Tripling the maximum imaging depth with third-harmonic generation microscopy.

Area of Science:

• Biomedical Optics
• Microscopy
• Tissue Imaging

Background:

• High-resolution, deep-tissue imaging is crucial for understanding tissue structures.
• Nonlinear imaging modalities, particularly three-photon microscopy (TPM), are advancing with longer excitation wavelengths.
• Challenges remain in achieving greater imaging depth in highly scattering and absorbing tissues.

Purpose of the Study:

• To investigate the potential of third-harmonic generation (THG) microscopy at 1552 nm for deep-tissue imaging.
• To compare the imaging depth of THG microscopy with two-photon microscopy (TPM).
• To assess and manage potential thermal damage during deep-tissue imaging.

Main Methods:

• Utilized third-harmonic generation (THG) microscopy with a 1552-nm excitation wavelength.
• Employed two-photon microscopy (TPM) with a 776-nm excitation wavelength for comparison.
• Conducted experimental, analytical, and Monte Carlo simulations for imaging depth and thermal analysis.
• Performed numerical simulations to determine depth-resolved temperature distribution and thermal management strategies.

Main Results:

• Achieved a threefold improvement in maximum imaging depth in ex vivo porcine vocal folds (420 μm vs. 140 μm).
• Reached an imaging depth of seven extinction lengths, nearly doubling previously reported normalized depths for three-photon modalities.
• Demonstrated a maximum temperature increase of approximately 2°C at the maximum imaging depth with effective laser shuttering (2τ).

Conclusions:

• THG microscopy at 1552 nm is a powerful modality for deep imaging in highly scattering and absorbing tissues.
• Effective thermal management ensures safety for deep-tissue imaging applications.
• This technique shows significant promise for imaging complex biological tissues like scarred vocal folds.