FIJI (ImageJ): Analysis of Scratch Assays, Wound Healing & Cell Migration [Wound Healing Size Tool]

[@comfortwilliams6131]

Dr. Thank you for this video. They are indeed helpful for my image analysis. I appreciate you.

[@johanna.m.dela-cruz]

Hi Comfort. I’m glad for the opportunity to help you.

[@samarsamy9890]

Thank you for your efforts dr ❤️🌸

[@johanna.m.dela-cruz]

It’s my pleasure.

[@claudiavanessafloresmedero7293]

Gracias! Me fue muy útil

[@johanna.m.dela-cruz]

I’m happy to help, @Claudia.

[@perrikaty2209]

Thank you for your video! I followed your instructions and it worked for my bright field images. However, no matter what parameter I put in, it didn't work for my fluorescence images as it appeared "Index (0) is outside of the 0-1 range in line 284 \ roiManager (“select” , 0 ;". Could you please let me know how to fix this? Thanks!

[@johanna.m.dela-cruz]

Hi @perrikaty2209. Are you working with time-lapse fluorescence stacks? Your variance window radius and threshold are probably too high.

[@job506]

Hi Johanna, it's the usual amazing you here again! Thank you extremely well. Please, is there a way I can save the image files with the main trace lines retained? Best!

[@johanna.m.dela-cruz]

Hello @JOB. Thanks for your nice comments. If you just want to burn the overlay on your image, you can go to Image>Overlay>Flatten. An RGB image with the overlay will be created. Or you can try right-clicking on the trace lines and select Add to Manager. From the Manager, you can Flatten the image. Saving the image with the overlay can also be done by just saving as a .tif. Hope this helps.

[@job506]

@@johanna.m.dela-cruz Hallo Johanna, it worked absolutely. Many thanks.

[@job506]

Furthermore, is there a way to change the color and width of the trace lines?

[@johanna.m.dela-cruz]

@@job506 You can change the trace line color and width in the ROI Manager. Highlight the ROI trace then go to Properties. You can also colorize the area where cells are not found by the Fill command under Properties.

[@job506]

@@johanna.m.dela-cruz Thumbs up for you, please. 👏✌💪

[@ravichandrans4564]

Hey, Thank you soo much for the clarification. ❤❤....I have a doubt, What value I can use to calculate the wound closure percentage. I don't know how to create a graph with these values....Kindly help me.

[@johanna.m.dela-cruz]

Hi! The area fraction in the Results table determines the wound closure percentage. These values can be found in the Results table.

[@edagultekin2566]

Hello, thank you for this video. I am running this code in my batch process to apply it to multiple images. How can I obtain a list of complete measurements with labels? I receive single results one by one. THank younin advance.

[@johanna.m.dela-cruz]

Hi there! Thanks for watching. Are you working with time lapse images or single images? The Results table that shows up after analysis of a stack is what needs to be saved, right? The one which gives measurements from each frame? Have you tested your code… on a single file?

[@khinaye5999]

How can I change the unit of width from pixel to micrometers

[@johanna.m.dela-cruz]

Hi! You need to scale your image. You can check out this tutorial: FIJI (ImageJ): Scaling & Spatial Calibration kzbin.info/www/bejne/g3TEpIqDnJ2ta6c Thanks for watching.

[@tremmble]

Hello Johanna, Thank you for the video! I have one error that I am dealing with (Error: Index (0) is outside of the 0--1 range) which I think occurs when the wound closes completely. Is there a way I can still output a results table with a 0 for the area fraction for all images in a sequence where the wound is closed?

[@johanna.m.dela-cruz]

Hi @tremmble. What if you cut out the last slice(s) of the stack where the wound closes? Do you still get the error?

[@rushilshanbhog9043]

Really helpful video. I was wondering if you don't have a time-lapse stack file and just two jpg files, is there any way to use the stacking tool in imageJ to see the relation between area and time?

[@johanna.m.dela-cruz]

Hi @rushilshanbhog9043. As long as the dimensions of your images are the same, you can put them together using Image>Stacks>Images to Stacks. Then go to Image>Properties, Use 1 as Slices and 2 as Frames --- this way, ImageJ will think that your stack is a time series.

[@putusixtinadewandari4043]

hi, i want to ask, how to make multiple circle analysis in one image?

[@johanna.m.dela-cruz]

Hi @putusixtinadewandari4043. Do you mean your image is made up of several circular objects? Have you tried thresholding?

[@putusixtinadewandari4043]

@johanna.m.dela-cruz not yet, how to do it? can you explain, please🙏

[@jessyhell3541]

Oi Johanna, tudo bem? O meu problema é que a extensão do Wound healing size não está instalando. Sempre que tento de acordo com a descrição do seu vídeo, aparece a mensagem: "There are no images open in line 5 Var width = [getWidth];

[@johanna.m.dela-cruz]

Hola @jessyhell3541. Sorry I don't speak Spanish. You need to have an image open first before running the tool.

[@NatashaAley]

Thank you for this! When I use my images I get several ROI's appear but only 1 measurement. How do I know which ROI matches to the measurement?

[@johanna.m.dela-cruz]

Hi @Natasha Aley. Unfortunately the plugin works in such a way that ROIs in a frame are overwritten by the ROIs in the next frame. The Results table, however, will show the overall Area, Area franction and width measurements for combined ROIs in a frame. So if your image has 50 frames, you will have 50 overall regions.

[@smitakapoor6527]

Hello Johanna I have one query while following all the steps as mentioned by you but the area of wound is not getting selected rather it is showing some other selected region than the scratch region what might be the reason. kindly resolve this. Thank you in advance.

[@johanna.m.dela-cruz]

Hi smita. You might have to change the radius of the variance filter and/or the threshold value. These parameters are image-dependent, so try playing with other values to see what works best. Check 2:58 of the video on how these values will affect your measurement.

[@pattarapoungcho5277]

Hello thank you for nice video and helpful. I would like to download wound healing tool. But I visited that site you posted I can’t find that plug in on zip file. Only have pdf file. Thank you

[@johanna.m.dela-cruz]

Hi @pattara poungcho. Thanks for watching my video. If you click on the link in my Description, you can download the Wound healing size tool by scrolling down to the new version: Wound_healing_size_tool_updated . You can then save the link as a zipped file under the macros folder of ImageJ (or Fiji app). Save in the toolsets subfolder. If you unzip this, you will get a pdf (manual) and an ijm file. The latter is the toolset. When you open FIJI, click on Plugins>Macros>Install and then select the ijm file. Hope this helps.

[@pattarapoungcho5277]

@@johanna.m.dela-cruz Thank you very much. I try to do that as you wrote me but imageJ say "There are no image open in line 5 var width =; How can I do? Thank you

[@johanna.m.dela-cruz]

@@pattarapoungcho5277 did you open an image first before you called the tool? If you’re still having a problem, can you send me a copy of your image so I can test it out?

[@pattarapoungcho5277]

@@johanna.m.dela-cruz I can do it now. Thank you very much for helping me. This video was really helpful.

[@job506]

In addition, once I re-open ImageJ software where will I find the installed Wound Healing Size Tool plugin?

[@johanna.m.dela-cruz]

Hello again. If you read the description below the video title, the instructions are there. :)

[@job506]

@@johanna.m.dela-cruz 🤣😍 😘Hearty gratitude!

[@suganiamalarchinapayan2075]

Hi how to save the images?

[@johanna.m.dela-cruz]

Hello. You can burn the overlay on your image, by going to Image> Overlay> Flatten. An RGB image with the overlay will be created. Or you can try right-clicking on the trace lines and select Add to Manager. From the ROI Manager, you can Flatten the image. Saving the image with the overlay can also be done by just saving as a .tif. Hope this helps.