In vivo spatially-targeted retinal stimulation enabled by closed-loop focus adjustment

The precise spatial control of photoreceptor stimulation is essential for understanding changes in retinal ganglion cell (RGC) function in neurodegenerative diseases, such as glaucoma. We present a fundus camera-guided system for spatially targeted retinal stimulation featuring closed-loop focus adjustment with real-time imaging feedback. This system uses a 415 nm stimulation pathway that effectively drives both S- and M-cones in the mouse retina, given their overlapping short-wavelength sensitivity. To project a spatially-modulated pattern, a digital micromirror device (DMD) is integrated into a custom fundus imaging pathway, enabling micrometer-scale alignment between the stimulation pattern and retinal features. Focus correction is achieved by measuring a segment perpendicular to a retinal blood vessel and iteratively adjusting an electronically tunable lens (ETL) to minimize its full width at half maximum (FWHM). System performance was validated by positioning a CMOS sensor at the eye plane, confirming a projected pattern resolution of 1.6μm. Moreover, the modulation transfer function (MTF) was derived from a slanted-edge projection, achieving a 50% MTF of 35-40 cycles/mm. These results demonstrate the feasibility of cellular-resolution stimulation in vivo and establish a foundation for future investigations of RGC activity and functional alterations in retinal disease models.