Spinning disk vs point scanner

[simon walker (BI)]

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A confocal question!
We have been imaging single live fluorescent cells in aqueous buffer held in
suspension at a distance of ~80 um from the coverslip surface.  We need to
visualise rapid changes in the 3D structure of the cells so have been trying to
capture image stacks using spinning disk confocal (EM-CCD camera, CSU-10,
60x water immersion 1.27 NA lens).  The images we obtain are useless - really
noisy, and the round cells apprear highly distorted (the fuzzy mass becomes
elongated in x and then y as you focus up and down through the cell).  We
then tried the same experiment on a point scanning confocal using exactly the
same microscope body and lens, and got really nice images (but not obtained
with sufficient speed to make the experiment viable).  So my (ignorant)
question is, why should the spinning disk microscope perform so badly imaging
a small fluorescent object at depth in an aqueous buffer?  I know (older)
spinning disk systems are not so good at imaging at depth into thicker
specimens due to out-of-focus light entering adjacent pinholes, but I thought
that imaging a small fluorescent object at depth with an RI-matched lens
would be ok.
Any explanations would be appreciated..
Thanks,
Simon
P.S. I tried playing with the correction collar on the lens, but that made no
difference.

[Mark Cannell-2]

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H Simon

The extended z is due to the effective pinhole size in the spinning disk. Can you use a higher magnification objective?

Cheers
On 25/04/2013, at 10:55 AM, Simon Walker <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> A confocal question!
> We have been imaging single live fluorescent cells in aqueous buffer held in
> suspension at a distance of ~80 um from the coverslip surface.  We need to
> visualise rapid changes in the 3D structure of the cells so have been trying to
> capture image stacks using spinning disk confocal (EM-CCD camera, CSU-10,
> 60x water immersion 1.27 NA lens).  The images we obtain are useless - really
> noisy, and the round cells apprear highly distorted (the fuzzy mass becomes
> elongated in x and then y as you focus up and down through the cell).  We
> then tried the same experiment on a point scanning confocal using exactly the
> same microscope body and lens, and got really nice images (but not obtained
> with sufficient speed to make the experiment viable).  So my (ignorant)
> question is, why should the spinning disk microscope perform so badly imaging
> a small fluorescent object at depth in an aqueous buffer?  I know (older)
> spinning disk systems are not so good at imaging at depth into thicker
> specimens due to out-of-focus light entering adjacent pinholes, but I thought
> that imaging a small fluorescent object at depth with an RI-matched lens
> would be ok.
> Any explanations would be appreciated..
> Thanks,
> Simon
> P.S. I tried playing with the correction collar on the lens, but that made no
> difference.

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Martin Wessendorf-2]

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Dear Simon--

On 4/25/2013 4:55 AM, Simon Walker wrote:

> We have been imaging single live fluorescent cells in aqueous buffer held in
> suspension at a distance of ~80 um from the coverslip surface.  We need to
> visualise rapid changes in the 3D structure of the cells so have been trying to
> capture image stacks using spinning disk confocal (EM-CCD camera, CSU-10,
> 60x water immersion 1.27 NA lens).  The images we obtain are useless - really
> noisy, and the round cells apprear highly distorted (the fuzzy mass becomes
> elongated in x and then y as you focus up and down through the cell).  We
> then tried the same experiment on a point scanning confocal using exactly the
> same microscope body and lens, and got really nice images (but not obtained
> with sufficient speed to make the experiment viable).

Can the spinning disk instrument image a fluorescent bead or other
similar test specimen without distortion?  Or is the problem only with
your thick specimen?

Good luck!

Martin


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