BG subtraction with flat-field correction

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BG subtraction with flat-field correction

Marie Schwebs
Hi,

We just installed the MM 2.0 gamma in combination to the upgrade to Windows 10. Before that, we were using MM 1.4 and we used the BF Corrector plugin to subtract the background during our recordings.

Now I’d like to subtract the background in the ROI + crop mode with the flat-field correction plugin. 
I generated an averaged intensity tiff image of the whole camera chip by using ZProject in FIJI (as recommended), but it does not subtract any background noise. If I choose only the ROI it works perfectly fine. Do I miss something?
I have also to note that I can’t select “Channel" for "Channel Group", can’t add any flat-field preset and the tick-box "Execute Flat Fielding is missing.

If the subtraction of the background with the flat-field correction plugin does not work for crop mode, I’d like to install the BF Corrector plugin. Putting it into the plugin folder of MM 2.0 did not result in the desired installation.

Regards,
Marie 
 

Marie Schwebs

Cell and Developmental Biology
Biocenter
Wuerzburg University
Am Hubland, 97074 Wuerzburg
Germany

Tel: +49 931 31 85228

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Re: BG subtraction with flat-field correction

Nico Stuurman-2
Hi Marie,

> We just installed the MM 2.0 gamma in combination to the upgrade to
> Windows 10. Before that, we were using MM 1.4 and we used the BF
> Corrector plugin to subtract the background during our recordings.
>
> Now I’d like to subtract the background in the ROI + crop mode with
> the flat-field correction plugin.
> I generated an averaged intensity tiff image of the whole camera chip
> by using ZProject in FIJI (as recommended), but it does not subtract
> any background noise. If I choose only the ROI it works perfectly
> fine. Do I miss something?

I am not super-familiar with "Crop" mode.  If I remember correctly, this
is an Andor specific setting that lets you speed up image transfer by
moving charge originating from a small fraction of the chip to the
read-out node without blanking the remainder of the chip (necessitating
"shielding" the rest of the chip).  The Flat-Field Correction plugin
asks the camera for its chip size, so if the Andor camera adapter
reports a different chip size when you apply Crop mode, then you will
need to provide a background image that matches that size rather than
the size of the complete chip.

When this happens, there should be a message in the main window
(bell-like icon, bottom left, you can click on it to see more
information) that explains the problem.

> I have also to note that I can’t select “Channel" for "Channel Group",
> can’t add any flat-field preset and the tick-box "Execute Flat
> Fielding” is missing.

Do you have any Configuration Groups defined?  If so, that sounds like a
but that manifests itself in your configuration.  Send a Truble Report.

The plugin has no tick-box "Execute Flat Fielding".  Instead, there is
the "On-The-Fly Processor Pipeline dialog that lets you enable/disable
any of the available "Processors" for MDA and/or Snap/Live.

> If the subtraction of the background with the flat-field correction
> plugin does not work for crop mode, I’d like to install the BF
> Corrector plugin. Putting it into the plugin folder of MM 2.0 did not
> result in the desired installation.

It should work with Crop mode, but you will need to provide differently
sized images for Crop mode and non-Crop mode (just like in the 1,4
plugin).  Plugins from 1.4 do not work in 2.0-gamma, since the API has
changed drastically.



Best,


Nico



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Re: BG subtraction with flat-field correction

Marie Schwebs
Hi Nico,

Thank you for your quick response!

>
>> We just installed the MM 2.0 gamma in combination to the upgrade to Windows 10. Before that, we were using MM 1.4 and we used the BF Corrector plugin to subtract the background during our recordings.
>>
>> Now I’d like to subtract the background in the ROI + crop mode with the flat-field correction plugin.
>> I generated an averaged intensity tiff image of the whole camera chip by using ZProject in FIJI (as recommended), but it does not subtract any background noise. If I choose only the ROI it works perfectly fine. Do I miss something?
>
> I am not super-familiar with "Crop" mode.  If I remember correctly, this is an Andor specific setting that lets you speed up image transfer by moving charge originating from a small fraction of the chip to the read-out node without blanking the remainder of the chip (necessitating "shielding" the rest of the chip).  The Flat-Field Correction plugin asks the camera for its chip size, so if the Andor camera adapter reports a different chip size when you apply Crop mode, then you will need to provide a background image that matches that size rather than the size of the complete chip.
>
> When this happens, there should be a message in the main window (bell-like icon, bottom left, you can click on it to see more information) that explains the problem.
>

We figured out how the BG subtraction is working with the Flat-Field Correction:
 
It is necessary to trick MM2.0, because the crop mode with frame transfer is only active when the image used for the BG correction is slightly smaller than the camera chip.
This means in our case: Camera chip is 512 x 512 px (width x height) and the ROI of BG image in FIJI  is 512 x 511 px  (width x height), 0 and 1 (X coor and Y coor).
Now it is important that the bottom left corner of the BG image overlays exactly with the bottom left corner of the latter ROI for the crop mode region on the camera chip.
Example: ROI shall be 120 x 120 px, settings in FIJI are 120 x 120 px and 0, 391 for the X coor and Y coor.

This is the only way how it works, otherwise we will have the error massage that the images are not of the same size, what you mentioned above.

>> I have also to note that I can’t select “Channel" for "Channel Group", can’t add any flat-field preset and the tick-box "Execute Flat Fielding” is missing.
>
> Do you have any Configuration Groups defined?  If so, that sounds like a but that manifests itself in your configuration.  Send a Truble Report.
>
> The plugin has no tick-box "Execute Flat Fielding".  Instead, there is the "On-The-Fly Processor Pipeline dialog that lets you enable/disable any of the available "Processors" for MDA and/or Snap/Live.
>

No, we don’t have them defined. Yes it would help. Okay, then I am relieved, because I was confused by the image of the window what is displayed in the Flat-Field correction description at the website.

>> If the subtraction of the background with the flat-field correction plugin does not work for crop mode, I’d like to install the BF Corrector plugin. Putting it into the plugin folder of MM 2.0 did not result in the desired installation.
>
> It should work with Crop mode, but you will need to provide differently sized images for Crop mode and non-Crop mode (just like in the 1,4 plugin).  Plugins from 1.4 do not work in 2.0-gamma, since the API has changed drastically.
>

Thank you a lot for the help!

Regards,
Marie



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