Single-Molecule Imaging With a Custom-Built Fluorescence Microscope

An apparatus for single-molecule imaging was constructed and used to examine nanomolar solutions of Bodipy Texas Red in water. The illumination source for this microscope is a synchronously pumped dye laser operating at a wavelength of 585 nm. The beam can easily be switched to give total internal reflection or wide field epi-illumination excitation of the sample. The instrument also has the capability of being used for single-molecule detection by confocal epiillumination and for fluorescence correlation spectroscopy. Movies of single molecules were analyzed using custom software written in Matlab. Molecules stuck to the surface revealed fluctuations in intensity, or blinking, for both forms of illumination. Possible causes for the fluctuations are discussed.