Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation

This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two- photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes uas accomplished using a mode-l ocked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 lab eled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our non cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximat ely 75%. We also demonstrate the ability to distinguish between different s ized DNA fragments in a mixture by their individual fluorescence burst size s by TPE, These studies indicate that using TPE for single molecule now cyt ometry experiments lowers the intensity of the background radiation try app roximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE .