Posted by
Kyle Michael Douglass on
URL: http://confocal-microscopy-list.275.s1.nabble.com/sCMOS-salt-n-pepper-issues-tp7583323p7583324.html
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Hi Neil,
On 01/21/2015 04:24 AM, Neil Anthony wrote:
>
> On the first camera we saw a bright cluster of 4 pixels that are
> between 2 and 10x greater than the surrounding pixels across nearly
> all imaging conditions (not seen in the other three cameras).
>
We have an Andor Zyla 4.2 and I've tested a separate Zyla and the Flash
4.0 and I've never observed the clustering of hot spots.
> Salt:
> In images with low signal and longer exposure times we see speckled
> bight pixels using both Volocity and ExCap/HDImage, with an intensity
> approx twice that of the background signal.
These hot spots I believe come from the "anonymously" noisy chip-level
amplifiers. In an sCMOS camera, each pixel has its own amplifier and it
seems that it's nearly impossible to ensure that all ~4 million pixels
are defect free. In contrast, an EMCCD does not have pixel-dependent
noise properties because the amplification is not pixel-dependent.
Therefore, it seems like you have to accept hot spots by thinking of
them as manufacturing defects that occur in making a large number of
single pixels.
We characterized our camera by measuring the pixel-dependent gain, noise
variance, and hot spot locations according to the procedure in the
supplement of this paper from the Bewersdorf lab:
http://www.ncbi.nlm.nih.gov/pubmed/23708387. We found that our
characterization matched the hot pixel map provided by Andor exactly, so
we know what the intensity statistics of those pixels are.
> Pepper:
> In almost the inverse situation, where we have higher signals with
> lower exposure times were also seen on all sCMOS cameras (the first
> camera with the clear hotspot had very bad 'peppering' compared to the
> others).
I'm not sure about this one. I haven't noticed the peppering. Is the hot
spot correction algorithm on in this case?
>
> I know that the pco cameras used on the Deltavision OMX have reference
> images applied to reduce the affects of these artifacts, and I was
> wondering if that's something that can be applied post-acquisition.
We work in STORM/PALM microscopy and incorporate the hot spots into our
noise model when performing the localization analysis, much like in the
paper I cited above. We therefore do not use the manufactuer's algorithm
for hot pixel smoothing.
For "normal" imaging, I think you either have to use the hot spot
correction algorithms to smooth over those pixels, or turn off the
algorithm and post-process the hot spots yourself.
I hope this helps!
Kyle
--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.iohttp://leb.epfl.ch