Vascular Density, Vascular Length Density and Diameter analysis of Blood Vessels using ImageJ Fiji

[@Zezezt]

Hello, nice and very useful video. I have a question: if you want to know the total value in the image, do you set ROI as the total of the image? because you choose some vessels but not al from the image

[@nrttaye4033]

Hello thanks for reaching out. In this video i used a particular selection area as you rightly pointed out. using the rectangular selection tool any large/small areas can be defined. when comparing between different images just make sure to keep the selection area the same. for example selection the entire image etc.

[@Zezezt]

@nrttaye4033 thank you a lot 😃😃

[@IkbalK.]

I am having some doubts of my data. Do I need to set scale before preforming this? I feel like my measurements are wrong. May I contact you?

[@nrttaye4033]

Hello, I think you can set scales. Have a look at this publication onlinelibrary.wiley.com/doi/10.1111/srt.12634 let me know if this helps.

[@nematuq]

Your videos have been very helpful. Can you please make one video/demo for segmentation and quantifying morphological features of endothelial junctions e.g VE-cadherin. I can share some images of such staining.

[@nrttaye4033]

Hello, thank you so much. I could try making a video on that. It would be great if you can donate some images for the tutorial.

[@merveemen3838]

Hi, thanks for this informative video. I would like to count number of fenestra in vessel sample. However, when I set the threshold, the fenestra is not seen. How could I solve that problem?

[@nrttaye4033]

Hi, Welcome. I am not sure if this plugins will help in counting the fenestra. Have you tried this way...change to 8 bit, threshold, analyze particles and count the numbers? Let me know if this works.

[@merveemen3838]

@@nrttaye4033 Hi, thank you for the response. Yes, I tried but when I set the threshold, the fenestra is not seen and not detectable. That's why analyze particles and count number did not work.

[@nrttaye4033]

sometimes when the fenestra color matches with the background color such issues can come up. If there is a slight change in color, a) a background corrrection or b) a weka segmentation can be performed (kzbin.info/www/bejne/qp6zo2h_rMRsjrs) Could you try this out? If it still does not work, send me a sample image and i could try the troubleshooting.

[@merveemen3838]

@@nrttaye4033 Hi, thank you so much for the response. Unfortunately, it does not work. If you could give me an e-mail address, I could send you.

[@nrttaye4033]

here is the email address [e-mail]

[@utaminoor2610]

Hi @nrtTAYE, your video is very helpful. But there's some problem in my MAC while I'm trying to install vascular density into FIJI. it says "Java.io.FileNotFOundException:/private bla bla bla" what should I do? I really need this vascular density features. Or can I use it in Image J only? how to measure vascular density from image J? Thank you so much!

[@nrttaye4033]

Hello, could you please check if the Fiji version is version 1.47g or higher (latest version is preferred )? I am checking in ImageJ and I will get back to you if this works.

[@nrttaye4033]

Good news. this process can be done in ImageJ too. please follow the same protocol for analysis as in Fiji. Local thickness plugins may not be present in ImageJ. It can be downloaded from this website www.optinav.info/Local_Thickness.htm All the best and let me know if it worked.

[@utaminoor2610]

Hi, okay I will try. Thank you so much!

[@utaminoor2610]

Hi@@nrttaye4033 , my FIJI version is "FIJI for Mac OS X", I don7t know whether it's the same version as 1.47 or not

[@utaminoor2610]

Hello@@nrttaye4033 , is it because I don't have JAVA on my Mac?

[@TheTran-yz9ic]

Currently I don't have Geometry to Distance Map. Can you tell me where I can download it? pls. thanks

[@nrttaye4033]

hello here is the site to download local thickness www.optinav.info/Local_Thickness.htm

[@divyagoyal8062]

Hi i liked the content. Thank you for the information But i have some problem I have two images : one image is having less number of vessels and the other is having more number. Although the vessel density calculated by plugin was low in the image having high number of vessels. Why so ?.. I am not getting

[@nrttaye4033]

Hello. Welcome. The density outcome may be affected on two conditions. 1) in the auto threshold please check on "White objects on black background", and 2) image pixel sizes are same for both the images. If image pixel sizes are different, then it can be adjusted using ImageJ. Here is a tutorial to change pixel sizes kzbin.info/www/bejne/qH-4nmOop897hMk Let me know if that worked at your end.

[@divyagoyal8062]

No actually with in same picture also I am facing this issue.. in small rounded vessels I am getting high vascular density . Can I share the picture?

[@divyagoyal8062]

In set measurements it is automatically set on area fraction when I am Tracing the vessel

[@nrttaye4033]

@@divyagoyal8062 sure. i will try to troubleshoot it

[@pratibhak95]

Hello..Very nice and informative video. i have a one question can we do angiogenesis analysis of CAM using this plugin? for example no. of blood vessel or vascular density count? please reply

[@nrttaye4033]

Hello, thanks and you are welcome. You can definitely use this for vascular density analysis. For vessel/branch length and numbers please have a look at these videos kzbin.info/www/bejne/rGa4g3SHfsSfhac kzbin.info/www/bejne/qYKviqKArcyIec0 kzbin.info/www/bejne/nJTUf6iqr7dqfJY

[@pratibhak95]

okay i will try to do by this method. but my mexican hat filter is not generating images like yours before 5 months ago when i saw all your videos that time i have done the whole analysis like you showed. but today when i trying to do the analysis the threshold 255 not generating the EDT images. Can you please guide me...Thank You..! can you please perform the above steps and check whether its a software problem or its something else. please rply

[@nrttaye4033]

Hello, I tried the mexican hat filter and i can confirm that it is working. Could you try changing the threshold value? depending on the image you may also need to select inverse case in the edt. let me know if that worked or i could troubleshoot it if you could send me a sample image.

[@pratibhak95]

Thank you for reply..😊 my problem was solved. But I have one request please make video on Angiotool software because no one actually made video and no one knows how to fo analysis using Angiotool. I will share you some images of CAM so it will be easy for you to make video. Please tell me how to share images of CAM?

[@nrttaye4033]

That would be great. I will get back to know once I am ready with the Angiotool software.

[@PatriciaNerydesiqueira]

hello, could you tell me if this plugin can be used to measure filaments? Is there already a more direct video published for this purpose? In this case, the images that I need to analyze have other bodies along with the filaments, but I just need to know the length of the filaments. thanks for the help :)

[@nrttaye4033]

hello, thanks for reaching out. Please check these videos kzbin.info/www/bejne/mGS9Y6dsZ85setU and kzbin.info/www/bejne/eYGVcmCAqZdqb8U

[@PatriciaNerydesiqueira]

@@nrttaye4033 Hello, thank you very much for the second video, I hadn't seen it yet. It will help me a lot :) Do you also have a video in which you exclude foreign bodies that are along with the filaments? I need to exclude them so that imagej only considers the filamentous ones.

[@nrttaye4033]

If the filament color is different from the forein bodies, then yes it is possible to first segment the filaments out and then perform the analysis. Here is the link to the video kzbin.info/www/bejne/qp6zo2h_rMRsjrs

[@PatriciaNerydesiqueira]

@@nrttaye4033 wow, you can't even imagine how much you're helping me

[@nrttaye4033]

Thanks and welcome. I am here to help. All the best with your analysis.

[@kelvynkennedy7580]

Where can I find the distance value in mm in my image? Thank you so much!

[@nrttaye4033]

That would be from the scale bar of your image.

[@kelvynkennedy7580]

@@nrttaye4033 In this case, would it be possible to convert pixels to mm? For example, 312 pixels = 82.55 mm? Thank you for your videos, they are very useful for analyzing my research data

[@nrttaye4033]

Hello, this formula is used for conversion mm = ( pixels X 25.4 ) / DPI. You can find this formula online

[@maninluv]

May I ask what is the unit for the vascular density results?

[@nrttaye4033]

Hello, vascular density here is calculated as vascular density = vessel area/total area X 100%. The unit of vessel area is pixels. Scale bar option can be used to set your own unit of measurement.

[@yamaha3859]

how did u know the distance in milimiter is 812?

[@nrttaye4033]

Hello, the pixel value is being converted to mm

[@vertikapathak9873]

How do you know the distance in mm?

[@nrttaye4033]

the distance to mm can be calculated using the scale bar in the image.

[@TanTan-ch3vq]

where is the 82.36mm coming from? thanks

[@nrttaye4033]

Hello, it is the width of the image from left to right in mm.

[@TanTan-ch3vq]

Thanks so much

[@nrttaye4033]

@@TanTan-ch3vq You are welcome