Commencing as a challenging problem in physics with the first detection of a single fluorescent molecule in condensed phase at temperatures of liquid helium single molecule fluorescence detection has diversified into a collection of methods applied in various scientific disciplines.
With the advent of ultra sensitive detectors and optical instrumentation and by combination with confocal and TIRFM techniques single molecule detection developed into a feasible approach in life science. Decisive for this adaptability is the possibility to detect single molecule fluorescence at room temperature in solution (e.g. FCS) or on surface membranes of even living cells (TIRFM).

The following experiment conducted on an inverse microscope using the Olympus UAPO150xO/TIRFM objective is an example of the many applications for TIRFM single molecule fluorescence detection:
Single stranded RNA hybridised to a complementary biotinylated DNA, which was immobilised on a BSA-Biotin-Streptavidin coated cover glass. The RNA was mono-labelled with Cy3. Imaging of single molecules was confirmed by single step photo bleaching of the dye. Emission intensity plotted versus time decays immediately after bleaching a single dye molecule (ROIs 2-5), contrary to a group of fluorescent molecules whose emission would decrease in a multistep exponential manner (ROI 1).

Figure 1: Fluorescence intensity (colour coded. Circles mark five regions of interest (ROI). Each ROI (except of ROI 1) contains one single fluorescent molecule as verified by single step photobleaching: See movie with fluorescence intensity recorded over time (to download the film click on figure 1) and corresponding emission intensity curves plotted vs. time for the selected ROIs (Figure 2, bottom of the page).

 

 

Data courtesy of Jesper Donsmark, University Leiden, NL