Miniature two-photon microscopes, such as MINI2P, are powerful tools to monitor the activity of brain cells in freely moving animals. To image deep in the brain, the MINI2P can be coupled to gradient index (GRIN) lenses, which are implanted in the tissue and relay the imaging beam to the target region of interest. However, GRIN lenses have intrinsic optical aberrations, which severely degrade imaging spatial resolution especially in lateral portions of the field-of-view (FOV) leading to restricted imaging FOV. Here, we tested the hypothesis that coupling MINI2P with aberration corrected GRIN lenses results in improved spatial resolution and extended FOV during two-photon fluorescence imaging. We coupled the MINI2P with a cylindrical GRIN lens (GRIN length, 4.07 mm; GRIN cross section, 0.5 mm), in which we corrected aberrations using a micro-fabricated polymer lens. We found that the axial dimension of both the on-axis and off-axis point-spread-function was significantly improved in MINI2P coupled with corrected GRIN lenses compared to MINI2P coupled with uncorrected GRIN lenses. Moreover, using corrected GRIN lenses enabled MINI2P imaging with > 3-fold larger FOV radius compared to using uncorrected GRIN lenses. We confirmed extended FOV imaging when MINI2P was coupled to corrected GRIN lenses by performing two-photon imaging of thalamic neurons expressing a fluorescent indicator in fixed brain tissue.
Graded index (GRIN) lens-based microendoscopes are widely used to perform two-photon fluorescence microscopy in deep (> 1 mm) regions of highly scattering biological tissue, such as the mammalian brain. However, GRIN microendoscopes are limited by intrinsic aberrations which severely restrict the usable field-of-view (FOV). The effect of aberrations is particularly relevant in ultrathin (diameter < 500 μm) microendoscopes which allow a less invasive insertion of the optical probe into the brain tissue but which are characterized by relatively small imaging FOV. Currently, there are limited commercially available solutions to correct aberrations in these ultrathin microendoscopes because of the difficulty in fabricating corrective optics at the small spatial scale corresponding to the microendoscope diameter. Here, we report the development and application of a new approach to correct aberrations in GRIN microendoscopes using microfabricated polymeric lenses. Corrective optical elements were first designed using optical simulation software, then fabricated by two-photon lithography, and finally coupled with the GRIN lens to generate aberration-corrected microendoscopic probes. The method that we developed was applied to several types of GRIN lenses that differed in length and diameter, and corrected microendoscopes had up to 9 folds larger FOV compared to uncorrected probes. We put corrected microendoscopes to the test by performing high-resolution functional imaging of hundreds of hippocampal or thalamic cells expressing genetically encoded fluorescent indicators in the mouse brain in vivo.
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