Re: widefield getting better images than spinning disk

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585194.html

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Which is the widefield image, the first one or the second one?

John Oreopoulos

> On May 20, 2016, at 5:14 PM, "Reece, Jeff (NIH/NIDDK) [C]" <[hidden email]> wrote:
>
> 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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> To join, leave or search the confocal microscopy listserv, go to:
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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:
>>
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>>
>> 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."
>>