1. M. J. Levene, W.W. Webb et al. Science (2003).
Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations
M. J. Levene, W.W. Webb et al. Science (2003).
Julia Hoscheit
March 11, 2004

2. Brief Outline of Presentation
Introduction
Describing zero-mode waveguides
Simulations
Applying the technology to DNA synthesis
Conclusion

3. Reasons for studying single molecules
Can reveal information about kinetic processes not normally accessible by ensemble measurements such as variances in kinetic rates
Measurements can reveal the distribution of molecular properties in inhomogeneous systems
Can visualize real-time observations of chemical reactions of biomolecules

4. Common Approaches
Fluorescence correlation spectroscopy (FCS) – Based on the measurement of fluctuations of the fluorescence
Direct observation of sparse molecules using diffraction-limited optics

5. What is a zero-mode waveguide?
In this case, a waveguide is a hollow metal conductor (hole) that provides a path to guide a 488 nm laser.
Single and multimode vs. zero-mode waveguide
More biologically relevant

6. Apparatus for single-molecule enzymology using zero-mode waveguide

7. What the coverslip looks like

8. Simulations

9. Autocorrelation analysis
Autocorrelation analysis is a very clever way of analyzing the measuring fluorescence data.
G(τ) contains information about equilibrium concentrations, reaction kinetics and diffusion rates of molecules in the sample .

10. FCS Using Zero-mode Waveguides

11. Applying this Technology
They used zero-mode waveguides to observe the enzymatic synthesis of dsDNA by DNA polymerase with the use of fluorescently tagged nucleotide analog coumarin-dCTP.
They immobilized mutant T7 DNA polymerase in the waveguides

12. Applying This Technology cont.

13. Conclusions
Studying single molecules can provide insight into the individual properties of the molecules that can be masked by ensemble averaging in experiments performed on several molecules.
This technology will find wide applicability in FCS and other spectroscopic techniques and will allow the techniques to be performed at higher, more biologically relevant concentrations.

14. Discussion
What other biochemical assays can be assessed with zero-mode waveguides?
What are the limitations of this technology?

15. Observation Volume in a waveguide