Re: widefield getting better images than spinning disk

Posted by RJ3 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585195.html

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Sorry I didn't specify. The top image is the spinning disk. The widefield is at the bottom, you can see it has much better contrast, but it's not crisp. Everyone says the light levels are much poorer with the spinning disk, but I hope something else is wrong if they're looking like that. It is unusable in the current state.

Rafael

On May 20, 2016 7:23:05 PM EDT, John Oreopoulos <[hidden email]> wrote:

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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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>posting.
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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."
>>>