A last decade tremendous effort has been put in achieving single molecule detection and making it useful for molecular and cellular biology. Illustrated at right is one of the methods, single-molecule fluorescence. To achieve single molecule fluorescence detection, we immobilize substrates (depicted as a tall grass) on a glass surface via conjugation between neutravidin (rock) and biotin attached at the end of a substrate. We label our protein (depicted as a butterfly) with dyes (Cy3 as donor and Cy5 as acceptor). After we inject the labeled proteins into a glass chamber, we wait and see how the proteins interact with the individual substrates immobilized. When a protein is floating around, it is not visible (see below, TIRF microscopy). When it interacts with its immobilized substrate, a protein is effectively localized and the signal is detected in real time (typically 10-1000msec time resolution). To prevent a protein from adsorbing onto the glass surface, we coat the surface with polymer (PEG: polyethylene glycol) (depicted as short grasses)
Artist's impression of single-molecule fluorescence measurement
FRET (Forster Resonance Energy Transfer)
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. R0 of fluorescent dyes is typically 5-10 nm which distance is biologically very useful since the size of typical proteins is ~1 - 10 nm and that of RNA and DNA is also an order of nanometers in width.
FRET distance dependence
TIRF (Total Internal Reflection Fluorescence) Microscopy
Eliminating background signal is crucial in achieving single molecule detection. There are several imaging methods developed. The commonly used ones are confocal microscopy, prism-type TIRF microscopy, and 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 a laser beam, the excitation beam does not reach the other part of the chamber, for example, labeled proteins floating around in solution.
Biotinylated substrates are immobilized on a glass surface inside a glass chamber assembled as below (left). Solution exchange is carried out via pipetting through holes.
Single Molecule Detection
Single molecules are observed through a CCD camera (iXon, Andor Technology) and a CMOS camera (BSI Prime, Photometrics). 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 this CCD camera is color-blind and the image below is drawn in the false color scheme. It is a dichroic mirror which distinguishes the color and splits an image into two separate windows.
Courtesy of Timothy Blosser