Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Imaging, Molecular, and Cellular Analysis
92
Novel Vibrational Nanoprobes for Microbiology at the Single Cell Level
Thomas Huser* (huser1@llnl.gov), Chad E. Talley, James W. Chan, Heiko Winhold, Ted Laurence, Anthony Esposito, Christopher W. Hollars, Christine A. Hara, Allen T. Christian, Michele H. Corzett, Rod Balhorn, and Stephen M. Lane
Lawrence Livermore National Laboratory, Livermore, CA
The measurement of intracellular chemical concentrations and molecular fluxes provides essential information for systems-biological models of cells. This information, however, is difficult to obtain at the single cell level – especially in living cells where chemical levels can change rapidly in response to external or internal events.
We are studying individual microbes by a combination of optical spectroscopy techniques to obtain dynamic chemical profiles at the single cell level. Raman spectroscopy in combination with optical tweezers is used to non-destructively capture individual microbes in their native environment and assess their chemical composition within seconds. We have used this technique to dynamically monitor changes in the total protein concentration of individual cells due to increased expression after external stimulation. By focusing on changes in particular Raman peaks of a microbe we can follow trends in the overall intensity of specific peaks on an even faster timescale – down to milliseconds – without the need for exogenous probes. We will present examples and applications of these powerful vibrational spectroscopy techniques.
To monitor chemicals at low concentrations or chemicals that cannot typically be measured by Raman spectroscopy we also present the development of nanoscale sensors based on functionalized metal nanoparticles and surface-enhanced Raman scattering (SERS). As an example, the SERS spectrum from individual silver nanoparticle (50-80 nm in diameter) clusters functionalized with 4-mercaptobenzoic acid (4-MBA) is shown to exhibit a characteristic response to the pH of the surrounding solution, and is sensitive to pH changes in the range of 6 to 8. Measurements from nanoparticles incorporated into individual cells demonstrate that these nanoparticle sensors retain their robust signal and sensitivity to pH when incorporated into a cell. These sensors can be probed almost entirely background-free and their signals do not suffer from photobleaching, which makes them attractive long-term probes for chemical concentrations that cannot be probed by conventional Raman spectroscopy.
This work was supported by the Office of Science of the U.S. Department of Energy. Work performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under contract number W-7405-ENG-48.
* Presenting author
