Posted by
Neil Anthony on
URL: http://confocal-microscopy-list.275.s1.nabble.com/sCMOS-salt-n-pepper-issues-tp7583323p7583334.html
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Hi Orla, thanks for offering to help with comparisons.
A set of three test movies under dark conditions are available in a
Volocity data set here:
https://drive.google.com/file/d/0BzfJAGNfrgieQklEN1lnQnBKMmM/view?usp=sharing(use Fiji to open the mvd2 file if you don't have Volocity)
The metadata should contain the details you need. For completeness, in
response to your questions:
1. Exposure Time
should be in the file name in the above
2. Frame rate (if time series taken)
maximum possible
3. Trigger mode (external or internal etc)
I assume internal as I don't have extra cables connected
4. Sensor Readout rate
don't know
5. Sensor Temperature
don't know
6. If you could provide a spec sheet for one of your cameras then I can
correlate sensitivities to replicate their photon flux
should be available from Hamamatsu
7. Are images single image or an image taken as part of a time series or
an accumulated image?
data above is time series without binning or accumulation etc.
8. Are you operating in Rolling Shutter or Global Reset Mode?
I assume global, but not sure
9. Are you using Light Sheet Readout mode?
no, not as far as I'm aware
The control of the camera is set in Volocity developed by Perkin Elmer,
and the majority of what you have requested above is not available to
the user.
I have not been able to catch a real big instance of ghost pixels in a
non-sample image set (i.e. a situation where it cannot be a piece of
biological schmutz jumping into and then out of the frame), but I have
seen a handful during my control data sets. They almost look like they
could be tiny pieces of dust jumping onto and off of the sensor, but I
really can't tell if they're real or electronic. They are ~5 pixels
wide and sometimes have a streak nature to them. A small version of
what I'm seeing can be seen on frame 118 of the 100ms data set and frame
10 of the 2s data set.
Once you've taken your data sets it would be great if you could share
them with the list for comparison.
Thanks
Neil
On 1/21/2015 7:04 AM, Orla Hanrahan wrote:
> Dear Neil,
>
> We would like to run our Zyla 4.2 under the exact same operating conditions as you ran your Flash 4 to see if we get the same salt 'n' pepper effect. In order for us to replicate your images precisely we would need the following data from you :
>
>
> 1. Raw images produced rather than screenshots (can you provide raw images?)
>
> 2. Details of the following for each image:
> 1. Exposure Time
> 2. Frame rate (if time series taken)
> 3. Trigger mode (external or internal etc)
> 4. Sensor Readout rate
> 5. Sensor Temperature
> 6. If you could provide a spec sheet for one of your cameras then I can correlate sensitivities to replicate their photon flux
> 7. Are images single image or an image taken as part of a time series or an accumulated image?
> 8. Are you operating in Rolling Shutter or Global Reset Mode?
> 9. Are you using Light Sheet Readout mode?
>
> 3. Also an image showing the "ghost bright spot" would be great.
>
> With this info we could get a better idea of what is going on with the cameras you are currently using and would be able to tell you more accurately if we have similar issues.
>
> I look forward to hearing from you,
>
> With my very best wishes,
>
> Orla Hanrahan
>
>
> ----------------------------------------------------------------------------------------
> Orla Hanrahan, PhD
> Application Specialist, Life Science Imaging
>
> Tel: +44 (0)74 256 27982
> W: www.andor.com
> Skype: orla.hanrahan1
>
>
>
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Neil Anthony
> Sent: 21 January 2015 03:24
> To:
[hidden email]
> Subject: sCMOS salt'n'pepper issues
>
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>
> Hi all, I hope the
> science/optics/hardware/software/students/post-docs/reviewers/managers/next-door-neighbors
> and FedEx are treating you well.
>
> We have a couple of issues with a recently installed Hamamatsu Flash 4.0 sCMOS camera and I was wondering if anybody has had any similar experiences, both in terms of feedback on what is expected from sCMOS hardware and what can be done to rectify any issues.
>
> Here's what we're seeing:
> Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're having three issues regarding hot and cold pixels. So far we have tried
> 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu and Perkin Elmer), to double check the subtleties, but the results are quite similar
>
> 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). Are these bright pixel clusters something that others have seen much? This issue is seen in both Volocity and ExCap/HDImage, but was not seen on the test sCMOS camera used to cross check. I also see 'ghost' clusters that look similar but only seem to last for one frame... Again, is that something seen in sCMOS cameras in general?
>
> 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. Exposure times ~secs; low signal <1000 counts per pixel. These speckled hot spots were seen with all sCMOS cameras to some degree.
> 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 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. Any thoughts and/or feedback on what's normal for an sCMOS, and how to get the best out of these cameras would be much appreciated. sCMOS cameras certainly are fast and have a great signal to noise, so am I expecting too much from this generation of hardware?
>
> Thanks
> Neil
>
>
> A couple of images to compare salt'n'pepper pixels seen on three cameras are here:
>
https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/view?usp=sharing>
https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/view?usp=sharing>