An adaptive approach for uniform scanning in multifocal multiphoton microscopy with a spatial light modulator

Area of Science:

• Optics and Photonics
• Biomedical Imaging
• Microscopy

Background:

• Multifocal multiphoton microscopy (MMM) enables deeper tissue imaging but requires precise control of excitation.
• Achieving uniform fluorescence intensity across multiple focal spots is a significant challenge in MMM.

Purpose of the Study:

• To develop and demonstrate a high-quality method for generating uniform multiple fluorescence spots (MFS).
• To enable uniform laser scanning calibration for multifocal multiphoton microscopy (MMM) prior to live tissue observation.

Main Methods:

• Utilized a spatial light modulator (SLM) to generate multiple fluorescence spots (MFS).
• Employed an iterative computer-generated hologram (CGH) correction method using coefficients to optimize fluorescence intensity distribution.
• Calibrated the system using a uniform dye solution to improve spot uniformity.

Main Results:

• Demonstrated significant improvement in the uniformity of a 10 × 10 grid of MFS.
• Successfully applied the calibration method to observe fluorescent polystyrene beads and guinea pig gastric gland tissue using two-photon excitation.

Conclusions:

• The proposed SLM-based MFS generation and CGH correction method provides effective calibration for MMM.
• This technique enhances the quality and reliability of multifocal multiphoton microscopy for biological sample imaging.