Autoscaling with ImageJ

I am using ImageJ for my analysis. I want to know when and when not to use autoscaling while acquiring images on the microscope.

I want to also now while images a 14 bit image I obtained without autoscaling appeared quite dark even when I set the Brightness and contrast in imageJ

Hi @turbine ,

When : you’re looking the focus

When not : when you want to acquire an image.

I prefer to fix the scaling to the maximal display range (0-255, …) and look at the histogram to avoid saturation.

If your images were acquired with a low exposure time / you’re making little use of the available range image might look dark…

Can you share some images?

Did you try different Image > Look Up Table > Fire ?

Best,

Romain

Hi Romain

Thanks, I set my image to 14-bit without autoscaling while acquiring the image. However when I set my B&C to 14 bit (0 -16383), the image appears a bit dark and does not show the signals as I expect.

Must I stick to 14-bit setting of brightness and contrast if I used 14-bit microscope setting in obtaining the image?

Hi @turbine ,

That’s why you need to look at the histogram, let’s take an example.

You can open the image m51 (File > Open Samples> m51), it is a 16-bit image, using the display range 0-65535, we see pretty much nothing.
Looking at the histogram (Analyze > Histogram) you see that the max is 10106.

So now if we change the display to 0-10106, you’ll get something like this (image below), which allow us to see a bit better. BUT if you click on the button “Log” in the histogram Window (to display the histogram with a log scale on the y axis) you will have a better idea of the pixel intensities distribution and you realize that even if the Max is 10106, there are not so many pixels above 3000.

So now we change the display to 0-3000 and we can see even better!

Eventually, you can also use a different Lookup Table (Image > Lookup Table ), I personally like the “Fire” image below) and you can use a “ramp” image and specify the limit of your display 0-3000. (you can also notice the update of the histogram)

Short answer: No
Longer answer: As shown above, you can change the display of your image, BUT if you want to compare images (between condition A and B) you have to acquire and display them with the same parameters.

Cheers,

Romain

PS : I’d suggest you have a look at this post to learn more :

Maybe you know already, but the brightness and contrast settings do not change your data, so (as long as you do not press the [Apply] button, which you never should) you can change the B&C as you wish. It is however highly recommended to use the same B&C settings for comparing different images that were acquired with the same microscope settings.

You could for example have a look at these tutorials (there are ImageJ based activities):