Open-source modular field-programmable gate array system for two-photon mesoscope enabling multiarea, multidepth...

Area of Science:

• Neuroscience
• Biomedical Engineering
• Optical Imaging

Background:

• Large field-of-view (FOV) two-photon microscopy is crucial for mesoscale neural recordings but faces limitations in temporal resolution due to long scan times.
• Existing systems often lack modularity and fluorescence-lifetime imaging capabilities, hindering broader accessibility for advanced neuroscience research.

Purpose of the Study:

• To develop and validate an open-source, modular platform combining field-programmable gate array (FPGA) and circular delay-path (CDP) modules.
• To enable high-throughput, multiarea, multidepth mesoscale two-photon imaging and large-FOV two-photon fluorescence lifetime imaging (2p-FLIM).

Main Methods:

• An FPGA system was built to digitize photomultiplier signals at 3.2 GS/s.
• A CDP module was integrated for temporal multiplexing of excitation across four focal planes, with FPGA handling demultiplexing and image reconstruction.
• 2p-FLIM was implemented on the same FPGA + CDP platform.

Main Results:

• The system achieved simultaneous recording of over 10,000 neurons across bilateral dorsal cortex at up to four depths.
• Demonstrated large-FOV 2p-FLIM capabilities.
• All hardware and software are open-source and compatible with existing two-photon microscopes.

Conclusions:

• The modular, open-source FPGA + CDP system significantly increases throughput for large-FOV two-photon imaging.
• It adds fluorescence lifetime contrast without requiring specialized lasers, broadening access to mesoscale neural measurements.
• Facilitates multiscale in vivo studies and diverse biomedical applications.