Fiji Assignment Help!

[alexwest99]

Hi everyone,

New to the community. I have a college project that requires me to use FIJI and ‘any plotting software’ to plot responses of neurons. This is way out of my depth as psychology is my speciality. Its now due tomorrow and im totally freaking out as I have no idea how to do it and if I don’t I won’t graduate. Is there anyone out there who would be able to help me? I’d even be willing to pay!

Attached is a screenshot of the first set of questions and also the tiff file!

GCaMP6_Responses_10stimuli.tiff

Q1.png

[CellKai]

Hi @alexwest99 , and welcome to the forum.

to receive more detailed help, could you also share the image on which the analysis should be done?
You might need to upload it to your google drive or dropbox and share the link here.

[alexwest99]

Thank you i’ve added them above! totally clueless

[CellKai]

Hi,
thanks for the image. Here are some instructions to hopefully get you started:

• download an install FIJI (Fiji is just ImageJ) here: Fiji/Downloads - ImageJ
• open Fiji, then open your image using File > open
  • you can zoom in and out of the image using ctrl + mousewheel or pressing the + and - buttons on your keyboard
  • note that the image will open and has a slider on the bottom that lets you flip through the time-laps
• in the toolbar, click on the third icon from the left (Polygon selection), mark the outline of cell1 and press “t” on your keyboard, then repeat the same for cell2. You can see the corresponding X and Y coordinates in the bottom of the Fiji toolbar, just underneath the “Polygon selection”, when you hover with your mouse over the image.
  • alternatively, I did this quickly as an example. Download RoiSet.zip (537 Bytes) and drag & drop the zip file onto Fiji.
  • the “ROI manager” window will open. There, tick “show all”
• in Analyze > Set Measurements, tick “Mean gray value” and press OK
• In the ROI manager window, click on More > Multi Measure, and tick “Measure all 637 slices” and also “One row per slice”, then press OK. The results-table with the mean intensities for cell1 and cell 2 will open.
• in the “Results”-window, you can go to File > save as to save the results as a text file so you can open it in Excel
• alternatively, in the “Results”-window, got to Results > plot and select
  • X: —, Y: Mean (cell1) and click OK. The “Plot of Results” window will open
• in the Plot window, got to Data > add from Table and select X: —, Y: Mean (cell2) , then press OK
  • you can add a legend to the plot using More > Legend
  • you can increase the quality of the Plot window using More > High resolution plot
• you can save the image of the plot by clicking on it, then using File > save as >
  Tiff

I hope this helps, and good luck with your assignment!

[biovoxxel (Biovoxxel (Jan Brocher))]

Alternatively to the suggestion from @CellKai,

automatically reading in the cell bodies might work with the following macro (at least on the provided image).

To use the macro do the following:

1. get Fiji as descibed by @CellKai
2. press [Shift] + N (now the script editor window should open)
3. paste the macro below in that window (make sure in the “Language” menu "IJ1Macro is selected.

originalImage = getTitle();
run("Select None");
run("Duplicate...", "duplicate");
duplicate = getTitle();
run("Gaussian Blur...", "sigma=1 stack");
run("Z Project...", "projection=[Max Intensity]");
projection = getTitle();
close(duplicate);
setOption("ScaleConversions", true);
run("8-bit");
run("Subtract Background...", "rolling=15");
run("Auto Threshold", "method=IsoData white");
run("Analyze Particles...", "size=10-Infinity pixel show=Nothing clear add");
close(projection);
selectWindow(originalImage);
roiManager("Select", 0);
run("Plot Z-axis Profile");

4. open your image stack
5. press the run button in the script editor
6. in the resulting plot window activate the [Live] button (turns red)
7. select your image (to activate it)
8. select the individual ROIs (the numbers in the ROI Manager and you should see the profile changing.

In addition, if you need the values you can then in the next step go to:
→Analyze →Set Measurements and activate the checkboxes related to intensity (Mean would be enough) as shown below.

Then go in the ROI Manager to >>More and Multi Measure

specify the popup dialog as shown

and press Ok and you get all values for all slices and ROIs

[alexwest99]

@biovoxxel @CellKai You guys are absolutely amazing thank you so much I’ve actually managed to answer the first question now… maybe I won’t need to drop out haha!

I dont think this is using Fiji but do you guys happen to know where to begin with this Question?