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Updated: Sep 25, 2025
Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering
Published on: July 6, 2019
Construction of a high-NFOM multiphoton microscope with large-angle resonant raster scanning
Bhaskar Jyoti Borah1, Chi-Kuang Sun1,2,3
1Department of Electrical Engineering and Graduate Institute of Photonics and Optoelectronics, National Taiwan University, Taipei 10617, Taiwan.
Abstract:
A resonant-scanning multiphoton optical microscope (MPM) with a millimeter-scale field-of-view (FOV) often encounters a poor Nyquist figure-of-merit (NFOM), leading to an aliasing effect owing to limited effective voxel-sampling rate. In this protocol, we provide a design guideline to enable high-NFOM MPM imaging while simultaneously securing a large FOV/digital-resolution ratio and a fast resonant raster-scanning speed. We further provide a free version of our custom acquisition software to assist with a smooth and easy construction process. For complete details on the use and execution of this protocol, please refer to Borah et al. (2021).
Insights
This study presents a design for multiphoton optical microscopes (MPM) that overcomes poor Nyquist figure-of-merit (NFOM) for clearer, faster imaging over a large field-of-view (FOV). A free software tool aids in building this high-performance microscopy system.
Area of Science:
- Biomedical Optics
- Microscopy Technology
- Image Processing
Background:
- Resonant-scanning multiphoton microscopy (MPM) often suffers from aliasing artifacts due to a low Nyquist figure-of-merit (NFOM).
- This limitation arises from an insufficient effective voxel-sampling rate, particularly in systems with a large field-of-view (FOV).
Purpose of the Study:
- To provide a design guideline for achieving high-NFOM MPM imaging.
- To enable simultaneous acquisition of large FOV/digital-resolution ratio and fast scanning speeds.
- To offer a free custom acquisition software to facilitate system construction.
Main Methods:
- Development of a design guideline for resonant-scanning MPM systems.
- Implementation of custom acquisition software for enhanced imaging control.
- Focus on optimizing voxel-sampling rate for improved image fidelity.
Main Results:
- Achieved high-NFOM MPM imaging capabilities.
- Successfully balanced large FOV/digital-resolution ratio with fast scanning speeds.
- Demonstrated a practical approach to constructing advanced MPM systems.
Conclusions:
- The presented design guideline and software enable high-performance MPM imaging.
- Aliasing effects can be mitigated, leading to improved image quality.
- This protocol facilitates the development of advanced microscopy tools for research.
