Large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and...

Area of Science:

• Neuroscience
• Biomedical Imaging
• Optical Microscopy

Background:

• Understanding complex brain functions requires high spatiotemporal resolution imaging of dynamic behaviors and neurochemical concentrations.
• Two-photon microscopy (TPM) offers high resolution and deep tissue penetration for in vivo brain imaging.
• Conventional TPM is limited by a small field-of-view (FOV), a trade-off between imaging area and temporal resolution, and insufficient data from intensity recording.

Purpose of the Study:

• To introduce large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter, quantitative brain function imaging.
• To overcome the FOV limitations and temporal resolution trade-offs of conventional TPM.
• To enable simultaneous, high-throughput imaging of multiple brain regions with high spatiotemporal resolution.

Main Methods:

• Extended FOV beyond commercial objective limits using adaptive optics.
• Implemented a temporal multiplexing system for simultaneous, flexible two-region imaging.
• Developed a field-programmable gate array (FPGA) module for high-throughput, dual-region fluorescence signal demultiplexing and FLIM.

Main Results:

• Achieved a large FOV of 3 × 3 mm², uniform 0.7 μm lateral resolution, and FLIM throughput up to 15.73 megapixels/s.
• Demonstrated simultaneous monitoring of neural activities across multiple cortical areas.
• Successfully recorded synchronous neurovascular coupling, microglial response to injury, and quantitative calcium concentrations in vivo.

Conclusions:

• LD-2P-FLIM significantly enhances in vivo brain imaging capabilities by expanding FOV and temporal resolution.
• The developed system enables multiparameter, quantitative analysis of neural dynamics and neurochemistry across large neuronal populations.
• This advanced imaging technique provides a powerful tool for investigating complex brain functions under physiological and pathological conditions.