A. Van Orden et al., Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation, ANALYT CHEM, 71(11), 1999, pp. 2108-2116
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
.