Real-time frame-registered resonant fluorescence scanning laser ophthalmoscopy for quantifying static and dynamic...

Fluorescence labeling offers excellent contrast for cell imaging within living mouse eyes. High-speed, high-resolution imaging with a large field of view (FOV) is always desirable. A high-speed scanning laser ophthalmoscopy (SLO) system has been developed, equipped with real-time desinusoiding correction and frame registration for fluorescence imaging of mouse retinas. Precise calibration using a standard raster grid compensates for scanning hysteresis and image lateral distortion caused by the sinusoidal motion of the resonant scanner. More importantly, a strip-based registration method has been developed to correct frame distortions induced by breathing and pupil drift, enabling effective real-time and post-processing frame averaging. This system captures images at 1024 × 1024 pixels, with a temporal resolution of 16 Hz and a lateral resolution of 1.8 µm, and a FOV of up to 50° (35 µm/degree), which facilitates accurate measurement of both static and dynamic cellular properties, such as microglia cell density, diameter, spacing, and blood hemodynamics, within living mouse eyes.