Detection of Single Molecules

Ribbe A E 1997 Laser scanning confocal microscopy in polymer science Trends Polym. Sc/. 5 333-7 Oliveira M J and Hemsiey D A 1996 Optical microscopy of polymers Sonderb. Prakt. Metallogr. 27 13-22 Nie Sh and Zare R N 1997 Optical detection of single molecules Ann. Rev. Biophys. Biomol. Struct. 26 567-96 Masters B R 1994 Confocal redox imaging of cells Adv. Mol. Cell Biol. 8 1-19... 

Fig. 11.13. Photon burst detection of single molecules in a focused laser beam.

Confocal microscopes (see Section 11.2.1.1) are well suited to the detection of single molecules. A photon burst is emitted when the molecule diffuses through the excitation volume (0.1-1 fL). An example is given in Figure 11.16. 

Two-photon excitation (TPE) fluorescence microscopy (Section 11.2.1.2) can be applied to the detection of single molecules in solution. By comparison with one-... 

Fig. 11.16. Detection of single molecules of Rhodamine 6G by confocal fluorescence microscopy. A solution of Rhodamine 6G 2 x 10 12 M in water B pure water (reproduced with permission from Mets and Rigler, 1994, Fluorescence 4, 259).

A good reference on the flow cytometry of DNA fragments is Kim Y, Jett JH, Larson EJ, et al. (1999). Bacterial fingerprinting by flow cytometry Bacterial species discrimination. Cytometry 36 324-332. The proposed technique for sequencing DNA is described in a paper by Jett JH, Keller RA, Martin JC, et al. (1989). High-speed DNA sequencing An approach based upon fluorescence detection of single molecules. J. Biomol. Struct. Dynamics 7 301-309. 

Fig. 4. Detection of single molecules by near-field techniques, (a), (b) SNOM images of single molecules embedded in a polymer film with random orientations, from [29]. (c) Sketch of the field distribution in the near field of the aperture, (d) Comparison of measured field distributions to calculated, once for both fundamental orientations, from [27]. (e) Effect of an optical antenna on the emission of a single emitter [36].

Despite these difficulties, considerable progress on TERS has been achieved in the last 5 years and single-molecule TERS spectra have been measured from an increasing number of analytes. The detection of single molecules by TERS usually requires a contribution from not only the enhanced electromagnetic fields of the tip but also resonant Raman effects as well as an additional contribution from a metallic substrate. 

Le Ru et al. demonstrated that a careful consideration of the minimum signal required for detection, the Raman cross section of the molecules involved, and the characteristics of the hot spot were required to determine the minimum enhancement factor necessary for single-molecule detection using SERS [95]. Through this process they concluded that a minimum enhancement factor of 2 x 10 was required for detection of single molecules with bare Raman cross sections of 10 cm sr This is 10 times smaller than the 10 " initially claimed to be essential for single-molecule SERS. The bare Raman cross section of many... 

R. Rigler, J. Widengren, Ultrasensitive detection of single molecules by Fluorescence Correlation Spectroscopy, in Bioscience, ed. by B. Klinge, C. Owman (Lund University Press, Lund, 1990), pp. 180-183... 

Nie, S.M., Chiu, D.T., and Zare, R.N. (1995) Real-time detection of single-molecules in solution by confocal fluorescence microscopy. Analytical Chemistry, 61, 2849-2857. 

It is known that some insects are able to detect a single molecule of a pheromone. The reported detection of single molecules of a dyestuff by repetitive laser pulsing shows that photochemists are now catching up with insects (Shera et al.). Luminescence-activated barometry in wind tunnels represents an unusual application of photophysics (Kavandi et al.). Wild and Renn have provided a useful review of new ideas for high-density information storage. 

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