Two-photon (2P) fluorescence imaging through gradient index (GRIN) lens-based endoscopes is fundamental to investigate the functional properties of neural populations in deep brain circuits. However, GRIN lenses have intrinsic optical aberrations, which severely degrade their imaging performance. GRIN aberrations decrease the signal-to-noise ratio (SNR) and spatial resolution of fluorescence signals, especially in lateral portions of the field-of-view (FOV), leading to restricted FOV and smaller number of recorded neurons. This is especially relevant for GRIN lenses of several millimeters in length, which are needed to reach the deeper regions of the rodent brain. We have previously demonstrated a novel method to enlarge the FOV and improve the spatial resolution of 2P microendoscopes based on GRIN lenses of length <4.1 mm (Antonini et al., 2020). However, previously developed microendoscopes were too short to reach the most ventral regions of the mouse brain. In this study, we combined optical simulations with fabrication of aspherical polymer microlenses through three-dimensional (3D) microprinting to correct for optical aberrations in long (length >6 mm) GRIN lens-based microendoscopes (diameter, 500 µm). Long corrected microendoscopes had improved spatial resolution, enabling imaging in significantly enlarged FOVs. Moreover, using synthetic calcium data we showed that aberration correction enabled detection of cells with higher SNR of fluorescent signals and decreased cross-contamination between neurons. Finally, we applied long corrected microendoscopes to perform large-scale and high-precision recordings of calcium signals in populations of neurons in the olfactory cortex, a brain region laying approximately 5 mm from the brain surface, of awake head-fixed mice. Long corrected microendoscopes are powerful new tools enabling population imaging with unprecedented large FOV and high spatial resolution in the most ventral regions of the mouse brain.

The structure of compound eyes in arthropods has been the subject of many studies, revealing important biological principles. Until recently, these studies were constrained by the two-dimensional nature of available ultrastructural data. By taking advantage of the novel three-dimensional ultrastructural dataset obtained using volume electron microscopy, we present the first cellular-level reconstruction of the whole compound eye of an insect, the miniaturized parasitoid wasp Megaphragma viggianii. The compound eye of the female M. viggianii consists of 29 ommatidia and contains 478 cells. Despite the almost anucleate brain, all cells of the compound eye contain nuclei. As in larger insects, the dorsal rim area of the eye in M. viggianii contains ommatidia that are believed to be specialized in polarized light detection as reflected in their corneal and retinal morphology. We report the presence of three ‘ectopic’ photoreceptors. Our results offer new insights into the miniaturization of compound eyes and scaling of sensory organs in general.