Correction of spherical aberration in multi-focal multiphoton microscopy with spatial light modulator

Area of Science:

• Biomedical optics
• Microscopy techniques
• Advanced imaging

Background:

• Deep imaging of thick biological samples is challenging due to light scattering and aberrations.
• Spherical aberration (SA) significantly degrades image quality and signal intensity in multiphoton microscopy.
• Existing methods often require compromises between imaging depth, resolution, and speed.

Purpose of the Study:

• To demonstrate the efficacy of multi-focal multiphoton microscopy (MMM) for high-quality deep imaging.
• To showcase the system's ability to correct for spherical aberration (SA) in thick samples.
• To evaluate the performance improvements of MMM over single-focal multiphoton microscopy (SMM).

Main Methods:

• Development of a multi-focal multiphoton microscopy (MMM) system utilizing a spatial light modulator.
• Implementation of wavefront modulation to correct for spherical aberration (SA) caused by refractive index mismatch.
• Comparative imaging of fluorescent beads and mouse brain vasculature using MMM and SMM.

Main Results:

• MMM achieved approximately 52 times higher fluorescence signal at depths up to 1100 μm with SA correction compared to uncorrected imaging.
• The scanning time for MMM was reduced by a factor of four relative to SMM.
• Improved fluorescence intensity and depth resolution were observed in MMM imaging of mouse brain blood vessels.

Conclusions:

• Multi-focal multiphoton microscopy (MMM) effectively overcomes limitations of deep tissue imaging.
• SA correction in MMM significantly enhances imaging quality and efficiency.
• MMM offers a promising advancement for high-resolution, deep-field microscopy in biological research.