Two-photon frequency division multiplexing for functional in vivo imaging: a feasibility study

Area of Science:

• Neuroscience
• Biophysics
• Optical Imaging

Background:

• High-speed amplitude modulation of femtosecond laser pulses enables multiplexing excitation beams.
• Two-photon microscopy is crucial for deep-tissue imaging and neural activity monitoring.

Purpose of the Study:

• To evaluate the utility of two-photon frequency-division multiplexing (2P-FDM) microscopy for recording calcium signals in brain tissue.
• To assess and mitigate cross-talk issues in frequency-multiplexed imaging.

Main Methods:

• Utilized high-speed amplitude modulation to tag multiple excitation beams with distinct frequencies.
• Applied phase information for signal alignment and recombination within regions of interest (ROIs).
• Conducted theoretical analysis, numerical simulations, and in vitro imaging experiments.

Main Results:

• 2P-FDM microscopy shows promise for recording average calcium signals from ROIs like neuronal cell bodies.
• Developed a phase-alignment procedure to narrow frequency detection windows and reduce noise.
• Demonstrated a >10-fold reduction in cross-talk between frequency channels.

Conclusions:

• Despite minor image quality trade-offs compared to conventional methods, 2P-FDM offers a viable approach for calcium imaging.
• The developed phase-alignment technique significantly enhances signal fidelity by reducing cross-talk.
• 2P-FDM microscopy is a promising tool for functional studies of brain activity.