Skip to main content

Single Molecule Studies

  • Chapter
  • First Online:
  • 675 Accesses

Part of the book series: Springer Theses ((Springer Theses))

Abstract

Single molecule detection (SMD) has become an important technique in recent years for studying dynamic processes such as chemical reactions and molecular motions at a fundamental level. Historically these processes are usually studied using methods based on ensemble averaging of a sample of molecules, however frequently the mean properties so found are insufficient. Studies on single molecules are thus advantageous as information, such as statistical distributions of particular quantities, is not lost by averaging. It should however be noted that even single molecule studies yield results that are temporally averaged over the course of the finite measurement time.

A physicist is just an atom’s way of looking at itself. Niels Bohr

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

Buying options

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Notes

  1. 1.

    The convention whereby an upper case letter denotes a random process and/or variable, whilst the lower case equivalent denotes a particular outcome is again used throughout this chapter.

  2. 2.

    The independence of each \(Z_j\) can be shown to follow from the assumed independence of \(\tau _j\) regardless of the independence of \(\gamma _j\) [15].

  3. 3.

    Although arguably a Fisher information analysis, as has been extensively utilised in this text, is possible using the derived PDFs, this is omitted here since consideration of the variance is adequate in highlighting the key points.

  4. 4.

    Strictly speaking the integration should be performed over the Gaussian reference sphere, however for a low numerical aperture lens the inter-component mixing caused by bending of rays of light is negligible and it is hence satisfactory to integrate over the back focal plane. The form of the image field of an electric dipole using two high numerical aperture lenses was considered earlier in Sect. 6.4.2.1 in which it was found the field on axis takes the form \(\mathbf{ E} _2 \propto (K_0^A p_x,K_0^A p_y, K_0^B p_z)\),

    where \(K_0^A\) and \(K_0^B\) are constants and \(K_0^B/K_0^A \ll 1\). This arrangement will be neglected in this chapter however due to its practical difficulties.

References

  1. T. Basché, W.P. Ambrose, W.E. Moerner, Optical spectra and kinetics of single impurity molecules in a polymer: spectral diffusion and persistent spectral hole burning. J. Opt. Soc. Am. B 9, 829–836 (1992)

    Article  ADS  Google Scholar 

  2. R.E. Dale, S.C. Hopkins, Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers. Biophys. J. 76, 1606–1618 (1999)

    Article  Google Scholar 

  3. P. Debye, Polar molecules. Ph.D. Thesis, Dover, New York, 1945

    Google Scholar 

  4. L.A. Deschenes, D.A. van den Bout, Single-molecule studies of heterogeneous dynamics in polymer melts near the glass transition. Science 292, 233, 255–258 (2001)

    Google Scholar 

  5. R.M. Dickson, D.J. Norris, W.E. Moerner, Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis. Phys. Rev. Lett. 81, 5322–5325 (1998)

    Article  ADS  Google Scholar 

  6. W. Feller, Probability Theory and its Applications (Addison-Wesley, New York, 1950)

    MATH  Google Scholar 

  7. M.R. Foreman, C. Macías Romero, P. Török, Determination of the three dimensional orientation of single molecules. Opt. Lett. 33, 1020–1022 (2008)

    Google Scholar 

  8. I.S. Gradshteyn, I.M. Ryzhik, Table of Integrals, Series and Products (Elsevier Academic Press, New York, 1980)

    MATH  Google Scholar 

  9. T. Ha, T. Enderle, D.S. Chemla, P.R. Selvin, S. Weiss, Single molecule dynamics studied by polarization modulation. Phys. Rev. Lett. 77, 3979–3982 (1996)

    Article  ADS  Google Scholar 

  10. T. Ha, T. Enderle, D.F. Ogletree, D.S. Chemla, P.R. Selvin, S. Weiss, Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl. Acad. Sci. U S A 93, 6264–6268 (1996)

    Article  ADS  Google Scholar 

  11. T. Ha, J. Glass, T. Enderle, D.S. Chemla, S. Weiss, Hindered rotational diffusion and rotational jumps of single molecules. Phys. Rev. Lett. 80, 2093–2096 (1998)

    Article  ADS  Google Scholar 

  12. Invitrogen, Fluorescence spectraviewer, http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html

  13. K. Itô, Introduction to Probability Theory (Cambridge University Press, Cambridge, 1984)

    MATH  Google Scholar 

  14. T.M. Jovin, M. Bartholdi, W.L.C. Vaz, R.H. Austin, Rotational diffusion of biological macromolecules by time-resolved delayed luminescence (phosphorescence, fluorescence) anisotropy. Ann. N Y Acad. Sci. 366, 176–196 (1981)

    Google Scholar 

  15. A. Leon-Garcia, Probability and Random Processes for Electrical Engineering (Addison-Wesley, New York, 1994)

    Google Scholar 

  16. H.P. Lu, L.Y. Xun, X.S. Xie, Single molecule enzymatic dynamics. Science 282, 1877–1882 (1998)

    Article  ADS  Google Scholar 

  17. W.E. Moerner, D.P. Fromm, Methods of single molecule fluorescence spectroscopy and microscopy. Rev. Sci. Instrum. 74, 3597–3619 (2003)

    Article  ADS  Google Scholar 

  18. I. Munro, I. Pecht, L. Stryer, Subnanosecond motions of Tryptophan residues in proteins. Proc. Natl. Acad. Sci. U S A 76, 56–60 (1979)

    Article  ADS  Google Scholar 

  19. D. Patra, I. Gregor, J. Enderlein, Image analysis of defocused single molecule images for three dimensional molecular orientation studies. J. Phys. Chem. A 108, 6836 (2004)

    Article  Google Scholar 

  20. G.H. Patterson, S.N. Knobel, W.D. Sharif, S.R. Kain, D.W. Piston, Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997)

    Article  Google Scholar 

  21. D.J. Pikas, S.M. Kirkpatrick, E. Tewksbury, L.L. Brott, R.R. Naik, M.O. Stone, W.M. Dennis, Nonlinear saturation and lasing characteristics of green fluorescent protein. J. Phys. Chem. B 106, 4831–4837 (2002)

    Article  Google Scholar 

  22. B. Sick, B. Hecht, L. Novotny, Orientational imaging of single molecules by annular illumination. Phys. Rev. Lett. 85, 4482–4485 (2000)

    Article  ADS  Google Scholar 

  23. P. Török, Propagation of electromagnetic dipole waves through dielectric interfaces. Opt. Lett. 25, 1463–1465 (2000)

    Article  ADS  Google Scholar 

  24. P. Török, P.D. Higdon, T. Wilson, On the general properties of polarised light conventional and confocal microscopes. Opt. Commun. 148, 300–315 (1998)

    Article  ADS  Google Scholar 

  25. P. Török, P.D. Higdon, T. Wilson, Theory for confocal and conventional microscopes imaging small dielectric scatterers. Opt. Commun. 45, 1681–1698 (1998)

    Google Scholar 

  26. H.C. van de Hulst, Light Scattering by Small Particles (Dover Publications, Dover, 1981)

    Google Scholar 

  27. P. Wahl, K. Tawada, J.C. Auchet, Study of tropomyosin labelled with a fluorescent probe by pulse fluorimetry in polarized light—interaction of that protein with troponin and actin. Eur. J. Biochem. 88, 421–424 (1978)

    Article  Google Scholar 

  28. D.M. Warshaw, E. Hayes, D. Gaffney, A.M. Lauzon, J.R. Wu, G. Kennedy, K. Trybus, S. Lowey, C. Berger, Myosin conformational states determined by single fluorophore polarization. Proc. Natl. Acad. Sci. U S A 95, 8034–8039 (1998)

    Article  ADS  Google Scholar 

  29. S. Weiss, Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat. Struct. Biol. 7, 724 (2000)

    Article  Google Scholar 

  30. K.D. Weston, L.S. Goldner, Orientation imaging and reorientation dynamics of single dye molecules. J. Phys. Chem. B 105, 3453–3462 (2001)

    Article  Google Scholar 

  31. J. Yguerabide, H.F. Epstein, L. Stryer, Segmental flexibility in an antibody molecule. J. Mol. Biol. 51, 573–590 (1970)

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Matthew R. Foreman .

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Foreman, M.R. (2012). Single Molecule Studies. In: Informational Limits in Optical Polarimetry and Vectorial Imaging. Springer Theses. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-28528-8_8

Download citation

  • DOI: https://doi.org/10.1007/978-3-642-28528-8_8

  • Published:

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-28527-1

  • Online ISBN: 978-3-642-28528-8

  • eBook Packages: Physics and AstronomyPhysics and Astronomy (R0)

Publish with us

Policies and ethics