Spiral scan and cylindrical deconvolution to maximize image volume and contrast of multiphoton GRIN microendoscopy

Optical microscopy provides sub-cellular and high-speed imaging to capture neuron dynamics in a living brain, but its penetration depth is limited by tissue scattering. Multiphoton excitation improves the depth to over 1 mm, while combining with a gradient refractive index (GRIN) lens enables centimeter penetration with minimal invasiveness. However, the system performance is compromised due to the intrinsic optical aberrations of GRIN lenses, which severely reduce the contrast, spatial resolution, and effective field of view (FoV). To address this issue, we developed a 3D aberration correction approach for GRIN lenses by combining spiral scanning with cylindrical deconvolution. This method leverages the cylindrical symmetry of GRIN-induced aberrations and incorporates the spatially varying point-spread function (PSF) across the imaging volume. Radially adaptive excitation implemented through spiral scanning expanded the usable FoV diameter by nearly 2-fold and achieved 30- and 10-fold improvement, respectively, in peripheral signal intensity and signal-to-noise ratio (SNR) compared to conventional raster scanning with uniform excitation, while cylindrical deconvolution improved spatial resolution by up to 3.5-fold. We further validated this method through 3D imaging of neuronal structures, demonstrating enhanced effective volume size and a 2-fold improvement in neuronal SNR. These results indicate that the spiral scanning and algorithm-augmented GRIN 2PF system is promising toward resolving structure/functional connectomics in deep brain regions.