Best Sampling for nucleus quantification in wide-field

[c.machu]

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Hello everyone !!

I have a very silly question. In our lab we would like to be able quantify
the whole signal intensity within a yeast nucleus. I know how to measure it
easily but I have doubts the Z sampling I am using. Should I sample using a
Nyquist criterion (but counting several time the same molecule in the
nucleus) or should I assume the theoretical Z resolution of my microscope
(knowing my objective specifics) ?

I would personally vote for the second option but I'd be glad to be
corrected if I am wrong.

Thanks a lot in advance for our inputs.

Best, Christophe

[Zdenek Svindrych-2]

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Hi Christophe,
congratulations for choosing widefield!

You don't need any z-sampling. A single image contains the fluorescence from
the whole nucleus, as long as the nucleus is at least approximately in focus
and the signal does not overlap with the fluorescence from other nuclei.

You may try it yourself - take a z-stack of a single nucleus in your FOV,
the total fluorescence should be constant over a fairly wide range of
defocus (of course you need to include all the fluorescence, including the
out-of-focus blur).

Depending on the intensity, size and density of your nuclei, you might
benefit from lower NA objective (you'll get lower intensity, but also less
blur).
Best, zdenek

Btw, not a silly question at all!

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Christophe Machu <[hidden email]>
Komu: [hidden email]
Datum: 5. 2. 2015 11:57:18
Předmět: Best Sampling for nucleus quantification in wide-field

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Hello everyone !!

I have a very silly question. In our lab we would like to be able quantify
the whole signal intensity within a yeast nucleus. I know how to measure it
easily but I have doubts the Z sampling I am using. Should I sample using a
Nyquist criterion (but counting several time the same molecule in the
nucleus) or should I assume the theoretical Z resolution of my microscope
(knowing my objective specifics) ?

I would personally vote for the second option but I'd be glad to be
corrected if I am wrong.

Thanks a lot in advance for our inputs.

Best, Christophe"

[c.machu]

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Hi Zdenek,

I never thought of that. I will test myself your solution ASAP. Can I then conclude from what you say that the integrated intensity I’ll be measuring is representative of the whole amount of tagged protein in the nucleus ?
I am trying to correlate fluorescence intensity with amount of protein.

And to reply to Peter, yes I would like to extract some morphological and localisation features from the images, so flow cytometry won’t bring me enough information on that aspect.

Thanks guys for your rapid input I really like this community and might come again with more questions in the near future.

Best, Christophe

> On 05 Feb 2015, at 19:22, Zdenek Svindrych <[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.
> *****
>
> Hi Christophe,
> congratulations for choosing widefield!
>
> You don't need any z-sampling. A single image contains the fluorescence from
> the whole nucleus, as long as the nucleus is at least approximately in focus
> and the signal does not overlap with the fluorescence from other nuclei.
>
> You may try it yourself - take a z-stack of a single nucleus in your FOV,
> the total fluorescence should be constant over a fairly wide range of
> defocus (of course you need to include all the fluorescence, including the
> out-of-focus blur).
>
> Depending on the intensity, size and density of your nuclei, you might
> benefit from lower NA objective (you'll get lower intensity, but also less
> blur).
> Best, zdenek
>
> Btw, not a silly question at all!
>
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> University of Virginia, Charlottesville, USA
> http://www.kcci.virginia.edu/workshop/index.php
>
>
>
> ---------- Původní zpráva ----------
> Od: Christophe Machu <[hidden email]>
> Komu: [hidden email]
> Datum: 5. 2. 2015 11:57:18
> Předmět: Best Sampling for nucleus quantification in wide-field
>
> "*****
> 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.
> *****
>
> Hello everyone !!
>
> I have a very silly question. In our lab we would like to be able quantify
> the whole signal intensity within a yeast nucleus. I know how to measure it
> easily but I have doubts the Z sampling I am using. Should I sample using a
> Nyquist criterion (but counting several time the same molecule in the
> nucleus) or should I assume the theoretical Z resolution of my microscope
> (knowing my objective specifics) ?
>
> I would personally vote for the second option but I'd be glad to be
> corrected if I am wrong.
>
> Thanks a lot in advance for our inputs.
>
> Best, Christophe"

[David Baddeley]

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Hi Cristophe,
Zdenek has pretty much nailed it, although it has the potential to get a bit (or maybe a lot) more complicated with very high NA lenses. These can have significant apodization of out of focus beam paths which means that you can no longer assume that the sum intensity is constant with depth (we see a 30-40% drop in the total integrated intensity of a bead which  is ~ 2um closer to the objective than the focal point when using our 1.49 NA objective). Testing whether the constant intensity assumption holds for your objective is pretty much as simple as taking a bead stack.  Assuming you're using a reasonable NA, and because yeast nuclei are relatively small, it's unlikely to be much of an issue, but it's one which is worth knowing about. If you do need to work with really high NAs, your best bet would be to take a Nyquist sampled image and then deconvolve it with an experimental PSF before quantifying the intensity.
cheers,David

     On Thursday, 5 February 2015 1:23 PM, Zdenek Svindrych <[hidden email]> wrote:
   

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Hi Christophe,
congratulations for choosing widefield!

You don't need any z-sampling. A single image contains the fluorescence from
the whole nucleus, as long as the nucleus is at least approximately in focus
and the signal does not overlap with the fluorescence from other nuclei.

You may try it yourself - take a z-stack of a single nucleus in your FOV,
the total fluorescence should be constant over a fairly wide range of
defocus (of course you need to include all the fluorescence, including the
out-of-focus blur).

Depending on the intensity, size and density of your nuclei, you might
benefit from lower NA objective (you'll get lower intensity, but also less
blur).
Best, zdenek

Btw, not a silly question at all!

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Christophe Machu <[hidden email]>
Komu: [hidden email]
Datum: 5. 2. 2015 11:57:18
Předmět: Best Sampling for nucleus quantification in wide-field

"*****
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.
*****

Hello everyone !!

I have a very silly question. In our lab we would like to be able quantify
the whole signal intensity within a yeast nucleus. I know how to measure it
easily but I have doubts the Z sampling I am using. Should I sample using a
Nyquist criterion (but counting several time the same molecule in the
nucleus) or should I assume the theoretical Z resolution of my microscope
(knowing my objective specifics) ?

I would personally vote for the second option but I'd be glad to be
corrected if I am wrong.

Thanks a lot in advance for our inputs.

Best, Christophe"

[Steffen Dietzel]

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Am 05.02.2015 um 20:59 schrieb Christophe Machu:
>  Can I then conclude from what you say that the integrated intensity I’ll be measuring is representative of the whole amount of tagged protein in the nucleus ?

Sure. Provided you do a few controls: Uniformity of illumination,
linearity of the detector, uniformity of labeling intensity (e.g.
antibody concentration and penetration), absence of quenching and
fluorescence saturation......

Seriously? if you have a 10x increase, no problem. If you have a 10%
increase, you need to work a lot on controls and provide huge numbers
(with good statistics) to convince a reviewer who knows the subject.
Have a look at Jim Pawley's 39 steps. Although written for confocal
quantitative fluorescence, much of it also applies to widefield. One link:
http://labs.pbrc.edu/cellbiology/documents/39steps.pdf

Good luck.

Steffen

>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Marchioninistr. 27
> D-81377 München
> Germany