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CELLPROFILER ANALYST MACHINE LEARNING TOOLS MANUAL

In a small-scale pre-clinical project with only 2 experimental groups, an investigator would be required to manually quantify RGCs in approximately 1000 images, necessitating at least 48 h of effort.

CELLPROFILER ANALYST MACHINE LEARNING TOOLS MANUAL

While the immunolabeling method is technically simpler than fluorogold labeling, manual quantification of immunolabeled murine RGCs is onerously time-consuming. The specificity of the β-III-tubulin antibody has been confirmed by colocalization of β-III-tubulin staining with fluorogold-labeled RGCs (Huihui et al., 2011). An alternative quantification method of RGC viability may be carried out with immunolabeling of RGCs in retinal flatmounts using an RGC-specific antibody directed against β-III-tubulin, which labels the RGC somata and nerve fiber extensions. However, this method is technically challenging, requiring intracranial surgery and, in a glaucomatous retina, retrograde labeling can result in the labeling of retinal microglia in addition to RGCs, due to the phagocytosis of degenerating RGCs (Peinado-Ramon et al., 1996, Thanos, 1991a, Thanos, 1991b). In a healthy, non-diseased retina, fluorogold retrograde labeling is a specific and accurate method to label and automatically quantify RGCs (Buckingham et al., 2008, Danias et al., 2003). Various techniques for visualizing and quantifying RGCs have been reported, including retrograde labeling and immunolabeling (Buckingham et al., 2008, Huihui et al., 2011). The ability to specifically identify and accurately count RGCs is essential to assess the death or survival of RGCs in models of the disease.

Animal models of glaucoma that simulate the optic neuropathy observed in human disease facilitate the elucidation of possible mechanisms of RGC loss and enable researchers to develop and evaluate neuroprotective therapies. While there are many forms of glaucoma, all are associated with an optic neuropathy characterized by the loss of retinal ganglion cells (RGCs) and their axons, resulting in optic nerve degeneration and irreversible vision loss. Glaucoma is the leading cause of irreversible blindness worldwide, affecting an estimated 60 million people (Tham et al., 2014).

Cellprofiler analyst machine learning tools