Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.
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