A versatile miniature two-photon microscope enabling multicolor deep-brain imaging

Area of Science:

• Neuroscience
• Biomedical Engineering
• Microscopy Technology

Background:

• Deep-brain imaging in freely behaving animals is crucial for understanding neural circuits.
• Existing two-photon microscopy systems face limitations in size, flexibility, and imaging depth.
• Multicolor imaging is essential for dissecting complex cellular and molecular processes in the brain.

Purpose of the Study:

• To introduce FHIRM-TPM 3.0, a novel, compact two-photon microscope for advanced in vivo brain imaging.
• To demonstrate the system's capability for multicolor deep-brain imaging in freely behaving mice.
• To showcase the system's versatility and high resolution for various neuroscience research applications.

Main Methods:

• Integration of a miniature two-photon microscope with a broadband anti-resonant hollow-core fiber.
• Correction of optical aberrations and optimization of fluorescence collection for deep tissue penetration.
• Engineering of interchangeable objectives to achieve scalable fields of view and high lateral resolution.
• Utilizing multicolor excitation wavelengths (780, 920, 1030 nm) for simultaneous cellular activity monitoring.

Main Results:

• Achieved cortical neuronal imaging at depths exceeding 820 μm.
• Enabled hippocampal Ca2+ imaging at single dendritic spine resolution using a GRIN lens.
• Provided a tenfold scalable field of view (up to 1 × 0.8 mm²) with resolutions from 0.68 μm to 1.46 μm.
• Successfully investigated mitochondrial and cytosolic Ca2+ activities relative to amyloid plaques in APP/PS1 mice.

Conclusions:

• FHIRM-TPM 3.0 is a versatile and powerful tool for multicolor deep-brain imaging in neuroscience research.
• The system's miniaturization and advanced optical design facilitate in vivo studies in freely behaving subjects.
• It enables high-resolution, deep-tissue imaging, advancing the study of neurological diseases and brain function.