Unless your imaging facility is in a clean room (and you never touch it), from time to time, we all end up with unsightly splotches on our transmitted light images. The best fix for this is to clean the microscope but sometimes you just have to do what you can with what you’ve got.
Thankfully there’s a fairly easy way to correct it post-acquisition. Let’s Flat-field correct!
In the last post, we looked at the following image to decide if an intensity of 2000 at the tip of the arrowhead was bright or dim. Without knowing the bit depth of the image it’s impossible to tell.
We posed that if this was a 12bit image, that’s fairly bright (2000/4096) but if it’s a 16bit image, then it’s very dim (2000/65536). But why would these two conditions look the same? The answer lies in the histograms and transfer functions…
In the list of things that everyone who uses a microscope should know, this has to be near the top and yet I find a surprising number of people are either never taught it or don’t fully grasp the idea of Bit Depth. In this post and Part 2, we will deal with (almost) everything you need to know about bit depth, dynamic range and image histograms.