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Single molecule spectroscopy with a combined atomic force, confocal, and total internal reflection microscope

William Heinz*, Kenneth Weston, Clemence Daeron, and Lori S. Goldner

Laser Application Group, Optical Technology Division, NIST,
Gaithersburg, MD 20899-8442 USA

This is an abstract for a presentation given at the
10th Foresight Conference on Molecular Nanotechnology

 

The interactions between biological macromolecules and macromolecular assemblies are central to the proper functioning of biological systems. Historically, these interactions have been measured by techniques that measure the averaged interactions of large ensembles of particles.

Recently, a number of experimental approaches have been developed that enable the study of single molecules.1, 2, 3, 4 Two advantages that single molecule experiments have over average measurements on ensembles are (1) the distributions that make up the average and the heterogeneity of the population can be determined and (2) the time-dependent behavior of molecules can be measured, whether they are in equilibrium, non-equilibrium, or undergoing some kind of reaction. Both types of information are masked by the average measurement. Understanding interactions on the level of single molecules provides framework for the design of new drugs, treatments for disease, and forms a basis for using those molecules as building blocks for new devices.

Interactions between individual molecules depend on many variables including orientation, conformation, local environment, and separation distance. Two single molecule techniques, atomic force microscopy and single-molecule fluorescence spectroscopy, are sensitive to those parameters. Specifically, the atomic force microscope5 (AFM) is a tool for measuring forces between nanometer-scale objects in physiological conditions (i.e.: in aqueous solutions) and has been used to measure interactions between individual biomolecules.6 Single-molecule fluorescence spectroscopy, including resonant energy transfer7 (sm-RET) and fluorescence polarization spectroscopy8 (FPS) are sensitive to the conformation, orientation, and dynamics of individual molecules.1 The combination of an AFM with sm-RET and FPS measurements can provide a more complete description of the interaction between single molecules, the effects of molecular orientation and conformation on the interaction forces (and vice versa) as a function of separation distance.

Here we present an instrument that combines AFM with confocal and total internal reflection microscopy and report on progress towards the simultaneous measurement of force and orientational and conformational dynamics of single antibody-antigen pairs during the binding and unbinding process in aqueous conditions.

References

  1. Bai, C., C. Wang, X. S. Xie, and P. G. Wolynes. 1999. PNAS 96:11075-11076.
  2. Gimzewski, J. K. and C. Joachim. 1999. Science. 283:1683-1688.
  3. Mehta, A. D., M. Rief, J. A. Spudich, D. A. Smith, and R. M. Simmons. 1999. Science. 283:1689-1695.
  4. Weiss, S. 1999. Science. 283:1676-1683.
  5. Binnig, G., C. F. Quate, and C. Gerber. 1986. Phys. Rev. Lett. 56:930-933.
  6. Heinz, W. F., and J. H. Hoh. 1999. Trends Biotechnol. 17:143-150.
  7. Ha, T., T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss. 1996. PNAS 93:6264-6268.
  8. Ha, T., T. A. Laurence, D. S. Chemla, and S. Weiss. 1999. JPCB. 103:6839-6850.

Abstract in Microsoft Word® format 22,342 bytes


*Corresponding Address:
William Heinz
Laser Application Group, Optical Technology Division, NIST
100 Bureau Dr., Gaithersburg, MD 20899-8442 USA
Phone: 301-975-8741 Fax: 301-840-8551
Email: wheinz@nist.gov



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