Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation

Area of Science:

• Nonlinear optical microscopy
• Biophotonics
• Fluorescence spectroscopy

Background:

• Maximizing fluorescent yield is crucial for high-resolution nonlinear imaging in biological tissues.
• Understanding fluorophore emission dynamics under pulsed laser excitation is key to improving imaging sensitivity.

Purpose of the Study:

• To investigate the effect of laser excitation pulse phase on the emission rates of various fluorophores.
• To determine optimal pulse conditions for maximizing signal-to-noise ratio in nonlinear imaging.
• To explore potential photoprotective effects in quantum dot samples.

Main Methods:

• Experimentally manipulated the phase of femtosecond laser excitation pulses at the focal plane.
• Measured the total fluorescent yield from rhodamine B, a red fluorescent protein, and cadmium selenide (CdSe) quantum dots.
• Analyzed emission rates and signal-to-noise ratios under different pulse phase conditions.

Main Results:

• A transform-limited pulse condition consistently maximized the total fluorescent yield across all tested fluorophores.
• The optimized pulse phase ensured the highest achievable signal-to-noise ratio for imaging.
• Evidence of fluorescence antibleaching was observed in cadmium selenide (CdSe) quantum dot samples.

Conclusions:

• Laser pulse phase manipulation is a critical parameter for optimizing fluorescent yield in nonlinear imaging.
• Transform-limited pulses offer a robust method for maximizing signal in diverse fluorophore systems.
• Quantum dots exhibit promising photostability characteristics, potentially benefiting from phase-controlled excitation.