I posted on here a while ago with the same problem. Right now my negative control keeps measuring as high or higher in intensity than my positive control when measuring the GFP channel.
The software I use to analyze is ImageJ and I am not converting the image after it has been output from the EVOS microscope.
Originally I saved the files as 8-bit .tif RGB and I realize that this is an issue since the bin goes from 0-4000 down to 0-255 leading to huge inaccuracies and basically unusable images for data.
I tried ticking the "always save .tif as 8-bit", tried instead saving as .jpg/.png, and the same issue occurs.
I was told to try saving it as a RAW format and not save it as RGB but I don't know if this is possible with the EVOS FL system and their website doesn't specific anything about this.
Does anyone know if that is possible to do? If not how to I save these images so I can actually analyze them with ImageJ properly?
The only thing I can think to do is save each channel separately but even then they're being saved the same way so I doubt it would change anything.
Here are some images for reference.
The first two have had EDU for 30min, no other treatments so the GFP signal is high on the microscope.
The next two images are cells that have not been treated with anything and viewing them on the microscope there is absolutely no GFP signal so it's absurd that the values are so high after measuring through imageJ.
P.S. NT = non-target, HU = Hydroxyurea
Dear Francesco,
Please take a look at this Evos manual:
https://tools.thermofisher.com/content/sfs/manuals/evos_fl_auto_man.pdf
(page 10/page 12 of the pdf)
I would recommend saving your images (acquired with the B/W camera) into individual 16bit black and white Tiffs. That should protect your 12bit image information.
Please give me a feedback if it does not work, I will ask a peer who owns a Evos FL.
Best Soenke
Hi all, I'm facing a similar problem. I also have a Evos FL Auto. I would like to analyse images of a scan of a whole 384 plate with imageJ. Unfortunately ImageJ does not know the sti-Fileformat. I discovered that it is possible to save images as 16bit monochrome tiff manually (capture, save well by well manually). But this function is not available for exporting images of a scan. Do you have any hint how to get hold of my images? Best Annegret
Hi Annegret,
When defining your scan parameters in the scan wizard, you have the option to save your images as a TIFF or PNG image. The STI file is a default file created by the system, you should still also have access to the TIFF or PNG file you selected when setting up your scan. IF you continue to run into additional issues, please feel free to reach out to our tech support group at [email protected]
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