Hi, I’m new to Image J and not sure I’m adjusting the threshold etc correctly. When I press OK after the particle analyzer, it comes up with a box telling me “A threshold has not been set using the Image->Adjust->Threshold tool or the setThreshold(min,max) macro function. I think it is having trouble defining the edge, my setting are currently 30; 50; 4; 10000. Sorry
Hey, there's no need to apologize! Asking questions helps us learn and grow together. I am here to help, so feel free to ask anything anytime. the last value is very large. try changing to a triple digit number. you may also adjust the threhold value. alternatively use the image, adjust and threhold to get an idea of the threshold value (usually between 10-100 in may images). just check if the image you have is in 8 bit format before starting. let me know if it worked, else you could send me a sample image and i can guide you with the parameters. Hope this helps.
Hello, thanks for the question. Here is the original description from the plugins website (link in description). Do let me know if you need more info. method: You can choose between a variance based method and a method based on find edges. Note that for find edges the parameters variance-filter-radius and threshold are not taken into account. Use for example the parameters 20, 1, 4 and 999999 on the example image for the variance case. variance filter radius: The radius of the variance filter that is applied to separate the zone occupied by tissue from the empty zone. The radius must be big enough so that the variance due to the tissue plays a role compared to the variance of the noise in the image. Note that the calculation time becomes longer with a bigger radius. threshold: The image resulting from the variance filter is converted to a mask by applying the given threshold. If the input images are 16bit the threshold 1 will probably work. radius open: This operation will close holes in the tissue. It must be big enough to close small holes in the tissue and small enough to not close the area of the wound in later images. min. size: Minimum size from which on the wound is taken into account. This excludes small remaining holes in the tissue. ignore spatial calibration: If checked the measurements will be in pixel otherwise the spatial calibration of the image if any is used.
Hello, thanks for the question. The initial steps for the stack images are same as single images. Once the scratch area is calculated, the output stack images with outline needs to be flattened. In the ROI manager click on Flatten, click "yes" on the next pop-up flatten all images? To save these images as individual images, click on Image, stacks, stack to images. Now the individual images can be saved separately with the outline. Go to file and save. Let me know if it worked.
Hello thanks for watching my videos. I just tried with a stack image and it is successful. The steps are same for either for a single image or stack images. Let me know if it works at your end.
Hello, I'm new to ImageJ and not good with it yet! I followed all of the steps in the video, but when I left click on "m" at the end it just comes up with an ROI manager - do you know what I am doing wrong?
Hello, thanks for watching my videos. This is not uncommon. It usually happens when ImageJ is unable to apply the parameters set in the "wound healing tool options" ie, "m". you may change the values particulalry the threshold and the min size and then try again. These values greatly vary according to the image brightness/constrast and the wound size/gaps in between and other parameters. the "radius" values may also need changes accordingly. Let me know if these changes still does not work.
Hi thank you for your great video. I have a problem at the very beginning steps, i have cropped my circular picture taken from microscope, turned into square inserted into image j. Then 8bit-> find edges-> smooth However when i click fill holes it fills the whole picture and if i dont do this after the scratch assay using steps from your video , it mistakenly circles around my cells instead of the scratch area. Could you please fell me what i should do
Hello, you are welcome. Could you please send me a sample of your cropped image? i can get back to you after troubleshooting, it is a bit difficult to answer without seeing the image.
Hi thank you for this wonderful macros. I have the very similar problem as one of the user and however I change the values it is not working. Once I give values in analyze particle and click ok, it is giving message as 'A threshold has not been set using the Image->Adjust->Threshold tool or the set Threshold(min, max) macro function'. My parameters after clicking m button are as follows i. Method: Variance ii. Variance filer radius: 10 iii. Threshold: 20 iv. Radius close: 4 v. Min. Size: 5000 As you suggested for another user I tried changing the last value to triple digit numbers but nothing changed. I am unable to measure these images. Please help! Thanks.
Hello, thanks for reaching out. Try changing the threshold value ranging from 50-150 in the wound healing tool options window and then run the macro again. The threshold value depends on the contrast of the cells and its background. sometimes the image dimensions are in pixels or inches and radius close value needs to be changed accordingly. If still it does not work out, send me a sample image to troubleshoot it.
Hello, I installed the macro and followed all the steps. However, when I clicked "m", the "Wound healing tool Options" box did not appear. Where might I be going wrong? I use Windows. Thank you.
Hi, thanks for this video. I have on issue with this method is at the last step when click on m, it calculates area of empty space that is wound but simultaneously it gives additional areas and yellow marks in between cells also. How I can remove/ avoid these areas which i dont want. Thanks!
hello, you are welcome. please try changing the parameters in the wound healing tool options especially the "min size" and "radius close". The parameters in the video is used with the unit inches. if you have unit of images in pixels, then need to change accordingly. let me know if that worked, esle you could send me a sample image to troubleshoot.
@@nrttaye4033 Hi thanks for your quick response. the unit of images are in pixels in my case. Would it be possible to change them into inches? I tried to change the "min size" and radius close but not successful in analyzing the data.
Hello, here is the protocol. The seeding density varies with the cell types, (MDA-MB 231 cells) is 5 X 10^5 cells in a 6 well plate. Mitomycin treatment was performed next day and a sterile 10 or 20 ul pipette tip was used to generate the scratch. Washed off the floating cells with complete medium once and scratch images were taken at 10X after 0 and 12 hours.
Hello, thanks for the comment. It is possible to measure the multiple outlines in an image. Are you planning to measure multiple pockets of scratch (cells are in patches with spaces between them) instead of one big scratch? Let me know if I got your question correct. Yes it is possible using ImageJ if this is the query you have raised.
hello, thanks for the question. From the analyze particle window once can get the idea of the image sizing either in pixels/inches and then define accordingly in the wound healing tool options. please check if these parameter are chosen. Analyze, analyze particles, and check display results and add to manager. Again go to analyze, set measurements, and check Area. try running the analysis again according to the image size. if you can share the image i could specifically troubleshoot. let me know if this helped.
Hi. Great tutorial! I have a problem- I am unable to save the image as overlay in the last step. My image with the outline is not getting saved. Could you help me out with this?
the output image with outline needs to be flattened before saving. In the ROI manager click on Flatten. To save the image Go to file and save as tiff format. Let me know if it worked. alternately, the overlay image can also be saved by clicking on image, overlay and flattened and then save as tiff.
Hello Sandhya, if you already have the existing macro installed from github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/Wound-Healing-Tool please delete it from the macro folder first. Re-installing the macro again may help. If your operating system is Windows the macro installation process may be slightly different. if needed please visit this link for macro installation demo and let me know if the m is still missing. Happy to help. ua-cam.com/video/l4BMB3pi9Oc/v-deo.html
![FIJI (ImageJ): Analysis of Scratch Assays, Wound Healing & Cell Migration [Wound Healing Size Tool]](/img/n.gif)
Hi, I’m new to Image J and not sure I’m adjusting the threshold etc correctly. When I press OK after the particle analyzer, it comes up with a box telling me “A threshold has not been set using the Image->Adjust->Threshold tool or the setThreshold(min,max) macro function. I think it is having trouble defining the edge, my setting are currently 30; 50; 4; 10000. Sorry
Hey, there's no need to apologize! Asking questions helps us learn and grow together. I am here to help, so feel free to ask anything anytime. the last value is very large. try changing to a triple digit number. you may also adjust the threhold value. alternatively use the image, adjust and threhold to get an idea of the threshold value (usually between 10-100 in may images). just check if the image you have is in 8 bit format before starting. let me know if it worked, else you could send me a sample image and i can guide you with the parameters. Hope this helps.
Thank you👍🏻
What does it mean, change the threshold according the image type? How should I know what to choose for it?
Hello, thanks for the question. Here is the original description from the plugins website (link in description). Do let me know if you need more info.
method: You can choose between a variance based method and a method based on find edges. Note that for find edges the parameters variance-filter-radius and threshold are not taken into account. Use for example the parameters 20, 1, 4 and 999999 on the example image for the variance case.
variance filter radius: The radius of the variance filter that is applied to separate the zone occupied by tissue from the empty zone. The radius must be big enough so that the variance due to the tissue plays a role compared to the variance of the noise in the image. Note that the calculation time becomes longer with a bigger radius.
threshold: The image resulting from the variance filter is converted to a mask by applying the given threshold. If the input images are 16bit the threshold 1 will probably work.
radius open: This operation will close holes in the tissue. It must be big enough to close small holes in the tissue and small enough to not close the area of the wound in later images.
min. size: Minimum size from which on the wound is taken into account. This excludes small remaining holes in the tissue.
ignore spatial calibration: If checked the measurements will be in pixel otherwise the spatial calibration of the image if any is used.
Many thanks🙂
Hello, How do I do this for a stack and save the images with the scratch area outline for all of them?
Hello, thanks for the question. The initial steps for the stack images are same as single images. Once the scratch area is calculated, the output stack images with outline needs to be flattened. In the ROI manager click on Flatten, click "yes" on the next pop-up flatten all images? To save these images as individual images, click on Image, stacks, stack to images. Now the individual images can be saved separately with the outline. Go to file and save. Let me know if it worked.
Hello, how can i set these for stack images ?
Hello thanks for watching my videos. I just tried with a stack image and it is successful. The steps are same for either for a single image or stack images. Let me know if it works at your end.
Hello, I'm new to ImageJ and not good with it yet! I followed all of the steps in the video, but when I left click on "m" at the end it just comes up with an ROI manager - do you know what I am doing wrong?
Hello, thanks for watching my videos. This is not uncommon. It usually happens when ImageJ is unable to apply the parameters set in the "wound healing tool options" ie, "m". you may change the values particulalry the threshold and the min size and then try again. These values greatly vary according to the image brightness/constrast and the wound size/gaps in between and other parameters. the "radius" values may also need changes accordingly. Let me know if these changes still does not work.
Hi thank you for your great video. I have a problem at the very beginning steps, i have cropped my circular picture taken from microscope, turned into square inserted into image j. Then 8bit-> find edges-> smooth
However when i click fill holes it fills the whole picture and if i dont do this after the scratch assay using steps from your video , it mistakenly circles around my cells instead of the scratch area. Could you please fell me what i should do
Hello, you are welcome. Could you please send me a sample of your cropped image? i can get back to you after troubleshooting, it is a bit difficult to answer without seeing the image.
@@nrttaye4033 thank you so much for your response. I was able to solve it🙏🏻
Hi thank you for this wonderful macros. I have the very similar problem as one of the user and however I change the values it is not working. Once I give values in analyze particle and click ok, it is giving message as 'A threshold has not been set using the Image->Adjust->Threshold tool or the set Threshold(min, max) macro function'. My parameters after clicking m button are as follows
i. Method: Variance
ii. Variance filer radius: 10
iii. Threshold: 20
iv. Radius close: 4
v. Min. Size: 5000
As you suggested for another user I tried changing the last value to triple digit numbers but nothing changed.
I am unable to measure these images. Please help! Thanks.
Hello, thanks for reaching out. Try changing the threshold value ranging from 50-150 in the wound healing tool options window and then run the macro again. The threshold value depends on the contrast of the cells and its background. sometimes the image dimensions are in pixels or inches and radius close value needs to be changed accordingly. If still it does not work out, send me a sample image to troubleshoot it.
@@nrttaye4033 Hi What's your email ID to send a sample image? I am still not getting
Hello,
I installed the macro and followed all the steps. However, when I clicked "m", the "Wound healing tool Options" box did not appear. Where might I be going wrong? I use Windows. Thank you.
Could you try making a right click (for windows) on "m"?
@@nrttaye4033 oh Lord. Thank you very much. It worked.
Hi, thanks for this video. I have on issue with this method is at the last step when click on m, it calculates area of empty space that is wound but simultaneously it gives additional areas and yellow marks in between cells also. How I can remove/ avoid these areas which i dont want. Thanks!
hello, you are welcome. please try changing the parameters in the wound healing tool options especially the "min size" and "radius close". The parameters in the video is used with the unit inches. if you have unit of images in pixels, then need to change accordingly. let me know if that worked, esle you could send me a sample image to troubleshoot.
@@nrttaye4033 Hi thanks for your quick response. the unit of images are in pixels in my case. Would it be possible to change them into inches? I tried to change the "min size" and radius close but not successful in analyzing the data.
@@nrttaye4033 Also, is there any option to upload the sample picture for you?
please mail it to... and i will try to troubleshoot it
@@nrttaye4033 image sent. Thanks so much for help!
Did you use 4x or 2x lens for taking scratch images?
Hello, here is the protocol. The seeding density varies with the cell types, (MDA-MB 231 cells) is 5 X 10^5 cells in a 6 well plate. Mitomycin treatment was performed next day and a sterile 10 or 20 ul pipette tip was used to generate the scratch. Washed off the floating cells with complete medium once and scratch images were taken at 10X after 0 and 12 hours.
Hey! Great informative tutorial mate, thanks. My requirement is to represent multiple outlines overlaid in a graph. Is there any method to that?
Hello, thanks for the comment. It is possible to measure the multiple outlines in an image. Are you planning to measure multiple pockets of scratch (cells are in patches with spaces between them) instead of one big scratch? Let me know if I got your question correct. Yes it is possible using ImageJ if this is the query you have raised.
When the area runnig to close appears various spaces between . How to put color liners in this areas simultanearly?
Is Set scale need to be filled? because i don't see any results after completing the procedure
hello, thanks for the question. From the analyze particle window once can get the idea of the image sizing either in pixels/inches and then define accordingly in the wound healing tool options. please check if these parameter are chosen. Analyze, analyze particles, and check display results and add to manager. Again go to analyze, set measurements, and check Area. try running the analysis again according to the image size. if you can share the image i could specifically troubleshoot. let me know if this helped.
Hi. Great tutorial! I have a problem- I am unable to save the image as overlay in the last step. My image with the outline is not getting saved. Could you help me out with this?
the output image with outline needs to be flattened before saving. In the ROI manager click on Flatten. To save the image Go to file and save as tiff format. Let me know if it worked. alternately, the overlay image can also be saved by clicking on image, overlay and flattened and then save as tiff.
@@nrttaye4033 oh wow it worked!! Thanks a ton. I had been struggling with it since last night. Have a great day!
glad it worked. you are welcome
Pls can you send me the protocol you used to induce scratch and type of microscope used to take images?
Hello, thanks for reaching out. I will reply to your comment with the protocol on Monday. Thanks for watching the video.
I don't find a m I.e method option in my toolbar. Wr do I find it ?
Hello Sandhya, if you already have the existing macro installed from github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/Wound-Healing-Tool please delete it from the macro folder first. Re-installing the macro again may help. If your operating system is Windows the macro installation process may be slightly different. if needed please visit this link for macro installation demo and let me know if the m is still missing. Happy to help. ua-cam.com/video/l4BMB3pi9Oc/v-deo.html