Photon Counting Histogram for One-Photon Excitation

An advance that counts! A method is presented for analyzing single‐molecule behavior using one‐photon excitation of fluorescence. It is based on the photon counting histogram (PCH) procedure that has enjoyed much success for two‐photon excitation, but has not previously been applied to one‐photon excitation. This corrected PCH model is able to resolve fluorescent species with different degrees of brightness, for example, a mixture of tetramethylrhodamine‐5′‐maleimide and Cy3‐maleimide (see graph). Because one‐photon excitation is often much easier to do than two‐photon excitation, this method has the potential to be widely used and represents an important advance in the field of single‐molecule spectroscopy.