There has been great effort to make single molecule detection possible and make it useful for molecular and cellular biology last 20 years since its first realization in 1989.

Described at right is one of the methods, single-molecule FRET (smFRET). To achieve single molecule FRET detection, we immobilize the substrates (depicted as a tall grass) on glass surface via the conjugation between neutravidin (rock) and biotin at the end of DNA. We label our protein (depicted as a butterfly) with dyes (Cy3 as donor, Cy5 as acceptor). After we inject the labeled proteins into a glass chamber, we wait and see how the proteins interact with individual substrates immobilized. When the protein is floating around, it is not visible (see below, TIRF microscopy). When it interacts with its substrate immobilized, the protein is effectively localized and the signal is detected in real time (typically 10-1000msec time resolution). Note: to prevent the protein from directly binding onto the glass surface, we coat the surface with polymer (PEG: polyethylene glycol) (depicted as short grasses)

 

Resonance energy transfer occurs via near-field radiation of dipoles. The transfer efficiency exhibits a strong distance dependence over R₀ which is determined by overlap between donor emission and acceptor excitation spectra (as well as other physical properties. See Box below). R₀ of fluorescent dyes is typically 5-10 nm which distance is biologically very useful considering that the size of proteins is ~1 - 100nm and that of RNA and DNA is also an order of nanometers in width.

Eliminating background signal is crucial in achieving single molcule detection. There are several imaging methods developed. The commonly used ones are 1) confocal microscopy, 2) prism-type TIRF microscopy, and 3) objective-type TIRF microscopy. Shown below is the prism-type TIRF microscope. The basic physics principle of total internal reflection is to excite only a thin layer of ~100nm from surface. While the immobilized molecules are effectively excited by external light such as laser beam, the excitation beam does not reach the other part of the chamber for example labeled proteins in solution.

Biotinylated substrates are immobilized on the glass surface inside the glass chamber assembled as below (left). Effective solution exchange is executed via pipetting through the holes either manually or automatically.

Once all the excitation and emission optics are successully aligned, single molecules are observed through CCD camera (iXon, Andor Technology). Shown below are single molecules, ~400 of them. The image at left (donor channel) is identical to the one at right (acceptor), except by its color. Note that the CCD camera is color-blind and the image below is drawn with false color. It is a dichroic mirror which distinguishes the color and split the image into the two separate screens.

The fluorescence microscopy of single molecule has become even more powerful after it was integrated with force spectroscopy. Illustrated below are the combination with optical trapping (Ha, Hohng, Lang, Block, Chu labs), with magnetic trapping (Bustamante, Liphardt, and Hohng labs), and with laminar flow (Greene, Kowalczykowski, Xie, Mizuuchi labs). With the collaboration with Hohng lab, we fully utilize the optical and magnetic trapping methods together with fluorescence.

1) ‘Single Molecule Techniques: A Laboratory Manual’ (2008) (ed P. Selvin and T. Ha) Cold Spring Harbor Laboratory Press

2) C. Joo et al (2008) “Advances in Single-Molecule Fluorescence Methodology” Annual Review of Biochemistry, 77, 51-76