Nov 24, 2022
Fluorescent image acquisition and processing using Axiovert 200M microscope and ImageJ software
- Electra Brunialti1,
- Alessandro Maria Villa1,
- Paolo Ciana1
- 1Department of Health Sciences, University of Milan, Milan, Italy
External link: https://doi.org/10.3390/cells12030343
Protocol Citation: Electra Brunialti, Alessandro Maria Villa, Paolo Ciana 2022. Fluorescent image acquisition and processing using Axiovert 200M microscope and ImageJ software. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1o98plr2/v1
Manuscript citation:
Brunialti E, Villa A, Toffoli M, Pozo SLD, Rizzi N, Meda C, Maggi A, Schapira AHV, Ciana P, Sex-Specific Microglial Responses to Glucocerebrosidase Inhibition: Relevance to GBA1-Linked Parkinson’s Disease. Cells 12(3). doi: 10.3390/cells12030343
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: November 24, 2022
Last Modified: November 24, 2022
Protocol Integer ID: 73221
Funders Acknowledgement:
The work was supported by the EU Joint Programme - Neurodegenerative Disease Research (JPND) project GBA-PaCTS, 01ED2005B
Abstract
Fluorescent image acquisition and processing using Axiovert 200M microscope and ImageJ software to analyze morphological and dynamic changes of primary fluorescent microglia.
Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
Insert the cell culture plate in the microscope holder, and set chamber parameters: T 37°C and CO2 5%.
Using X20 magnification chose 20 random fields;
Set exposition of the fluorescent channel to have a sharp image of microglia bodies and branches;
Records the live fluorescent microglia for 2 h taking a picture every 5 min.
Save the recorded file as a “.zvi”.
Step 2: Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
Step 2: Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
Open ".zvi" file with Fiji software as hyperstack and tick the “split channel” option;
close the bright field channel and start to elaborate the fluorescent channel (microglia);
subtract the background using “Process › Subtract Background”(identifier: legacy:ij.plugin.filter.BackgroundSubtracter);
defined threshold (foreground) that corresponds to green fluorescent objects using “Image › Adjust › Threshold” (identifier: legacy:ij.plugin.frame.ThresholdAdjuster). Keep the threshold consistent between acquisitions;
apply despeckle function “Process › Noise › Despeckle” (identifier:legacy:ij.plugin.filter.RankFilters("despeckle"));
apply smoothing function “Process › Smooth” (identifier: legacy:ij.plugin.filter.Filters("smooth"));
set the measurement: “Analyze › Set Measurements” (identifier: legacy:ij.plugin.filter.Analyzer("set")), and tick “Area”, “Center of Mass”, “Feret’s Diameter” and “Shape Descriptors”;
for each microglia select the area that contains the microglia in each time-frames using “Edit › Options › Roi Defaults” (identifier:legacy:ij.gui.RoiDefaultsDialog);
run analyze particles “Analyze › Analyze Particles” (identifier:legacy:ij.plugin.filter.ParticleAnalyzer), set size (micron^2):130-infinity, circularity: 0.00-1.00; tick “display results”;
process “all images”;
copy the data in an Excell file;
repeat the steps from 13 to 16 for each microglia.
Among the "Shape Descriptors", keep the values of “Area”, “Solidity”, “FeretAngle”, “XM” and “YM”;
to calculate the distance covered by the cell during the time-lapse use the coordinates of the center of mass and sum the distance covered in each time frame assuming that the distance between frames corresponds to the cathetus of a right triangle made by X-axis and Y-axis displacement;
to calculate the rotation sum the “FeretAngle” taking into account that it is the angle among Ferret’s diameter and parallel line to the cell contour only on x-axis;
calculate the median and the CV% of "Solidity" and "Area";
to perform the cluster analysis use each parameter obtained from the analysis; use the values of the vehicle and treated cells and identify the median parameter for the experiment;
use the identified median as a threshold to cluster the cells in two groups (over or under the median);
combine two parameters to generate four different clusters.