Efficient Detection of Single DNA Fragments in Flowing Sample Streams by Two-Photon Fluorescence Excitation

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• 作者：Alan Van Orden ; Hong Cai ; Peter M. Goodwin ; and Richard A. Keller
• 刊名：Analytical Chemistry
• 出版年：1999
• 出版时间：June 1, 1999
• 年：1999
• 卷：71
• 期：11
• 页码：2108 - 2116
• 全文大小：110K
• 年卷期：v.71,no.11(June 1, 1999)
• ISSN：1520-6882

文摘

This paper reports the demonstration of efficient singlemolecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation(TPE) to detect single DNA fragments as small as 383base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNAcomplexes was accomplished using a mode-locked, 120fs pulse width Ti:sapphire laser operating at 810 nm.POPO-1 labeled DNA fragments of 1.1 kilobase pairs(kbp) and larger were sequentially detected in our flowcytometry system with a detection efficiency of nearly100%. The detection efficiency for the 383 bp DNAfragments was approximately 75%. We also demonstratethe ability to distinguish between different sized DNAfragments in a mixture by their individual fluorescenceburst sizes by TPE. These studies indicate that using TPEfor single molecule flow cytometry experiments lowers theintensity of the background radiation by approximately anorder of magnitude compared to one-photon excitation,due to the large separation between the excitation andemission wavelengths in TPE.