Two-dimensional electro-optical multiphoton microscopy

• Published in: Neurophotonics (Print) Vol. 11; no. 2; p. 025005
• Main Authors: Farinella, Deano M., Stanek, Samuel, Jayakumar, Harishankar, Newman, Zachary L., Gable, Jacob, Leger, James, Kerlin, Aaron
• Format: Journal Article
• Language: English
• Published: United States Society of Photo-Optical Instrumentation Engineers 01.04.2024; SPIE
• Subjects: Research Papers; electro-optical deflector; voltage imaging; imaging; multiphoton microscopy; in vivo imaging
• ISSN: 2329-423X; 2329-4248
• DOI: 10.1117/1.NPh.11.2.025005

The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging . To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast imaging of genetically encoded indicators with millisecond dynamics. The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals from ASAP3, a genetically encoded voltage indicator. Optimal KTN-EOD deflection of laser light with a central wavelength of 960 nm was obtained up to the highest average powers and pulse intensities tested (power: 350 mW; pulse duration: 118 fs). Up to 32 resolvable spots per line at a 560 kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a by field-of-view at over 10 kHz frame rate with lateral resolution. We demonstrate imaging of neurons expressing ASAP3 with high temporal resolution. We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging , providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.