Current biomarkers of ALS used by clinicians to help care for ALS patients have many drawbacks that may include cost, accessibility, and invasiveness of procedures. An imaging biomarker that is non-invasive, non-contact, and widely accessible would be a welcome addition to the current armamentarium of clinical tools used to diagnose, monitor and treat ALS patients. Axonal spheroids are a pathological hallmark of axonal injury and are found in ALS brain and spinal cord motor neurons. We have recently discovered the presence of axon spheroids in the retinas of ALS patients, and believe that the eye may be an important window into ALS axon injury through the detection and monitoring of these retinal spheroids. Thus, the primary goal of this proposal is to develop a method to reliably image retinal spheroids for clinical use in ALS patients to support and advance their care with attention to quality-of-life considerations. A multi-disciplinary will determine their optical features, understand their molecular composition, define an imaging signature based on combined optical and morphometric properties, and validate imaging models to estimate ALS disease severity. The widely available clinical eye imaging devices such as scanning laser ophthalmoscope (SLO) and optical coherence tomography (OCT) are Health Canada approved and will be used to define a surrogate retinal marker of spheroids. These will be validated neuropathologically using molecular markers for spheroids to ensure that the optical signature obtained matches the profile of interest before proceeding to train, test and validate the imaging signature of spheroids using machine learning techniques. This project will establish the groundwork for a safe, non-contact, non-invasive, rapid and relatively inexpensive biomarker for ALS. This imaging test for ALS would be highly accessible in academic and most ophthalmology offices. In combination with other biochemical, functional and neuroimaging biomarkers, we are hopeful that this eye biomarker will advance care management and facilitate clinical trials to find a cure for ALS.