TITLE: Fluorescent image acquisition and processing using Axiovert 200M microscope and ImageJ software
AUTHORS: Electra Brunialti, Alessandro Maria Villa, Paolo Ciana

[DESCRIPTION]
Fluorescent image acquisition and processing using Axiovert 200M microscope and ImageJ software to analyze morphological and dynamic changes of primary fluorescent microglia.

[STEPS]
SECTION: Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
1. Insert the cell culture plate in the microscope holder, and set chamber parameters: T 37°C and CO2 5%.
SECTION: Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
2. Using X20 magnification chose 20 random fields; 
SECTION: Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
3. Set exposition of the fluorescent channel to have a sharp image of microglia bodies and branches;
SECTION: Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
4. Records the live fluorescent microglia for 2 h taking a picture every 5 min.
SECTION: Step 1: Acquire live microglia images using Axiovert 200M microscope with dedicated software (AxioVision Rel 4.9, Zeiss)
5. Save the recorded file as a “.zvi”. 
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
6. Open ".zvi" file with Fiji software as hyperstack and tick the “split channel” option;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
7. close the bright field channel and start to elaborate the fluorescent channel (microglia);
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
8. subtract the background using “Process › Subtract Background”(identifier: legacy:ij.plugin.filter.BackgroundSubtracter);
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
9. 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;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
10. apply despeckle function “Process › Noise › Despeckle” (identifier:legacy:ij.plugin.filter.RankFilters("despeckle"));
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
11. apply smoothing function “Process › Smooth” (identifier: legacy:ij.plugin.filter.Filters("smooth"));
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
12. 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”;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
13. for each microglia select the area that contains the microglia in each time-frames using “Edit › Options › Roi Defaults” (identifier:legacy:ij.gui.RoiDefaultsDialog);
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
14. 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”;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
15. process “all images”;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
16. copy the data in an Excell file;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
17. repeat the steps from 13 to 16 for each microglia.
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
18. Among the "Shape Descriptors", keep the values of “Area”, “Solidity”, “FeretAngle”, “XM” and “YM”;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
19. 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;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
20. 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; 
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
21. calculate the median and the CV% of "Solidity" and "Area";
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
22. 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;
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
23. use the identified median as a threshold to cluster the cells in two groups (over or under the median);
SECTION: Step 2:  Elaborate the acquired images using Fiji software (ImageJ, NIH, version 2.0.0)
24. combine two parameters to generate four different clusters.