Dear fellows i have images of meniscus sections stained with Safranin-O-fast green. I need to quantify the images for Safranin-O-fast green intensity and any other parameter if available.
Presently i did it in by Image J but i am not sure i am doing it correctly, moreover i do not have a reference for the method i am using. I will be very thankful if anyone can help
Present method:
Safranin O stained section images: 20x ( 20x was covering most of the injury area)
ROI selection= selected 5 random ROIs ( 200 size, square box) on every images
Set mearurement: Gray value and other relevant parameters
mearure=reults
sample
Mean StdDev Min Max Integrated density Raw Integrated density SD
127.007 21.791 45 253.2 5080296.2 5080296.2 6.538
I would highly recommend you to read:
- Tutorial: guidance for quantitative confocal microscopy, Nature Protocols volume 15, pages1585–1611(2020)
A fantastic review of the Do's and Don'ts for quantitative confocal microscopy. Image capture is equally important to that of image analysis.
Regarding quantification:
- Make sure that you are drawing randomized ROI not biased in any way
- There is a flat field and shading correction image to avoid the 'edge effects'
- The imaging parameters/ sample preparations are set the same from one image/ sample to the other
- Area of the original tissue to avoid the 'reference trap'
- When possible, use Systematic Random Sampling as with Stereology methods
- Intensity quantification has quite a bit of variability even between images captured on the same session and all the factors has to be taken into account
Good luck.
@Sathya
Thank you so much , i have taken care of most of the parameters that could add bias or give us flaws, however variability oresults for same magnification images and even the same image if checked again is making me worried of my method.
Also removing the backgroung or keeping it as it is , or to do thresholding or not to do, all are confusing
Hi Sayed,
The variability may be due to few factors:
- If you cold start the microscope (eg. if you are the first user) the lamp intensity will be different resulting in varying image intensities. If you had another user before you (like in a core facility or another user from your lab) and the lamp has already warmed up, you will get a different intensity. Check the stability of the lamp intensity over time along with the camera variability due to warming up. If possible capture a standard slide whose intensity does not vary much due to storage and use it as a reference before capturing your images before each session and keep that slide as a reference.
- Maintain a constant exposure time (or gain) for all your images and make sure to maintain similar parameters as much as possible. Do not over/ under - saturate your pixels. If your microscope has a saturated pixels indicator, use it.
- Depending on what post-processing you perform, the results will vary, so minimize post-processing as much as possible. It varies from software to software too. Each time you save your image (depending on format like .jpeg, .tiff, .png, etc.) there is going to be some changes to the images. Use lossless image formats like .tiff. Try to keep a note of what post-processing that you are performing and find out what it does to the image. The intensity value also varies of how much dark area, over/ under-saturated pixels, where you draw your ROIs, how many ROIs, etc.
- The variability can occur at sample preparation, imaging, post-processing and anywhere in-between.
Good luck.
@Sathya
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining