Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue.

Area of Science:

• Biomedical imaging
• Fluorescence microscopy
• Biophotonics

Background:

• Autofluorescence from biological samples and fixatives limits sensitivity in fluorescence imaging.
• High probe concentrations are often needed to overcome autofluorescence, which is not always feasible.
• Traditional time-gated detection suppresses autofluorescence but sacrifices probe signal.

Purpose of the Study:

• To develop a method for enhancing the signal of long-lived fluorescence probes.
• To improve signal-to-background ratio in fluorescence imaging without increasing background noise.
• To increase detection sensitivity for cellular and tissue imaging.

Main Methods:

• Utilized controllable bursts of closely spaced laser excitation pulses.
• Employed a commercial confocal microscopy system with a laser diode and time-correlated single photon counting (TCSPC) detection.
• Applied pulsed laser excitation to a long-lived Ruthenium (Ru)-based probe with a 380 ns lifetime.

Main Results:

• Achieved a many-fold increase in the intensity of a long-lived probe without increasing background fluorescence.
• Enhanced the signal of a Ru-based probe by nearly an order of magnitude.
• Demonstrated that using 10 pulses in a burst increased the Ru signal almost 10-fold.

Conclusions:

• The pulsed laser excitation technique effectively enhances long-lived probe signals.
• This method significantly improves detection sensitivity by overcoming autofluorescence limitations.
• The approach offers a practical solution for sensitive fluorescence imaging in biological samples.