Measurements of multiphoton action cross sections for multiphoton microscopy

Area of Science:

• Biophotonics
• Microscopy
• Quantum Mechanics

Background:

• Multi-photon microscopy offers advantages for deep tissue imaging.
• Longer excitation wavelengths are desirable for deeper tissue penetration.
• Understanding excitation cross sections is crucial for optimizing imaging parameters.

Purpose of the Study:

• To quantitatively measure two-, three-, and four-photon excitation action cross sections.
• To determine optimal repetition rates for 3-photon microscopy.
• To demonstrate the feasibility of 4-photon fluorescence microscopy at 1700 nm.

Main Methods:

• Measurement of excitation action cross sections for common fluorophores and fluorescent proteins.
• Utilizing excitation wavelengths of 800 nm, 1300 nm, and 1680 nm.
• In vivo 4-photon fluorescence microscopy of GFP-labeled microglia in mouse brain.

Main Results:

• Measured cross section values align with quantum mechanical predictions.
• Optimal repetition rate for deep tissue 3-photon microscopy identified as 1-2 MHz.
• Successful in vivo 4-photon fluorescence microscopy demonstrated at 1700 nm.

Conclusions:

• Quantitative cross section data guides microscopy optimization.
• 4-photon excitation enhances accessibility of fluorophores in the 1700 nm spectral window.
• This work supports advanced deep tissue imaging applications.