Hi Alison,

I also experienced the same thing in the past. Do you do an individual analysis of each image? If so, sometimes individual analyzes can produce errors like this. I also don’t understand why this error can occur. However, I have a solution to avoid this error with batch processing.

Batch processing is a mechanism within ImageJ that allows users to perform multiple image analysis at once. To do so, try to follow my protocol:

1. You need to prepare the image that you will process. This means that you need to “crop to a region of interest to exclude background” on all images first.

2. After all the images are ready. Put the image in one or more folders according to your needs.

3. Click Process> batch> macro and the batch process window will appear.

4. Select the input folder containing the image you wish to analyze. Remember, you can only batch process one folder at a time! So if you are working in multiple folders, you need to do batch processing equal to the number of your folders.

5. Select the output folder where you want it. Basically, this output folder will contain the logs and histograms of the analysis of all the images in your input.

6. Set the output format according to your needs. I usually don’t change the settings for “output format”, “add macro code”, and “file name contain”.

7. Under the file name contains, there will be a large empty space. It’s where you put your batch processing code. Each protocol has a different code. I have coded your protocol as the following:

run (“IHC Profiler”, “mode = [Cytoplasmic Stained Image] vectors = [H DAB]”);

8. After that click process, than the log and histogram window will start batch processing all the images in your input file.

This batch processing has two advantages. The first is to streamline your work on image analysis. Second, for some reason, if you use this batch processing, the error that you asked about earlier will not occur.

I hope my answer helps. I’ve also included my publications if you’d like to read more ( https://www.phcogj.com/article/1326 ). Contact me if you have further questions. Cheers!