Single-molecule detection using continuous wave excitation of two-photon fluorescence

Area of Science:

• Biophotonics
• Molecular Imaging
• Laser Physics

Background:

• Two-photon fluorescence (TPF) is crucial for biological imaging, but its application with continuous wave (cw) lasers is limited by perceived low excitation efficiency.
• Existing TPF imaging typically relies on pulsed lasers, restricting certain applications.

Purpose of the Study:

• To investigate the feasibility of achieving single-molecule TPF sensitivity using a cw infrared laser.
• To demonstrate the potential of cw lasers for advanced biological imaging techniques.

Main Methods:

• Direct excitation of fluorophores using an infrared (IR) cw laser, commonly used for optical trapping.
• Observation and analysis of discrete stepwise photobleaching in enhanced green fluorescent proteins (eGFPs).
• Single-molecule fluorescence intensity analysis and on-time distribution measurements.

Main Results:

• Achieved single-molecule fluorescence sensitivity with a cw IR laser.
• Observed discrete stepwise photobleaching, a hallmark of single-molecule events.
• Fluorescence intensity and on-time data confirmed cw laser-induced TPF at the single-molecule level.

Conclusions:

• Continuous wave lasers are capable of generating detectable two-photon fluorescence at the single-molecule level.
• This finding overcomes previous limitations and opens possibilities for single-molecule TPF imaging using cw lasers.
• Enables new applications in biological imaging where cw lasers are preferred or necessary.