Skip to main content

Advertisement

SpringerLink
Log in

Single-molecule fluorescence characterization in native environment

  • Review
  • Published:
Biophysical Reviews Aims and scope Submit manuscript

Abstract

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the “missing information” problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ∼1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

AOM:

Acousto-optic modulator

AS:

Activatable shutter

BFP:

Back focal plane

BS:

Beam stop

CCD:

Charge-coupled device

Cys707:

Myosin head domain highly reactive thiol

DM:

Dichroic mirror

ELC:

Myosin essential light chain

GFP:

Green fluorescent protein

HCRLC:

Human cardiac myosin regulatory light chain

NA:

Numerical aperture

NSOM:

Near-field scanning optical microscope

PA:

Photoactivatable

PA-FP:

Photoactivatable fluorescent protein

PA-GFP:

Photoactivatable green fluorescent protein

PALM:

Photoactivated localization microscopy

PC:

Pockels cell

PEG:

Polyethylene glycol

PMMA:

Polymethylmethacrylate

PSF:

Point spread function

RLC:

Myosin regulatory light chain

S1:

Myosin subfragment 1

SAF:

Supercritical angle fluorescence

SI:

Structured illumination

SMD:

Single-molecule detection

STED:

Stimulated emission depletion

STORM:

Stochastic optical reconstruction microscopy

TIRF:

Total internal reflection microscopy

References

  • Arata T (1990) Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers. J Mol Biol 214:471–478

    Article  CAS  PubMed  Google Scholar 

  • Ashkin A, Dziedzic JM, Yamane T (1987) Optical trapping and manipulation of single cells using infrared laser beams. Nature 330:769–771

    Article  CAS  PubMed  Google Scholar 

  • Axelrod D (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89:141–145

    Article  CAS  PubMed  Google Scholar 

  • Axelrod D (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764–774

    Article  CAS  PubMed  Google Scholar 

  • Axelrod D, Thompson NL, Burghardt TP (1983) Total internal reflection flourescence microscopy. J Microsc 129:19–28

    CAS  PubMed  Google Scholar 

  • Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:108101

    Article  PubMed  Google Scholar 

  • Betzig E, Chichester RJ (1993) Single molecules observed by near-field scanning optical microscopy. Science 262:1422–1425

    Article  CAS  PubMed  Google Scholar 

  • Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  CAS  PubMed  Google Scholar 

  • Bobroff N (1986) Position measurement with a resolution and noise-limited instrument. Rev Sci Instrum 57:1152–1157

    Article  Google Scholar 

  • Borejdo J, Ushakov DS, Akopova I (2002) Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle. Biophys J 82:3150–3159

    Article  CAS  PubMed  Google Scholar 

  • Borejdo J, Talent J, Akopova I, Burghardt TP (2006) Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy. Biochim Biophys Acta 1763:137–140

    Article  CAS  PubMed  Google Scholar 

  • Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ (1997) Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci USA 94:2306–2311

    Article  CAS  PubMed  Google Scholar 

  • Burghardt TP, Axelrod D (1981) Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics. Biophys J 33:455–467

    Article  CAS  PubMed  Google Scholar 

  • Burghardt TP, Ajtai K, Borejdo J (2006a) In situ single molecule imaging with attoliter detection using objective total internal reflection confocal microscopy. Biochemistry 45:4058–4068

    Article  CAS  PubMed  Google Scholar 

  • Burghardt TP, Charlesworth JE, Halstead MF, Tarara JE, Ajtai K (2006b) In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy. Biophys J 90:4662–4671

    Article  CAS  PubMed  Google Scholar 

  • Burghardt TP, Hipp AP, Ajtai K (2009a) Around-the-objective total internal reflection fluorescence microscopy. Appl Opt 48:6120–6131

    Article  PubMed  Google Scholar 

  • Burghardt TP, Li J, Ajtai K (2009b) Single myosin lever-arm orientation in a muscle fiber detected with photoactivatable GFP. Biochemistry 48:754–765

    Article  CAS  PubMed  Google Scholar 

  • Corrie JET, Brandmeier BD, Ferguson RE, Trentham DR, Kendrick-Jones J, Hopkins SC, van der Heide UA, Goldman YE, Sabido-David C, Dale RE, Criddle S, Irving M (1999) Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction. Nature 400:425–430

    Article  CAS  PubMed  Google Scholar 

  • Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Luhrmann R, Jan R, Eggeling C, Hell SW (2006) Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci USA 103:11440–11445

    Article  CAS  PubMed  Google Scholar 

  • Eisenberg E, Hill TL (1985) Muscle contraction and free energy transduction in biological systems. Science 227:999–1006

    Article  CAS  PubMed  Google Scholar 

  • Finer JT, Simmons RM, Spudich JA (1994) Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368:113–119

    Article  CAS  PubMed  Google Scholar 

  • Fulbright RM, Axelrod D (1993) Dynamics of nonspecific adsorption of insulin of erythrocyte membranes. J Fluoresc 3:1–16

    Article  CAS  Google Scholar 

  • Geeves MA, Holmes KC (1999) Structural mechanism of muscle contraction. Annu Rev Biochem 68:687–728

    Article  CAS  PubMed  Google Scholar 

  • Gugel H, Bewersdorf J, Jakobs S, Engelhardt J, Storz R, Hell SW (2004) Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. Biophys J 87:4146–4152

    Article  CAS  PubMed  Google Scholar 

  • Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102:13081–13086

    Article  CAS  PubMed  Google Scholar 

  • Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100

    Article  CAS  PubMed  Google Scholar 

  • Harrick NJ (1979) Internal reflection spectroscopy. Wiley Interscience, New York

    Google Scholar 

  • Hellen EH, Axelrod D (1987) Fluorescence emission at dielectric and metal-film interfaces. J Opt Soc Am B 4:337–350

    Article  CAS  Google Scholar 

  • Hirschfeld T (1976) Optical microscopic observation of single small molecules. Appl Opt 15:2965–2966

    Article  CAS  PubMed  Google Scholar 

  • Holtzer L, Meckel T, Schmidt T (2007) Nanometric three-dimensional tracking of individual quantum dots in cells. Appl Phys Lett 90:053902-1–053902-3

    Article  Google Scholar 

  • Huxley HE (1969) The mechanism of muscular contraction. Science 164:1356–1366

    Article  CAS  PubMed  Google Scholar 

  • Inoué S, Shimomura O, Goda M, Shribak M, Tran PT (2002) Fluorescence polarization of green fluorescence protein. Proc Natl Acad Sci USA 99:4272–4277

    Article  PubMed  Google Scholar 

  • Irving M, Allen TSC, Sabido-David C, Cralk JS, Brandmeler B, Kendrick-Jones J, Corrie JET, Trentham DR, Goldman YE (1995) Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle. Nature 375:688–690

    Article  CAS  PubMed  Google Scholar 

  • Ishijima A, Doi T, Sakurada K, Yanagida T (1991) Sub-piconewton force fluctuations of actomyosin in vitro. Nature 352:301–306

    Article  CAS  PubMed  Google Scholar 

  • Ishijima A, Kojima H, Funatsu T, Tokunaga M, Higuchi H, Tanaka H, Yanagida T (1998) Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92:161–171

    Article  CAS  PubMed  Google Scholar 

  • Katz A (1967) Principles of statistical mechanics: the information theory approach. Freeman, San Francisco, pp 14–22

    Google Scholar 

  • Kishino A, Yanagida T (1988) Force measurements by micromanipulation of a single actin filament by glass needles. Nature 334:74–76

    Article  CAS  PubMed  Google Scholar 

  • Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW (2003) Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299:682–686

    Article  CAS  PubMed  Google Scholar 

  • Lewis A, Isaacson M, Harootunian A, Muray A (1984) Development of a 500 Å spatial resolution light microscope. I. Light is efficiently transmitted through λ/16 diameter apertures. Ultramicroscopy 13(3):227–231

    Article  Google Scholar 

  • Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91

    Article  CAS  PubMed  Google Scholar 

  • Lord SJ, Conley NR, Lee HL, Samuel R, Liu N, Twieg RJ, Moerner WE (2008) A photoactivatable push−pull fluorophore for single-molecule imaging in live cells. J Am Chem Soc 130(29):9204–9205

    Article  CAS  PubMed  Google Scholar 

  • Minton AP (2001) The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J Biol Chem 276:10577–10580

    Article  CAS  PubMed  Google Scholar 

  • Moerner WE, Kador L (1989) Optical detection and spectroscopy of single molecules in a solid. Phys Rev Lett 62:2535–2538

    Article  CAS  PubMed  Google Scholar 

  • Mortensen KI, Churchman LS, Spudich JA, Flyvbjerg H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Meth 7:377–381

    Article  CAS  Google Scholar 

  • Muthu P, Mettikolla P, Calander N, Luchowski R, Gryczynski I, Gryczynski Z, Szczesna-Cordary D, Borejdo J (2010) Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart. J Mol Cell Cardiol 48(5):989–998

    Article  CAS  PubMed  Google Scholar 

  • Neher E, Sakmann B (1976) Single channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799–802

    Article  CAS  PubMed  Google Scholar 

  • Neuweiler H, Sauer M (2005) Exploring life by single-molecule fluorescence spectroscopy. Anal Chem 77:179A–185A

    Article  PubMed  Google Scholar 

  • Nguyen DC, Keller RA, Jett JH, Martin JC (1987) Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser-induced fluorescence. Anal Chem 59:2158–2161

    Article  CAS  PubMed  Google Scholar 

  • Ober RJ, Ram S, Ward ES (2004) Localization accuracy in single-molecule microscopy. Biophys J 86:1185–1200

    Article  CAS  PubMed  Google Scholar 

  • Orrit M, Bernard J (1990) Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys Rev Lett 65:2716–2719

    Article  CAS  PubMed  Google Scholar 

  • Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877

    Article  CAS  PubMed  Google Scholar 

  • Pavani SRP, Thompson MA, Biteen JS, Lord SJ, Liu N, Twieg RJ, Piestun R, Moerner WE (2009) Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci USA 106(9):2995–2999

    Article  CAS  PubMed  Google Scholar 

  • Peck K, Stryer L, Glazer AN, Mathies RA (1989) Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin. Proc Natl Acad Sci USA 86:4087–4091

    Article  CAS  PubMed  Google Scholar 

  • Peyser YM, Shaya S, Ajtai K, Burghardt TP, Muhlrad A (2003) Cosolvent induced aggregation inhibits myosin ATPase activity by stabilizing the predominant transition intermediate. Biochemistry 42:12669–12675

    Article  CAS  PubMed  Google Scholar 

  • Pierobon P, Achouri S, Courty S, Dunn AR, Spudich JA, Dahan M, Cappello G (2010) Velocity, processivity, and individual steps of single myosin V molecules in live cells. Biophys J 96(10):4268–4275

    Article  Google Scholar 

  • Pohl DW, Denk W, Lanz M (1984) Optical stethoscopy: image recording with resolution λ/20. Appl Phys Lett 44:651–653

    Article  Google Scholar 

  • Ram S, Prabhat P, Chao J, Ward ES, Ober RJ (2008) High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells. Biophys J 95:6025–6043

    Article  CAS  PubMed  Google Scholar 

  • Rocheleau JV, Edidin M, Piston DW (2003) Intrasequence GFP in class I MHC molecules, a rigid probe for fluorescence anisotropy measurements of the membrane environment. Biophys J 84:4078–4086

    Article  CAS  PubMed  Google Scholar 

  • Rosell FI, Boxer SG (2003) Polarized absorption spectra of green fluorescent protein single crystals: transition dipoles moment directions. Biochemistry 42:177–183

    Article  CAS  PubMed  Google Scholar 

  • Ruckstuhl T, Seeger S (2003) Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens. Appl Opt 42:3277–3283

    Article  PubMed  Google Scholar 

  • Ruckstuhl T, Seeger S (2004) Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy. Opt Lett 29:569–571

    Article  PubMed  Google Scholar 

  • Ruckstuhl T, Verdes D (2004) Supercritical angle fluorescence (SAF) microscopy. Opt Express 12:4246–4254

    Article  PubMed  Google Scholar 

  • Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Meth 3:793–795

    Article  CAS  Google Scholar 

  • Simmons RM, Finer JT, Chu S, Spudich JA (1996) Quantitative measurements of force and displacement using an optical trap. Biophys J 70:1813–1822

    Article  CAS  PubMed  Google Scholar 

  • Stoneking MR, Den Hartog DJ (1997) Maximum-likelihood fitting of data dominated by Poisson statistical uncertainties. Rev Sci Instrum 68:914–917

    Article  CAS  Google Scholar 

  • Thompson NL, Burghardt TP, Axelrod D (1981) Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery and correlation spectroscopy. Biophys J 33:435–454

    Article  CAS  PubMed  Google Scholar 

  • Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783

    Article  CAS  PubMed  Google Scholar 

  • Tikunov BA, Sweeney HL, Rome LC (2001) Quantitative electrophoretic analysis of myosin heavy chains in single muscle fibers. J Appl Physiol 90:1927–1935

    CAS  PubMed  Google Scholar 

  • Timasheff SN (2002) Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components. Proc Natl Acad Sci USA 99:9721–9726

    Article  CAS  PubMed  Google Scholar 

  • Wagner PD, Weeds AG (1977) Studies on the role of myosin alkali light chains. Recombination and hybridization of light chains and heavy chains in subfragment one preparations. J Mol Biol 109:455–470

    Article  CAS  PubMed  Google Scholar 

  • Whitten WB, Ramsey JM, Arnold S, Bronk BV (1991) Single-molecule detection limits in levitated microdroplets. Anal Chem 63:1027–1031

    Article  CAS  Google Scholar 

  • Yanagida T, Nakase M, Nishiyama K, Oosawa F (1984) Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307:58–60

    Article  CAS  PubMed  Google Scholar 

  • Yanagida T, Harada Y, Ishijima A (1993) Nano-manipulation of actomyosin molecular motors in vitro: a new working principle. Trends Biochem Sci 18:319–324

    Article  CAS  PubMed  Google Scholar 

  • Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) grant R01AR049277; the National Heart, Lung, Blood Institute (NHLBI) grant R01HL095572; and the Mayo Foundation.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN, 55905, USA

    Thomas P. Burghardt & Katalin Ajtai

  2. Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, MN, 55905, USA

    Thomas P. Burghardt

Authors
  1. Thomas P. Burghardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katalin Ajtai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas P. Burghardt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Burghardt, T.P., Ajtai, K. Single-molecule fluorescence characterization in native environment. Biophys Rev 2, 159–167 (2010). https://doi.org/10.1007/s12551-010-0038-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12551-010-0038-z

Keywords

Search

Navigation