Very high intensity images during a screen

[Pascal Lorentz-2]

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Dear list

Please apologize, this is not a confocal question but I was hopping to
get some hints from you.
We encountered strange images during a screen of multi-well plates on
our high content screener.
Please lock at the images at the link below:
https://imgur.com/a/PJCvzjw
- The first image shows the first 96 well plate in a overview. The first
7 rows are fine but then we imaged these strange images.
- The second image shows an other plate with the same effect but only in
the last row.
- The third image shows a single field of view from a bad well.
- The forth image shows an image of a normal image.
For example the YFP signal in the bad image is very high at around
16000, while the normal signal from the YFP positiv cells is only around
250.
We checked the plates on an other system. They are fine. I even
reacquired the plates on the screener and had no problems to acquire
them in the second run. However we were wondering what can cause such an
effect. Could this be a camera issue? I have no explanation why gray
levels increase to such an extend but no real signal is visible.
Thanks for looking at it.

Best regards

Pascal

[Sylvie Le Guyader]

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To me it looks like a slow focus drift that leads to a reflection at the bottom of the plate. The last row before the reflection appears is already a bit out of focus. Do you use a hardware autofocus?

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7C,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Pascal Lorentz
Sent: den 3 maj 2018 09:30
To: [hidden email]
Subject: Very high intensity images during a screen

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Dear list

Please apologize, this is not a confocal question but I was hopping to get some hints from you.
We encountered strange images during a screen of multi-well plates on our high content screener.
Please lock at the images at the link below:
https://imgur.com/a/PJCvzjw
- The first image shows the first 96 well plate in a overview. The first
7 rows are fine but then we imaged these strange images.
- The second image shows an other plate with the same effect but only in the last row.
- The third image shows a single field of view from a bad well.
- The forth image shows an image of a normal image.
For example the YFP signal in the bad image is very high at around 16000, while the normal signal from the YFP positiv cells is only around 250.
We checked the plates on an other system. They are fine. I even reacquired the plates on the screener and had no problems to acquire them in the second run. However we were wondering what can cause such an effect. Could this be a camera issue? I have no explanation why gray levels increase to such an extend but no real signal is visible.
Thanks for looking at it.

Best regards

Pascal

[Pascal Lorentz-2]

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Dear Sylvie

Yes the hardware autofocus is supposed to look for the two peaks of the
plate bottom for every image. Usually the system indicates the images
where the autofocus failed and does not acquire an image at all in that
case. Because of that I doubt that the autofocus had a problem. I also
tried to slightly misplace the plate (could happen by the robot) but in
that case the autofocus indeed failed. But I agree a reflection might
cause these high intensity values. I just don't have an explanation how
this could happen.

Best regards

Pascal

Am 03.05.2018 um 10:19 schrieb Sylvie Le Guyader:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> To me it looks like a slow focus drift that leads to a reflection at the bottom of the plate. The last row before the reflection appears is already a bit out of focus. Do you use a hardware autofocus?
>
> Med vänlig hälsning / Best regards
>  
> Sylvie
>  
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7C,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Pascal Lorentz
> Sent: den 3 maj 2018 09:30
> To: [hidden email]
> Subject: Very high intensity images during a screen
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear list
>
> Please apologize, this is not a confocal question but I was hopping to get some hints from you.
> We encountered strange images during a screen of multi-well plates on our high content screener.
> Please lock at the images at the link below:
> https://imgur.com/a/PJCvzjw
> - The first image shows the first 96 well plate in a overview. The first
> 7 rows are fine but then we imaged these strange images.
> - The second image shows an other plate with the same effect but only in the last row.
> - The third image shows a single field of view from a bad well.
> - The forth image shows an image of a normal image.
> For example the YFP signal in the bad image is very high at around 16000, while the normal signal from the YFP positiv cells is only around 250.
> We checked the plates on an other system. They are fine. I even reacquired the plates on the screener and had no problems to acquire them in the second run. However we were wondering what can cause such an effect. Could this be a camera issue? I have no explanation why gray levels increase to such an extend but no real signal is visible.
> Thanks for looking at it.
>
> Best regards
>
> Pascal

[jerie]

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Hi Pascal, filter/cube not positioning correctly? then excitation light
leaks through in the yfp channel. would need more info about the setup.
Good success!


On Thu, May 3, 2018 at 1:14 PM, Pascal Lorentz <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Sylvie
>
> Yes the hardware autofocus is supposed to look for the two peaks of the
> plate bottom for every image. Usually the system indicates the images where
> the autofocus failed and does not acquire an image at all in that case.
> Because of that I doubt that the autofocus had a problem. I also tried to
> slightly misplace the plate (could happen by the robot) but in that case
> the autofocus indeed failed. But I agree a reflection might cause these
> high intensity values. I just don't have an explanation how this could
> happen.
>
> Best regards
>
> Pascal
>
>
> Am 03.05.2018 um 10:19 schrieb Sylvie Le Guyader:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> To me it looks like a slow focus drift that leads to a reflection at the
>> bottom of the plate. The last row before the reflection appears is already
>> a bit out of focus. Do you use a hardware autofocus?
>>
>> Med vänlig hälsning / Best regards
>>   Sylvie
>>   @@@@@@@@@@@@@@@@@@@@@@@@
>> Sylvie Le Guyader, PhD
>> Live Cell Imaging Facility Manager
>> Karolinska Institutet- Bionut
>> <https://maps.google.com/?q=olinska+Institutet-+Bionut+&entry=gmail&source=g>
>> Dpt
>> Hälsovägen 7C,
>> Room 7362 (lab)/7840 (office)
>> 14157 Huddinge, Sweden
>> mobile: [phone]
>> LCI website
>> Follow our microscopy blog!
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List <[hidden email]> On
>> Behalf Of Pascal Lorentz
>> Sent: den 3 maj 2018 09:30
>> To: [hidden email]
>> Subject: Very high intensity images during a screen
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear list
>>
>> Please apologize, this is not a confocal question but I was hopping to
>> get some hints from you.
>> We encountered strange images during a screen of multi-well plates on our
>> high content screener.
>> Please lock at the images at the link below:
>> https://imgur.com/a/PJCvzjw
>> - The first image shows the first 96 well plate in a overview. The first
>> 7 rows are fine but then we imaged these strange images.
>> - The second image shows an other plate with the same effect but only in
>> the last row.
>> - The third image shows a single field of view from a bad well.
>> - The forth image shows an image of a normal image.
>> For example the YFP signal in the bad image is very high at around 16000,
>> while the normal signal from the YFP positiv cells is only around 250.
>> We checked the plates on an other system. They are fine. I even
>> reacquired the plates on the screener and had no problems to acquire them
>> in the second run. However we were wondering what can cause such an effect.
>> Could this be a camera issue? I have no explanation why gray levels
>> increase to such an extend but no real signal is visible.
>> Thanks for looking at it.
>>
>> Best regards
>>
>> Pascal
>>
>

[Pascal Lorentz-2]

*****
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*****

Hi Jens

Thanks, good point. Since it is a closed system it's very hard to check
if the filters are correctly positioned. But I will keep that in mind
and discuss it with the manufacturer.

Best regards

Pascal

Am 04.05.2018 um 22:39 schrieb jens rietdorf:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Pascal, filter/cube not positioning correctly? then excitation light
> leaks through in the yfp channel. would need more info about the setup.
> Good success!
>
>
> On Thu, May 3, 2018 at 1:14 PM, Pascal Lorentz <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear Sylvie
>>
>> Yes the hardware autofocus is supposed to look for the two peaks of the
>> plate bottom for every image. Usually the system indicates the images where
>> the autofocus failed and does not acquire an image at all in that case.
>> Because of that I doubt that the autofocus had a problem. I also tried to
>> slightly misplace the plate (could happen by the robot) but in that case
>> the autofocus indeed failed. But I agree a reflection might cause these
>> high intensity values. I just don't have an explanation how this could
>> happen.
>>
>> Best regards
>>
>> Pascal
>>
>>
>> Am 03.05.2018 um 10:19 schrieb Sylvie Le Guyader:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> To me it looks like a slow focus drift that leads to a reflection at the
>>> bottom of the plate. The last row before the reflection appears is already
>>> a bit out of focus. Do you use a hardware autofocus?
>>>
>>> Med vänlig hälsning / Best regards
>>>    Sylvie
>>>    @@@@@@@@@@@@@@@@@@@@@@@@
>>> Sylvie Le Guyader, PhD
>>> Live Cell Imaging Facility Manager
>>> Karolinska Institutet- Bionut
>>> <https://maps.google.com/?q=olinska+Institutet-+Bionut+&entry=gmail&source=g>
>>> Dpt
>>> Hälsovägen 7C,
>>> Room 7362 (lab)/7840 (office)
>>> 14157 Huddinge, Sweden
>>> mobile: [phone]
>>> LCI website
>>> Follow our microscopy blog!
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List <[hidden email]> On
>>> Behalf Of Pascal Lorentz
>>> Sent: den 3 maj 2018 09:30
>>> To: [hidden email]
>>> Subject: Very high intensity images during a screen
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> Dear list
>>>
>>> Please apologize, this is not a confocal question but I was hopping to
>>> get some hints from you.
>>> We encountered strange images during a screen of multi-well plates on our
>>> high content screener.
>>> Please lock at the images at the link below:
>>> https://imgur.com/a/PJCvzjw
>>> - The first image shows the first 96 well plate in a overview. The first
>>> 7 rows are fine but then we imaged these strange images.
>>> - The second image shows an other plate with the same effect but only in
>>> the last row.
>>> - The third image shows a single field of view from a bad well.
>>> - The forth image shows an image of a normal image.
>>> For example the YFP signal in the bad image is very high at around 16000,
>>> while the normal signal from the YFP positiv cells is only around 250.
>>> We checked the plates on an other system. They are fine. I even
>>> reacquired the plates on the screener and had no problems to acquire them
>>> in the second run. However we were wondering what can cause such an effect.
>>> Could this be a camera issue? I have no explanation why gray levels
>>> increase to such an extend but no real signal is visible.
>>> Thanks for looking at it.
>>>
>>> Best regards
>>>
>>> Pascal
>>>