Posted by
RJ3 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585187.html
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1. Live neonatal rat cardiomyocyte-fibroblast layers stained with Fluo-4
for calcium. They are field stimulated and imaged >100 fps.
2. My crest is the dual pattern type: 40 and 70um pinholes. I use the 40um
for the 10x (0.45 NA) and 20x (0.8) objectives, and 70um for the 60x
(1.45) objective. This was at the recommendation by Nikon.
3. I tried stopping the disk to look at the holes. They look pretty good to
my untrained eye (I have not used a spinning disk before). I can certainly
try to optimize the focus, are there any specific tricks to doing this? For
example having a particular sample, light, or objective in place that would
affect the focusing process?
4. I will evaluate this, but the excitation light looked to me as it is
centered. I don't think I have a scan head? But I won't disregard the
possibility of some other component being out of alignment.
5. Since this disk is new to me, I don't have a good grasp on what an image
should look like for my typical samples. I was just surprised that the
widefields look better than when the disk is in place. Though the widefield
images do not look very crisp to me so I figured the disk would improve the
image quality. I will look into purchasing these slides, they sound like a
great thing to have around for testing the setup.
Thanks for all your help troubleshooting this.
Rafael Jaimes, PhD
Staff Scientist
Children's National Medical Center
Washington, DC
On Fri, May 20, 2016 at 12:29 AM, John Oreopoulos <
[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> *****
>
> There will be some sample-dependent situations where this might be the
> case, that confocal yields an image that is no better than widefield
> imaging. In fact, you should always ask yourself if confocal is even
> necessary to answer the question you're after in an imaging experiment. See
> for example:
>
> Murray, J.M., et al., Evaluating performance in three-dimensional
> fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p.
> 390-405.
>
> Also, if the specimen is really thin, confocal might not be so
> beneficial... But having said that, a few basic questions about your setup
> are in order:
>
> 1. What are you imaging?
>
> 2. What pinhole size are you using and with what objective lens
> (magnification and NA)? If the pinhole size is too large relative to the
> lateral and axial resolution afforded by the objective, you'll end up with
> a widefield-like image. Also consider that confocal imaging generally
> breaks down at low magnification (see end sections of Amos, B., G.
> McConnell, and T. Wilson, Confocal microscopy, in Comprehensive biophysics,
> E.H. Egelman, Editor. 2012, Elsevier: Amsterdam. p. 3-23.)
>
> 3. Is the camera well focused to the pinholes of the disk? Check by
> stopping the disk.
>
> 4. Is the scan head well aligned to the microscope? Check by looking for
> eyepiece parfocality and centration, and also check that the excitation
> light enters the back of the objective centred (be laser safe when looking
> for this).
>
> 5. Can you try imaging a "standard" sample in your lab as a sanity check
> (ie: a positive control)? Do you obtain good optical sectioning behaviour
> with a sample like that? Pollen grains (Carolina, no commercial interest),
> or the mouse kidney section (Life Technologies, no commercial interest),
> are good for a confocal sanity check. Imaging the autofluorescence of sheet
> of white paper is also an option.
>
> Just some thoughts,
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research
> A Division of Andor Technologies
> www.spectral.ca
>
>
>
> On 2016-05-19, at 11:45 PM, Rafael Jaimes 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.
> > *****
> >
> > This has me stumped. I suspected it was a lack of light but I turned up
> the
> > excitation light significantly and still haven't gotten good images with
> the
> > spinning disk in place. The widefield mode is giving much better images
> > which doesn't make any sense to me. I figured I'd ask for
> recommendations in
> > case anyone has been in a similar boat, especially with the hardware I'm
> > running:
> >
> > Nikon Ti w/ Crest X-Light v1 spinning disk. Detector is a Andor Zyla 4.2
> > PLUS sCMOS.
>