Jan 24, 2024
Quantification of area and optical density of intracellular neuromelanin with Image J
- Camille Guillard-Sirieix1,2,
- Thais Cuadros1,2,
- Miquel Vila1,2,3,4
- 1Neurodegenerative Diseases Research Group, Vall d’Hebron Research Institute (VHIR)-Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), 08035 Barcelona, Spain;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 3Institute of Neurosciences, Autonomous University of Barcelona (INc-UAB), Bellaterra, Barcelona, Spain;
- 4Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain
Protocol Citation: Camille Guillard-Sirieix, Thais Cuadros, Miquel Vila 2024. Quantification of area and optical density of intracellular neuromelanin with Image J. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxer1lpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2024
Last Modified: January 24, 2024
Protocol Integer ID: 94081
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020505
Abstract
Quantification of area and optical density of intracellular neuromelanin with Image J
Image Acquisition
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
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