(a) Schematic illustration of the optical path within the GRIN lens. The ray diagram shows telecentricity at the image plane. (b) Optical setup of the LNTS system. Obj, objective lens; L1-4, optical lenses; DM, dichroic mirror; Si-PMT, silicon photomultiplier tube; F, emission filter. (c) Conventional wide-field imaging of fluorescent beads of 4 µm in diameter. (d) LNTS imaging of fluorescent beads of 4 µm in diameter. (e) Point spread function (PSF) quantification. The bead imaging was repeated independently 3 times with similar results.

(a) Wide-field image of microglia in the brain of a live Cx3cr1-eGFP mouse acquired through a GRIN lens of 0.5 mm in diameter and 6.7 mm in length. The in vivo Cx3cr1-eGFP imaging was repeated independently in 2 mice with similar results. (b) LNTS image through the same GRIN lens. (c) Zoomed-in view of the ROIs in a and b. (d) Normalized intensity profiles across the dashed lines in c for image contrast comparison.

(a) Large FOV calcium image acquired through a GRIN lens of 0.6 mm in diameter and 8.4 mm in length in a Tbr1-creER × Ai162D transgenic mouse. (b) Raw calcium fluorescence traces from six selected ROIs over a 30-minute imaging session. (c) Quantification of fluorescence intensity baseline during early (0–5 min) and late (26–30 min) periods of the session. Each dot and gray line in the center of the chart represents the comparison of the same neuron. The violin plot illustrates the data distribution, with the solid black line indicating the median, and the dashed lines above and below indicating the upper and lower quartiles. The Mean ± SEM is shown next to the violin plot. P = 0.0348. Two-tailed paired t-test, t = 2.874, df = 5, n = 6 ROIs from 1 mouse. The in vivo photobleaching imaging was repeated independently in 5 mice with similar results. (d) Experimental timeline illustrating the key stages: surgical implantation, recovery, and behavioral testing. ITI: inter-trial interval. (e) Heatmaps of normalized ΔF/F0 traces for all 15 ROIs on Day 31 (pre-training) and Day 33 (post-training). The in vivo calcium GCaMP6s imaging was repeated independently in 3 mice with similar results. (f) Calcium traces from four ROIs before and after training.

Simulated wavefront through a GRIN lens of 0.5 mm in diameter 6.7 mm in length.

(a) Beam path configuration. The exit location is 238 µm from the center of the FOV (b) GRIN lens induced aberration as a function of input NA.

Comparison of wide-field imaging and the LNTS method for imaging through a GRIN lens of 0.5 mm in diameter 6.7 mm in length.

(a-c) Wide-field imaging with a detection NA of 0.4, 0.2 and 0.05, respectively. (d) LNTS based imaging of the same sample. The illumination energy over the 500 µm FOV required for the beads in the middle to have ∼1,000 emission photons collected is shown for each image.

In vivo wide-field imaging of microglia through a GRIN lens of 0.5 mm in diameter 6.7 mm in length.

Images of microglia through the same GRIN lens at a detection NA of 0.4 and 0.05, respectively.

In vivo comparison of LNTS and ultra-large flat FOV two-photon system for imaging neurons through a GRIN lens of 0.6 mm in diameter 8.4 mm in length.

(a, b) Images of neurons acquired by LNTS and by ultra-large flat FOV two-photon system, respectively.

Non-sequential ray tracing through a GRIN lens of 0.5 mm in diameter 6.7 mm in length and a uniform H-ZLAF78B glass light pipe of identical dimensions.

(a) The radiant intensity distribution of the light collected by the GRIN lens. The signal source is a uniform cylinder of 0.5 mm in diameter and 0.1 mm in length located right under the GRIN lens facet. Among the isotropic emissions, 15.7% is captured through the GRIN lens. (b) The radiant intensity distribution of the light collected by the H-ZLAF78B glass light pipe under the same condition. The collection efficiency is 27.6%.