Asked 13th Jan, 2023

How to compare fluorescent images taken at different exposure times?

I have images stained with different immunofluorescent markers (DAPI + many different markers) and captured images. However I recently discovered when I went to Metamorph to compare that the exposure times were different (300 vs 350ms). I know that in an ideal world, all the settings would have remained the same, however I am trying to salvage this data as re-doing the experiment is not an option at this time.
I have read the Filkins paper attached here talking about normalizing exposure times (Dark pixel intensity determination and its applications in normalizing different exposure time and autofluorescence removal) but my question is - how do I actually go about applying these changes to the images in question? Is there a journal I run it through?

All Answers (1)

14th Jan, 2023
Nicolò M. Villa
Swedish University of Agricultural Sciences
The Filkins paper you have mentioned provides a method for normalizing exposure times in fluorescent images by using the dark pixel intensity values. This method can be applied using image processing software such as ImageJ, which is a free and open-source image processing software.
Here are the general steps you can follow to apply the method to your images:
  1. Open your images in ImageJ and convert them to 8-bit grayscale images (Image > Type > 8-bit)
  2. Measure the dark pixel intensity values of each image by selecting a region of the image that does not contain any fluorescent signal, such as the background or a blank area (Analyze > Histogram)
  3. Calculate the ratio of the dark pixel intensity values for each image (i.e. the ratio of the dark pixel intensity value for the image with the longer exposure time to the dark pixel intensity value for the image with the shorter exposure time)
  4. Multiply the pixel intensity values of the image with the longer exposure time by the calculated ratio to normalize the exposure times (Image > Math > Multiply)
  5. Compare the normalized images using the Metamorph software, or analyze the images as desired.
It's important to note that this method assumes that the autofluorescence is constant across the images, and that the dark pixel intensity is directly proportional to the exposure time. If this is not the case, the method will not work and may lead to inaccurate results. Additionally, the method is just one way to tackle the problem of exposure time normalization, and it's important to validate the results obtained by this method, by comparing them with other methods and by analyzing the images with different parameters.
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