Question of generating thresholded image

[Nan]

Dear all,

I have multi-photon microscopy(MPM)  stack images of epidermis that I would like to process with Image J to quantify the melanin. The two-photon excited fluorescent(TPEF) image was processed with Image J by, first crop out the ROI for each picture, and then, converted into binary images. However, during converting, the threshold value is automatically set, how can I adjust it manually and apply it on my images? After converting into binary image, I would like to apply this binary image as a mask on the original image, how to achieve this?(As shown in images below)

(a,c) raw TPEF images  (b,d) thresholded TPEF images shown in (a,c).  

In my case there’s an article(J. Biomed. Opt. 20(6), 066005 (Jun 12, 2015). doi:10.1117/1.JBO.20.6.066005, Page 4) that used this approach, but I could not repeat it. Is there any way around this?

Thank you very much for your help!

Best regards,

Nan

[Krs5]

Dear Nan,

If you use Image > Adjust > Threshold you get a menu that allows you to adjust the threshold settings manually.

To create the mask you can use Analyze > Analyse particle and select "Add to Manager". This will add all selections to the ROI Manager what allows you to overlay them on the original image using "Show All" from the ROI Manager menu while the original image is selected.

Best wishes

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
Centre for Core Biotechnology Services
University of Leicester
http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif


-----Original Message-----
From: Nan [mailto:[hidden email]]
Sent: 22 December 2016 07:22
To: [hidden email]
Subject: Question of generating thresholded image

Dear all,

I have multi-photon microscopy(MPM)  stack images of epidermis that I would like to process with Image J to quantify the melanin. The two-photon excited fluorescent(TPEF) image was processed with Image J by, first crop out the ROI for each picture, and then, converted into binary images. However, during converting, the threshold value is automatically set, how can I adjust it manually and apply it on my images? After converting into binary image, I would like to apply this binary image as a mask on the original image, how to achieve this?(As shown in images below)

(a,c) raw TPEF images  (b,d) thresholded TPEF images shown in (a,c).  

In my case there’s an article(J. Biomed. Opt. 20(6), 066005 (Jun 12, 2015). doi:10.1117/1.JBO.20.6.066005, Page 4) that used this approach, but I could not repeat it. Is there any way around this?

Thank you very much for your help!

Best regards,
Nan

page4image40096.jpeg (299K) <http://imagej.1557.x6.nabble.com/attachment/5017805/0/page4image40096.jpeg>




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[Nan]

Dear Dr. Straatman,

Thank you very much for your reply.
Indeed, what you told me worked well by overlay the mask on the original image, but how to extract the image of mask position from the original image? Is that even possible?
However, there's still problem with adjusting the threshold.
1. For example, in the article, the auther use a threshold corresponding to the mean value of non-zero pixels in the image. How can the non-zero pixel intensity be obtained? (By using the Analyze --> Measure function of ImageJ it's possible to get the mean intensity of the image, but not the non-zero pixel intensity)
2. Attached Image1.bmp shows the approach used in this article. They indicated to set the pixel with intensity values larger than the threshold to 1, and the rest to 0. How would this be achieved in manual adjustment of threshold?

Best regards,
Nan


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