Construction of a high-NFOM multiphoton microscope with large-angle resonant raster scanning

• Published in: STAR protocols Vol. 3; no. 2; p. 101330
• Main Authors: Borah, Bhaskar Jyoti, Sun, Chi-Kuang
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.06.2022; Elsevier
• Subjects: Biotechnology and bioengineering; Cell Biology; Microscopy; Microscopy, Fluorescence, Multiphoton - methods; Protocol; Radionuclide Imaging; Software; Microscopy; Biotechnology and bioengineering; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101330

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). [Display omitted] •Critical guidelines to build a high-NFOM laser-scanning multiphoton optical microscope•An optimized optical design for large-angle resonant-galvo raster scanning•A simplified electronic design for high-speed raster scanning•A free-version of C++ based control and acquisition software (LASERaster+) Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.