Hi, trying to figure out if this cell culture looks above or below 50% confluent. If anybody can give me a rough estimate? ImageJ says this is only about 32% confluent which seems a bit off to me.. but since I am fairly new, all the input is appreciated.
Hi Aarohi,
Confluency is a term that means the percentage of the growth medium area/plate surface covered by adherent cells.
Suppose, you need 50% confluency, so select a particular area in the image and look whether the 50% places of plate has been covered by cells or not. ImageJ also works in same way to measure confluency.
But from naked eye, I would have called it as 40% confluent.
Hope this will help!
Ayan Mandal thank you for responding! I used ImageJ for calculating cell confluency but it was showing the result only around 32% where as visually it seemed more than that. So I needed some opinion before leaning towards ImageJ results.
Aarohi Shah imageJ is showing low confluency because your thresholding is not optimum. The cells are not completely different from the background, as you can see that pixel value some where whithin the cell is similar to that of background, ImageJ is calculating that as the background. that is why visually it looks around 45% confluent but imageJ shows 32% only as it is only calculating the dark areas in the image.
You can try calculating the number of cells in the image by imageJ multiply it by diameter of a single cell (which you can calculate in imageJ by simply drawing a straight line which will give you average diameter) and then divide the total image area by the above obtained value.
Key point to be noted for this type of calculation is that you ROI should be different from the background. As imageJ works on pixel value it will take the ROI pixel as background if it have same pixel value as the background. Plus try to take evenly and nicely lit pictures. Dim images tends to create problem during Thresholding the image.
I have not worked with cell culture experiment but I have some experience with ImageJ hope this helps.
- Badges
- Science method