Pixel value becomes inaccurate after exceeding certain values

Dear micro manager community,

I noticed that the pixel value for the images I acquired show unexpected behavior: the same field of cells were acquired at 1,000ms vs 250ms and some of the bright pixels become black and the value is reduced, not to zero, but to something like 20,000~25,000. Any explanation for this? Is that due to the camera (iXon Ultra DU897_BV 12451) readout error? Thanks~


Kai

With iXon cameras, I have seen very saturated pixels turn black (often a very bright glob of something will be black in the center with a smeared bright donut around it). This looks somehow more sharp, but I don’t recall saturating a sample that looked quite like that.

I definitely think you’re saturating the camera, since the bins are nearly full in the 250 ms image. So I’d reduce the gain and/or the exposure time.

Sam

Hi Sam,

Thanks for the information! I tested this possibility but it is not like the case: I tried serial increment of exposure, from 450ms to 650ms, with 20ms as step. The pixels turned gray/black and reduced value before reaching >50,000. I screenshot two of them: 500ms and 600ms. If the saturation is the cause of this result, probably the readout of camera is not really 16 bits so it caps at <50,000 or so?


Saturation on the iXons is not actually 65535, but slightly lower. 50,000 sounds right.

When the analog to digital converters on your camera are set up right, a full well of electrons in your camera will result in pixel values that are less then the maximum digital value of the bitdepth, so 50,000 sounds about right (the exact value will probably vary a bit with settings like gain and EM gain of the camera).

Also, I am quite sure that the dropping off to lower values with higher saturation is due to the EM amplification (try to imagine what happens when you are filling up wells in the EM register, my mind goes into catastrophy mode). In general, I would avoid this situation, as it is likely not good for your camera to image like this long-term.

I see~ This is new to me as my previous experience with hamamatsu EMCCD is different and I can see pixel value all the way to 65535.