Apotome for intensity measurements?

[mcammer]

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Dear microscopists,

I recall from years ago discussions with Zeiss during an one-site demo that while the Apotome produces beautiful images, the deconvolution method of the sequential grid images may not accurately reflect relative intensity values for quantification of fluorescence.  Recently I mentioned this to colleagues who have been using the Apotome specifically to look at quantification of chemical species and they got worried about their data.  However, there appear to be many peer reviewed published papers where people have used the Apotome for intensity quantification.  Does anyone know the real answer?

Sincerely,

Michael


               
_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

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[Daniel James White]

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Hi Michael,

On Oct 29, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> Date:    Thu, 28 Oct 2010 21:26:14 -0400
> From:    "Cammer, Michael" <[hidden email]>
> Subject: Apotome for intensity measurements?
>
> Dear microscopists,
>
> I recall from years ago discussions with Zeiss during an one-site demo that=
> while the Apotome produces beautiful images, the deconvolution method of t=
> he sequential grid images may not accurately reflect relative intensity val=
> ues for quantification of fluorescence.  

Correct, its not really a linear process... and the intensities in the resultl image
then might not be proportional in different parts of the image...
there may be feint residual striped... or not.

I would use widefield plus deconvolution instead....
but deconv is also somewhat non linear....

> Recently I mentioned this to colle=
> agues who have been using the Apotome specifically to look at quantificatio=
> n of chemical species and they got worried about their data.  However, ther=
> e appear to be many peer reviewed published papers where people have used t=
> he Apotome for intensity quantification.  Does anyone know the real answer?

the real answer is to do the right controls....

image fields of fluorescent beads that as ground truth known objects
can be compared in widefield and apotome versions of the image.

you can then check the linearity and noise problems.

Just measure the problem , then you dont have to guess!!!

cheers

Dan

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Processing and Analysis
Light Microscopy Facility
Max Planck Institute of Molecular Cell Biology and Genetics
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01307 DRESDEN
Germany

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[Barlow, Andrew]

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Hi Michael,
This is an issue that we were concerned about when we added support for a similar structured illumination device, the Optigid to Volocity acquisition. This device is based on the work of Tony Wilson's group (Optics Letters 22: 1905-1907). We ran tests using Invitrogen Inspeck beads, which have known relative intensities.  We concluded that the Optigrid is indeed suitable for quantitative studies, and the fact that the algorithm subtracts background actually makes the images ideal for quantitation.  Our results are available in Microscopy Research and Technique 70(1) 76-84.
The Apotome is a device that is physically identical to the Optigid, although the algorithms used by Zeiss are, at least as far I know, not in the public domain.  Zeiss apparently do not infringe on Wilson's patent for the technique so they may do something unusual.
I would suggest that your colleague buys a set of Inspeck beads and checks for a linear response from the Apotome for themselves. I've yet to see any reliable tests either way for the Apotome.
Regards,
Andrew



Andrew Barlow PhD | Applications Specialist
PerkinElmer | For the Better
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> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Cammer, Michael
> Sent: 29 October 2010 02:26
> To: [hidden email]
> Subject: Apotome for intensity measurements?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear microscopists,
>
> I recall from years ago discussions with Zeiss during an one-site demo
> that while the Apotome produces beautiful images, the deconvolution
> method of the sequential grid images may not accurately reflect
> relative intensity values for quantification of fluorescence.  Recently
> I mentioned this to colleagues who have been using the Apotome
> specifically to look at quantification of chemical species and they got
> worried about their data.  However, there appear to be many peer
> reviewed published papers where people have used the Apotome for
> intensity quantification.  Does anyone know the real answer?
>
> Sincerely,
>
> Michael
>
>
>
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: [phone]  Cell: [phone]
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of
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> sender by return email and delete the original message. Please note,
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[G. Esteban Fernandez]

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As it was explained to me by Zeiss during Apotome training, relative
intensities can be compared within one image but not across different
images.

Also I wonder how appropriate beads are for validating the technique.
I don't know enough about the math to know...is final intensity after
grid deconvolution affected by neighboring pixels (in Z as well as
XY)?  If beads are point-like and well-separated are they are an
unrealistic representation of an actual specimen?  If it were me doing
this, I'd validate the method by taking pictures of real specimens
with known relative intensities, e.g. cells titrated to express
different relative levels of GFP.  Can someone comment on differences
between beads vs. real specimens, if any?

Thanks,
Esteban


On Thu, Oct 28, 2010 at 6:26 PM, Cammer, Michael
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear microscopists,
>
> I recall from years ago discussions with Zeiss during an one-site demo that while the Apotome produces beautiful images, the deconvolution method of the sequential grid images may not accurately reflect relative intensity values for quantification of fluorescence.  Recently I mentioned this to colleagues who have been using the Apotome specifically to look at quantification of chemical species and they got worried about their data.  However, there appear to be many peer reviewed published papers where people have used the Apotome for intensity quantification.  Does anyone know the real answer?
>
> Sincerely,
>
> Michael
>
>
>
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: [phone]  Cell: [phone]
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
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>

[Carol Heckman]

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Michael
i suspect the "real" answer might be that there is hardly any way of quantifying fluorescence relative intensity values or at least relating these values to the quantity of fluorochrome at any given subcellular site.  I think the scientists prefer such methods as FRET and FLIM because the relevant binding of fluorochrome to something appears more reliable as an indicator of what the experiment is directed at (not grammatical but you know what I mean?).
Carol Heckman
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Cammer, Michael [[hidden email]]
Sent: Thursday, October 28, 2010 9:26 PM
To: [hidden email]
Subject: Apotome for intensity measurements?

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Dear microscopists,

I recall from years ago discussions with Zeiss during an one-site demo that while the Apotome produces beautiful images, the deconvolution method of the sequential grid images may not accurately reflect relative intensity values for quantification of fluorescence.  Recently I mentioned this to colleagues who have been using the Apotome specifically to look at quantification of chemical species and they got worried about their data.  However, there appear to be many peer reviewed published papers where people have used the Apotome for intensity quantification.  Does anyone know the real answer?

Sincerely,

Michael



_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

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This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
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