Re: widefield getting better images than spinning disk

Posted by Reece, Jeff (NIH/NIDDK) [E] on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585193.html

Hi Rafael,

How thick do you think the sample is?
The pinhole is ~3 Airy Units, which means the confocal section thickness is ~20 microns.
So if you had great SNR in the confocal image, you may not see a difference in the two images.  By eye they look very similar, except for the reduced # of photons in the confocal image.

The formula can be found here (Eq. 5):
http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html

I have it in an Excel spreadsheet if anyone is interested (but can't vouch for its complete accuracy).

Cheers,
Jeff


-----Original Message-----
From: Rafael Jaimes III [mailto:[hidden email]]
Sent: Friday, May 20, 2016 4:54 PM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

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Thanks Sam,

http://imgur.com/a/KQysd​

Let me know if the above album works for you. I uploaded two images: one in widefield and one in spinning disk confocal. All things else are equal. For this example I used a fixed sample with 80 ms exposure. The image is at 10x, 0.45 NA with confocal pinhole of 40 um. Is this amount of image degradation to be expected? I also tried turning up the light source intensity significantly, with not much difference.

Rafael

On Fri, May 20, 2016 at 12:57 PM, Sam Lord <[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.
> *****
>
> On Fri, 20 May 2016 12:46:37 -0400, Rafael Jaimes III
> <[hidden email]
> >
> wrote:
> >1. Live neonatal rat cardiomyocyte-fibroblast layers stained with
> >Fluo-4 for calcium. They are field stimulated and imaged >100 fps.
>
> At 10 ms exposure time, I doubt you're getting enough fluorescence
> hitting the camera to get a sufficient signal to noise. Wide field is
> always much brighter than confocal because both the excitation light
> and the emission intensity is reduced going through the pinholes.
>
> Why don't you send images of wide field vs confocal so we can see what
> you mean by "better."
>