Multimodal-capable, multiphoton miniaturized microscope for mice brain imaging

A multiphoton, multimodal miniaturized microscope for mice brain imaging is developed. The optical design to provide a compact and lightweight probe uses small lenses, of less than 4mm diameter, selected for performance using ray-tracing software. The microscope is designed to maintain diffraction-limited resolution for imaging reporters GCAMP6 and eYFP or performing near-infra-red reflection confocal microscopy. A Mai-Tai femtosecond laser is used to provide a 920nm pulse laser, an Insight X3 laser to provide a 960nm pulse laser, and a separate 785nm continuous wave (CW) laser to support NIR reflection with optical sectioning capability with a high signal- to-noise ratio. The miniaturized system was tested as a benchtop prototype using a reflection target, green fluorescent protein, and enhanced yellow fluorescent protein. The resolution for all three wavelengths is less than 2 mu m. A customized parametric resonance electrostatic MEMS scanner provided the beam scanning of 1.8mm in diameter, resulting in a 350 mu m by 350 mu m field of view with a numerical aperture of 0.42, at a working distance of about 350 mu m. A 4 mu m core single-mode fiber and a photodiode collect the reflection beam in the confocal mode. Two dichroic mirrors out of the probe are used to combine all three beams toward the probe. Two dichroic mirrors are used for two emission wavelength separations.