 168 laser trap, 223 law of action and reaction, 279 lazy phase, 93 LH2, 514 ligand, 189 ligand exchange, 196 light microscopy, 399 light scattering, 177–179 limping mode, 19 lipase, 497 lipase B, 91 lipid, 380 lipid monolayer, 461 liposomes, 164 living cells, 230, 489, 537 localization, 386 localization accuracy, 400, 538, 544 low expression levels, 98 low temperatures, 69 lowest frequency normal modes, 15 lubricant-like phase, 544 MF1 , 271, 277, 281 M10, 82 M13 DNA, 85 M20, 82 M41S hexagonal phase, 538 macromolecular crowding, 466 magnet, 274, 276 magnetic resonance, 32, 38 manifold of conformational states, 89 Markov processes, 93 master equation, 127, 556 Mechalis–Menten, 471 Index mechanical, 317, 321 mechanical fingerprint, 318 mechano-chemical energy conversion, 280 mechanochemical, 247 mechanochemical cycle, 243, 245, 247, 258, 262 mechanochemistry, 264 memory landscapes, 93 memory period, 95 mercaptoethanol, 195 mesoporous M41S systems, 542 mesoscale structure, 451 metabolic network, 119 metal nanoparticles, 177 metastable, 382 MgADP inhibition, 281 MHCII, 46 Michaelis–Menten, 243, 245, 251, 258, 260, 262, 327, 471 Michaelis-Menten (MM) enzymatic reaction, 457 Michaelis-Menten equation, 439 microcrystal, 71 microelectrofusion, 459 microemulsion, 451 microfluidic, 419 microinjection, 150 micromanipulation, 453 4Pi microscopy, 372 microtubule-dependent transport, 549 microtubules, 405 miRNA, 150 mitochondria, 449 mitochondrial membranes, 11 modulated excitation, 168 molecular coordinates, 294 molecular dynamics (MD) simulation, 481 molecular dynamics simulations, 295 molecular fingerprint, 294 molecular motors, 3, 238, 253, 256 molecular transcription factor activation, 112 molecular transcription factor activation in the cell nucleus, 112 molten globule, monodisperse, 191 monomode or multimode glassfiber, 82 567 monomolecularly thin fluid film, 461 monovalent, 192 morphogen gradients, 312 mosaic, 73 motor proteins, 7, 548, 549 movement with drift, 549 MreB, 47 multi-color STORM, 406 multichannel analyzer, 80 multidentate, 188 multifunctional, 188 multilamellar vesicle, 463 multiphoton, 369 multiphoton laser scanning microscopy, 108 multiple conformational states, 227 multiple steady states, 120 multiplexing, 206 muscle contraction, muscle system, 229, 290 myosin, 221 myosin NMY-2, 140, 143 myosin V, 225 myosin VI, 225 n-Alkanes, 71 NALMS, 386 nano- or mesoporous silica structures, 538 nanocrystal, 66 nanocrystal molecules, 175, 178, 180, 182, 184 nanocrystalline, 197 nanocrystals, 187 nanofluidic device, 450 nanofluidic surface reactor, 460 nanofluidic transporter, 449 nanoparticles, 300 nanoparticles (metal), 68 nanoporous materials, 537 nanoprobe, 33 nanoscale, 370 nanoscopy, 371, 379, 391 nanosecond anisotropy, 476 nanostructured systems, 537 nanotube, 451 nanotube-vesicle network, 451 near-field, 366 near-field light, 40 568 Index near-field scanning optical microscopy (NSOM), 41 network morphology, 456 network nodes, 454 neurodegenerative disease, 111 Ni-NTA, 274 non-adiabatic system, 557 non-Arrhenius temperature dependence, non-equlibrium processes, 94 non-exponential decay, 497 non-hydrolyzable ATP, 249 non-Markovian function, 93 non-viral vectors, 548 nonequilibrium, 120, 471 nonequilibrium kinetics, 292 nonequilibrium steady states (NESSs), 120, 122–124, 127, 129, 132, 135 nonhydrolyzable ATP analogs, 246 nonlinear reactions, 120 nonlinearity, 390 nonspecific interaction, 479 novel drug delivery systems, 538, 546 nuclear coordinate, 483 nucleophiles, 327 NV centers, 380 Olympus, 82 on-off, 66 one-dimensional generalized Langevin equation, one-dimensional multiple-step random walk, 483 one-dimensional random walk, 483 oocyte, 140 optical bistability, 384 optical mapping, 208 optical switch, 43 optical transfer function, 384 optical trapping, 10 optical tweezers, 240, 339, 340, 358 optical wide-field microscope, 540 orbital, 392 order parameter, orientational fluctuations, 78 oriented single molecules, 542 oscillating field, 96 oscillator strength, 194, 520 oscillatory conformational motions, 472, 480 Ostwald ripening, 197 overlay of the optical and electron microscopic images, 540 overstretching phase transition, 264 oxygen exchange, 275 packaging motor, 239 pair-wise distance distribution, 250 PALM with independently running acquisition (PALMIRA), 387 pancreatic porcine lipase, 81 PAR proteins, 143 PAR-2 protein, 140 parabolic reflector, 79 patch clamp tips, 96 pentacene, 388 pentacene in p-terphenyl, 30, 34, 38 periodic solutions, 94 periodicity in the NMF, 94 perylene, 37 pH-sensitive dyes, 159 phenotypes, 119, 132, 443 phenotypic, 129 phenotypic noise, 126 phospholipase, 504 phospholipid bilayer, 503 photo-activation, 307, 310 photo-induced charge transfer, 165 photo-induced transient states, 164 photo-switchable, 402 photo-switching, 165 photoactivatable, 371, 385 photoactivatable localization microscopy (PALM), 49, 386, 400 photoactivatable proteins, 385 photoactivation, 49 photobleaching, 86, 192 photolithographic microfabrication, 465 photon distribution, 98 photon noise, 62 photon time-stamping, 477 photon-stamping spectroscopy, 476 photostability, 68, 191 photoswitchable fluorescent proteins, 44 photoswitching, 32, 36, 44, 371, 385 photosynthesis, 513 Index photothermal, 64 Pi rebinding, 277 Pi release, 275, 277, 278 picoliter injector, 488 pili, 338, 343, 359 P, 338, 352–358 rod, 343, 351, 356 type 1, 338, 348, 352–356, 358, 359 plasmon coupling, 184 plasmon resonance, 177, 178 plasmon rulers, 178, 180, 182, 184 plastic bead, 274 point-spread-function PSF, 34, 366, 400 Poisson, 486 Poisson process, 97 Poisson statistics, 37 polarization, 72, 522 polarization-modulated confocal microscopy, 542 poly(acrylamide) gels, 42 polymer elasticity, 294 polyplex, 548 polyprotein engineering, 318 polysaccharide, 471 polytenic chromosomes, 99 position accuracy, 542 positioning accuracy, 540, 544 potential surfaces, 492 power laws, 66 power stroke, 279 power-law memory kernel, processive enzymatic reaction, 509 product-releasing, 480 promoter, 203 protein conformational changes, 472 protein folding, protein structure fluctuations, 111 proton and ion gradients, proton exchange, 160, 161 proton transport, 162 proton-collecting antenna, 163 proton-collecting antennae, 162 proton-motive force, 15 protonization kinetics of Fluorescein, 87 protonization reaction, 87 pseudofree-trajectory, 53 PufX, 515 pulse protocols, 333 pump-and-probe, 293 569 pump-probe, 64, 68 purple photosynthetic bacteria, 513 push–pull mechanism, 280, 282 PWD, 251, 256 pyelonephritis, 338 quadruple trap, 96 quantum dot (QD), 175, 183, 187, 206 quantum yield, 191 quantum-limited detection, 30 quasi-elastic light scattering (QELS), 107 radius of gyration, Rg , Raman scattering, 63 random coil, random movement, 549 Ras, 229 rate process, 486 RC-LH1, 514, 520 reaction networks, 122 reaction order, 455 reaction-diffusion model, 459 reaction-diffusion process, 455 reactor shape, 451 rebinding of CO to myoglobin, refolding, 338, 339, 343, 345, 347, 350, 353 force, 347, 348, 352 rate, 348, 350 regulatory proteins, 47 relaxation kinetics, 86 relaxation of the NMF, 95 repChIP, 205 reporters, 402 RESOLFT, 51, 377, 400 resolution, 365, 399 response force-vs.-contraction, 349 force-vs.-elongation, 339–342, 345, 349, 350, 352, 353, 359 force-vs.-elongation speed, 346, 353 force-vs.-time, 348 retinoic acid, 312 reversible hydrolysis, 275 rheology, 73 rhodopsin, 425 ring-ATPase, 238 RNA polymerase (RNAP), 203 570 Index RNA-induced silencing complex, 147 RNAi, 97 RNAP, 209, 214 room-temperature fluorescence spectroscopy, rotary molecular motor, 271 rotational diffusion, 78 rotational ellipsoid, 78 rotational motions, 487 salivary gland cells, 99 saturated pattern excitation microscopy (SPEM), 377 saturated structured illumination microscopy (SSIM), 377, 400 saturation, 32, 66 scale-free, 454 scanning FCS, 143, 145 scanning speed, 98 scanning transmission electron microscope (STEM), 417 scanning tunneling microscope (STM), 418, 420 scattered field, 65 Schlogl, 122 Schlogl reaction, 124, 126 second harmonic generation (SHG), 111 self-assembly, 189, 300, 451, 452 self-inhibition, 459 self-organization, 451, 452 self-trapping, 67 SeqChIP, 205 sequential model, 247 γ shaft, 19 shear geometry, 298 SHRIMP, 386 signal transduction processes, 229 signal-to-background, 156 signal-to-noise ratio, 62, 64, 66, 249 single cell physiology, 314 single colour cross correlation, 86 single enzyme molecule kinetics, 88 single exponential, 330 single exponential phase, 93 single molecule biophysical, 182 single molecule detection, 30, 238 single molecule detection in single cells, 97 single molecule enzymology, 436 single molecule experiments, single molecule imaging, 54, 81 single molecule manipulation, 238 single molecule simulations, single molecules, 537, 544 single molecules in the electric field, 95 single particle biophysics, 180 single particle tracking, 549 single protein, 317 single- molecule enzymatic turnover assays, 439 single-molecule active-control microscopy (SMACM), 49 single-molecule anisotropy, 476 single-molecule cut-and-paste, 296 single-molecule detection at room temperature, 435 single-molecule diffusion, 109 single-molecule dynamics, 554 single-molecule force spectroscopy, 426–428 single-molecule FRET, 226 single-molecule gene switch, 555 single-molecule placing, 489 single-molecule spectroscopy, 26, 69 single-molecule spectroscopy illuminating the molecular dynamics of life, 114 single-molecule statistical physics, 554 single-molecule tracking experiments, 538 single-molecule trajectory, 475, 538, 540 siRNA, 150 site occupancy, 276, 277 sliding friction, 465 SMS, 26 solid-like, 73 solvent substitution, 323 spatial resolution, 247 spatial resolution of 2–3 nm, 544 specific binding site, 299 specific heat, 65 specificity, 89 spectral diffusion, 32, 34, 35 spectral hole, 28 spectral hole-burning, 27 spectral trajectory, 34 spin-boson, 555 spontaneous epigenetic switching, 559 Index spreading power, 465 stall torque, 282 standard deviation, 483 standard free-energy change (ΔGo ), 16 Stark shifts, 41 static disorder, 498 static network geometries, 456 stationary excitation, 79 statistical fine structure, 29, 32 statistical theories, 330 steady state, 120, 126, 129 steady state probability distribution, 557 STED, 400 STED-4Pi, 379 steered molecular dynamics, 322 STEM, 418, 420 step size, 247 stepping rate, 273 stimulated emission depletion (STED), 51, 193, 194, 370, 400 stochastic and deterministic processes, 94 stochastic behavior, 121 stochastic networks, 129 stochastic optical reconstruction microscopy STORM, 50, 386, 399 stochastic resonance, 559 stochastic switching, 375 stochastic system, 127 stochasticity, 554 3D STORM, 408 strain-sensor, 226 strain-tunable, 194 streptavidin, 300 stretch parameter, 84 stretched exponentials, 83, 89 structure helixlike, 338, 342, 345–348, 350, 352, 353, 358 quaternary, 342 structured geometries, 450 student’s t-test, 251 SU-8, 461 sub-diffraction-limit, 404 subdiffraction, 375 subdiffraction resolution, 298 substep, 221, 273, 274 substrate, 474 571 subunit, 338, 342, 358 FimA, 338 PapA, 338 α and β subunits, 14 β subunits, 14 super-resolution image, 411 super-resolution imaging, 193, 297 supercooled liquids, 72 superlocalization, 48 superresolution, 48, 49, 370 surface tension, 461 surface-based catalysis, 463 surface-immobilized vesicle, 451 surfactant membrane, 451 survival time of photobleaching, 85 switch stability, 556 switchable, 371 switchable mobility, 538, 542 switching, 384, 390 symmetry, 276 synapse, 379 synthetic viruses, 538, 547, 548 T7 bacteriophage, 204, 209 targeted switching, 375 targeting, 546 TDI, 539 template synthesis, 542 terphenyl, 388 terrylendiimide, 539 terrylene, 70 tetra methyl rhodamin, 83 texas red, 81 TF, 205, 206 TF1 , 271, 277, 280 TFs, 204 the jigglings and wigglings, the nonexponential time dependence, the potential of mean force, the randomness parameter, 254 thermal fluctuation, 219, 279 thermophilic bacterium, 19 thioredoxin, 325 three-Dimensional, 408 tight binding, 245, 260 time amplitude converter, 80 time-dependent friction, 486 titin kinase, 290 T-jump, 77 572 Index T4 lysozyme, 472 tobacco mosaic virus (TMV), 53 topology transformation, 459 torque generation, 282 total internal reflection fluorescence (TIRF), 43, 220 total internal reflection microscopy, 298 toxic drugs, 547 trajectory, 506 trans-cis isomerization, 165 transcription, 203, 204, 215 transcription factors (TFs), 99, 203, 441 transcriptional efficiency, 126 transfection, 144 transient state, 165, 166 transient state imaging, 168 transition state, 320, 343–346, 348, 355 transition state theory, 557 translational diffusion, 157 translational efficiency, 126 translocation along DNA/RNA, transmembrane portion (Fo ), 12 transmission electron microscopy (TEM), 538 transport model, 455 transport processes, 548 trapping of a molecule, 546 treadmilling, 48 tri-site, 277 triplet (bottleneck), 39 triplet kinetics, 86 triplet scheme, 87 triplet states, 166, 168 tryptophan, 277 t-test, 255 tuberculosis, 447 tumbling, 72 turnover followed, 89 two-color excitation, 505 two-level system (TLSs), 36 two-photon excitation, 141, 143, 306, 309 two-photon FCS, 140 two-state kinetics, 555 two-state switch behavior, 556 ubiquitin, 329 ultrasmall, 197 unfolding, 338, 339, 342, 343, 345–348, 350, 352, 353 force, 347, 348, 350–353, 355, 357 rate, 347, 356 sequential, 342, 345, 346 unidimensional channels, 538 unidimensional Nanochannels, 542 unisite hydrolysis, 16 unzip geometry, 298 UPEC, 338 van der Waals, 485 Van’t-Hoff –Le Chatelier principle, 457 velocity trajectories, 486 vibrational spectroscopy, 32 vibrations, 68 viral particles, 97 virtual reactions, 125 visual proteomics, 419, 421, 428 weakly non-adiabatic, 558 weighted histogram analysis method, wide field, 503 wide-field imaging set-up, 539 worm-like chain, 242 yeast cell cycle, 124, 125 yoctomole, 27 z-resolution, 372 zero-phonon lines, 69 zinc blende, 198 ... The impact of single- molecule spectroscopy and imaging spans areas of chemistry, physics, and biology (B) Schematic of a focused optical beam pumping a single resonant molecule in a cell or other... scaling as N Optical detection and spectroscopy of a single molecule in a solid Optical temperature-dependent dephasing, nonlinear optical saturation Imaging of a single molecule in space and. .. I Introductory Lecture: Molecular Dynamics of Single Molecules How Biomolecular Motors Work: Synergy Between Single Molecule Experiments and Single Molecule Simulations Martin Karplus and Jingzhi