Improving Signal Levels in Intravital Multiphoton Microscopy using an Objective Correction Collar.

Area of Science:

• Biomedical optics
• Microscopy techniques
• In vivo imaging

Background:

• Multiphoton microscopy offers high-resolution deep tissue imaging.
• Imaging depth is limited by factors degrading illumination focus, such as spherical aberration.
• Spherical aberration particularly affects multiphoton fluorescence excitation due to its quadratic dependence on illumination intensity.

Purpose of the Study:

• To investigate the impact of spherical aberration on multiphoton microscopy.
• To evaluate methods for correcting spherical aberration in deep tissue imaging.
• To assess the improvement in signal levels achievable by aberration correction in living animal kidneys.

Main Methods:

• Examined spherical aberration in fixed and live kidney tissues using multiphoton microscopy.
• Evaluated spherical aberration via axial asymmetry in the point spread function.
• Adjusted the correction collar of a water immersion objective lens to introduce compensatory spherical aberration.

Main Results:

• Spherical aberration was identified as a limiting factor for deep imaging.
• Correction collar adjustment effectively corrected spherical aberration.
• Compensating for spherical aberration increased signal levels by up to 50% at depth in live animal kidneys.
• Reduced depth-dependence of signal levels was observed.

Conclusions:

• Spherical aberration significantly impacts multiphoton microscopy performance in biological tissues.
• Water immersion objective lens correction collars can effectively mitigate spherical aberration.
• Aberration correction enhances deep tissue imaging capabilities, particularly in vivo, by improving signal penetration and consistency.