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

Here we present FHIRM-TPM 3.0, a 2.6 g miniature two-photon microscope capable of multicolor deep-brain imaging in freely behaving mice. The system was integrated with a broadband anti-resonant hollow-core fiber featuring low transmission loss, minimal dispersion from 700 nm to 1,060 nm and high tolerance of laser power. By correcting chromatic and spherical aberrations and optimizing the fluorescence collection aperture, we achieved cortical neuronal imaging at depths exceeding 820 μm and, using a GRIN lens, hippocampal Ca²⁺ imaging at single dendritic spine resolution. Moreover, we engineered three interchangeable parfocal objectives, allowing for a tenfold scalable field of view up to 1 × 0.8 mm², with lateral resolutions ranging from 0.68 μm to 1.46 μm. By multicolor imaging at excitation wavelengths of 780 nm, 920 nm and 1,030 nm, we investigated mitochondrial and cytosolic Ca²⁺ activities relative to the deposition of amyloid plaques in the cortex of awake APP/PS1 transgenic mice. Thus, FHIRM-TPM 3.0 provides a versatile imaging system suitable for diverse brain imaging scenarios.