Quantification of area and optical density of intracellular neuromelanin with Image J

Scan sections using 20x objective (NA=0.8) with pre-set focusing and exposure parameters for optimal NM signal quality with an automated Slide Scanner Olympus (SLIDEVIEW VS200, Tokyo, Japan).

Acquire SNpc images with Qupath v0.5.0 software

Upload images at Image J software

Adjust canvas size at 1596x1198

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With the free hand selections, draft a neuromelanin-pigmented neuron (excluding the nucleus) and measure the optical density (pixel brightness) and the cell area

With the free hand selections, draft the neuromelanin pigment of the neuron and measure the optical density (pixel brightness) and the neuromelanin-occupied area

With the free hand selections, draft 15-25 non-pigmented neurons (excluding the nucleus) and measure the optical density and calculate mean

Normalize (i.e., subtract) the values of the neuromelanin pigmented neuron’s optical density with the mean value of the optical density of the non-pigmented neurons

Additional: calculate the percentage of occupied area diving the neuromelanin pigment area by the neuron’s area