Quantification of area and optical density of intracellular neuromelanin with Image J
Miquel Vila, Camille Guillard-Sirieix, Thais Cuadros
Abstract
Quantification of area and optical density of intracellular neuromelanin with Image J
Steps
Image Acquisition
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
Neuromelanin Quantification
Upload images at Image J software
Adjust canvas size at 1596x1198
Invert image
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
