widefield getting better images than spinning disk

[RJ3]

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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.

[John Oreopoulos]

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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.

[RJ3]

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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:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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.
>

[samjlord]

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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."

[Andrea Latini]

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*****

Dear Rafael,
thank you for raising the question to the Confocal List and sorry for my late response. Luckily
John Oreopoulos answers/explanations are a very good starting point on how to optimize your
confocal system and better evaluate your experimental results and of course I fully agree with
him.
I'll be pleased to help you on pinholes focusing and overall system setup and to directly
evaluate your acquisitions.

regards.

Andrea Latini
President
CrestOptics srl

[RJ3]

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Hi Andrea,

Thanks for your reply! It means a lot to hear from the president of the
company.

I was also wondering -- is there an easy way to determine the maximum
acquisition speed for my disk? I have the 10,000 RPM variant. One of the
other users mentioned that 10ms exposure / 100 fps may be too fast, at
least regarding the light levels.​ Do you typically use EMCCD for these
disks to accommodate the loss of light? I am worried that my sCMOS is not
sensitive enough.

As some others requested, I will certainly post some pictures to imgur when
I get a chance.. please stand by.

Rafael

On Fri, May 20, 2016 at 2:45 PM, Andrea Latini <[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.
> *****
>
> Dear Rafael,
> thank you for raising the question to the Confocal List and sorry for my
> late response. Luckily
> John Oreopoulos answers/explanations are a very good starting point on how
> to optimize your
> confocal system and better evaluate your experimental results and of
> course I fully agree with
> him.
> I'll be pleased to help you on pinholes focusing and overall system setup
> and to directly
> evaluate your acquisitions.
>
> regards.
>
> Andrea Latini
> President
> CrestOptics srl
>

[Andrea Latini]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Rafael,

Confocal System limitation:
your confocal system max acquisition speed is determined by disk rotation speed and total
number of even detector FOV pinholes overlap for single disk revolution;
in your case: 10.000 RPM, 24 even overlaps per revolution,  means 6ms/24= 250us for an
even sample pinholes overlap.
hence, exposure time set at minimum of 250us gives you a maximum frame rate of 4000fps
and exposure time set at 250us * N (N integer), gives you a striping free acquisition.

the disk motor has an accuracy of 10.000 RPM +/- 50RPM (0.5%) and the 250us must be
fine tuned on your system's precise rotation speed.

with that said, in more than 7 years I've seen only one experimental setup and sample whit
a fluorescence signal bright enough to be detected at 1ms exposure time on an EMCCD
detector with Region of Interest and Readout Time fine tuning.

in other words, the maximum frame rate you will achieve will depend on:
- sample fluorescence photon flux
- optical power of your excitation source
- Magnification and NA of your objective
- throughput of your Confocal System (for both excitation and signal collection)

it's a balance and optimization of each of the above variables, mostly, that will give you a
good experimental result and an increased knowledge of your Confocal System capabilities
and limitations.

Andrea Latini
President
CrestOptics Srl    


On Fri, 20 May 2016 15:40:59 -0400, Rafael Jaimes III <[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.
>*****
>
>Hi Andrea,
>
>Thanks for your reply! It means a lot to hear from the president of the
>company.
>
>I was also wondering -- is there an easy way to determine the maximum
>acquisition speed for my disk? I have the 10,000 RPM variant. One of the
>other users mentioned that 10ms exposure / 100 fps may be too fast, at
>least regarding the light levels.​ Do you typically use EMCCD for these
>disks to accommodate the loss of light? I am worried that my sCMOS is not
>sensitive enough.
>
>As some others requested, I will certainly post some pictures to imgur when
>I get a chance.. please stand by.
>
>Rafael
>
>On Fri, May 20, 2016 at 2:45 PM, Andrea Latini <[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.
>> *****
>>
>> Dear Rafael,
>> thank you for raising the question to the Confocal List and sorry for my
>> late response. Luckily
>> John Oreopoulos answers/explanations are a very good starting point on how
>> to optimize your
>> confocal system and better evaluate your experimental results and of
>> course I fully agree with
>> him.
>> I'll be pleased to help you on pinholes focusing and overall system setup
>> and to directly
>> evaluate your acquisitions.
>>
>> regards.
>>
>> Andrea Latini
>> President
>> CrestOptics srl
>>

[RJ3]

*****
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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."
>

[Reece, Jeff (NIH/NIDDK) [E]]

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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Post images on http://www.imgur.com and include the link in your posting.
*****

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."
>

[John Oreopoulos]

*****
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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
>
> *****
> 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.
> *****
>
> 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."
>>

[RJ3]

*****
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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:

>*****
>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.
>*****
>
>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
>>
>> *****
>> 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.
>> *****
>>
>> 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."
>>>

[Andrea Latini]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Rafael,
from your images it looks like there's clearly a poor S/N in your confocal acquisition.

please check:
- pinholes pattern correct focusing on sCMOS detector (we can do that together remotely,
please connect through email)
- excitation launcher optical optimization (fiber, focal plane) (remotely, as well)

regards.

Andrea Latini
President
CrestOptics Srl

On Fri, 20 May 2016 22:01:32 -0400, Rafael Jaimes III <[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.
>*****
>
>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:

>>*****
>>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.
>>*****
>>
>>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
>>>
>>> *****
>>> 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.
>>> *****
>>>
>>> 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."
>>>>

[samjlord]

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I would recommmend setting the exposure time to 100 ms or however long an exposure it
takes to get a good image. If the image gets bright but the contrast still looks worse than
wide field, then maybe there's an alignment issue. But I suspect your sample is just too dim
to image with confocal at 10 ms per frame. Not many samples are bright enough for that.
Remember that confocal is designed to throw out light in order to improve optical slicing.

[Guy Cox-2]

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Repeat 100 times:

CONFOCAL ONLY THROWS OUT
OUT OF FOCUS LIGHT!  

The reason spinning disk systems lose light is to attain speed, and has nothing to do with the confocal principle.  

                                   Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Lord
Sent: Sunday, 22 May 2016 10:45 AM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

*****
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I would recommmend setting the exposure time to 100 ms or however long an exposure it takes to get a good image. If the image gets bright but the contrast still looks worse than wide field, then maybe there's an alignment issue. But I suspect your sample is just too dim to image with confocal at 10 ms per frame. Not many samples are bright enough for that.
Remember that confocal is designed to throw out light in order to improve optical slicing.

[Ian Dobbie]

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Except in the excitation path where it throws out loads of lift which don't make it through the pinholes.

The excitation might be 10x or even 100x less than the widefield case. You really need to measure the light intensity at the sample.

Ian

Sent from my iPhone

> On 22 May 2016, at 07:17, Guy Cox <[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.
> *****
>
> Repeat 100 times:
>
> CONFOCAL ONLY THROWS OUT
> OUT OF FOCUS LIGHT!  
>
> The reason spinning disk systems lose light is to attain speed, and has nothing to do with the confocal principle.  
>
>                                   Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis,
> Madsen, F09, University of Sydney, NSW 2006
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Lord
> Sent: Sunday, 22 May 2016 10:45 AM
> To: [hidden email]
> Subject: Re: widefield getting better images than spinning disk
>
> *****
> 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.
> *****
>
> I would recommmend setting the exposure time to 100 ms or however long an exposure it takes to get a good image. If the image gets bright but the contrast still looks worse than wide field, then maybe there's an alignment issue. But I suspect your sample is just too dim to image with confocal at 10 ms per frame. Not many samples are bright enough for that.
> Remember that confocal is designed to throw out light in order to improve optical slicing.

[Reece, Jeff (NIH/NIDDK) [E]]

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I was wondering how long it would take before Guy responded.  There have been discussions on the listserv in years past about the effect of pinhole on brightness, and Guy has always stressed this basic concept.  I can't find those previous discussions, but there is a nice graph here that illustrates the concept (scroll down to Figure 6, the graph on the right):
http://www.leica-microsystems.com/science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/ (disclaimer: this is the only place I could find it on the web; thanks Leica)
which makes sense if you look at Figure 3 on the same page, just visually estimating how much of the total light from the psf must be inside the Airy Disk.
So, another way to state the same concept: only 16% of in-focus light is thrown away when the pinhole is ~1 AU (i.e. "confocal" in the general sense).  
If anyone has the original references that show the graph of integrated intensity vs AU, or another place it might be on the web for free, I would be interested.  Perhaps those links are with the previous discussion on the listserv that I can't find.

With the 10x/0.45 lens, and the pinhole at ~3 AU, you are collecting more like 94% of the in-focus light.
And since the FWHM z-resolution is ~20 microns for that pinhole and lens (assuming 500nm as the emission wavelength), then you are collecting ~47% of the out-of-focus light that originates from 10 microns away from the focal plane.

All theoretical of course, but usually not far off when the system is aligned properly.  ;-)

Hope that helps.
Cheers,
Jeff


-----Original Message-----
From: Guy Cox [mailto:[hidden email]]
Sent: Sunday, May 22, 2016 1:55 AM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Repeat 100 times:

CONFOCAL ONLY THROWS OUT
OUT OF FOCUS LIGHT!  

The reason spinning disk systems lose light is to attain speed, and has nothing to do with the confocal principle.  

                                   Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis, Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Lord
Sent: Sunday, 22 May 2016 10:45 AM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

*****
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.
*****

I would recommmend setting the exposure time to 100 ms or however long an exposure it takes to get a good image. If the image gets bright but the contrast still looks worse than wide field, then maybe there's an alignment issue. But I suspect your sample is just too dim to image with confocal at 10 ms per frame. Not many samples are bright enough for that.
Remember that confocal is designed to throw out light in order to improve optical slicing.

[Reece, Jeff (NIH/NIDDK) [E]]

*****
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*****

I was wondering how long it would take before Guy responded.  There have been discussions on the listserv in years past about signal as a function of confocal pinhole, and Guy has always stressed this basic concept.  I can't find those previous discussions, but there is a nice graph here that illustrates the concept (scroll down to Figure 6, the graph on the right):
http://www.leica-microsystems.com/science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/ (disclaimer: this is the only place I could find it on the web; thanks Leica) which makes sense if you look at Figure 3 on the same page, just visually estimating how much of the total light from the psf must be inside the Airy Disk.
So, another way to state the same concept: only 16% of in-focus light is thrown away when the pinhole is ~1 AU (i.e. "confocal" in the general sense).  
If anyone has the original references that show the graph of integrated intensity vs AU, or another place it might be on the web for free, I would be interested.  Perhaps those links are with the previous discussion on the listserv that I can't find.

With the 10x/0.45 lens, and the pinhole at ~3 AU, you are collecting more like 94% of the in-focus light.
And since the FWHM z-resolution is ~20 microns for that pinhole and lens (assuming 500nm as the emission wavelength), then you are collecting ~47% of the out-of-focus light that originates from 10 microns away from the focal plane.  

My understanding of the Crest Disk, from the info I see on their web site, is that throughput on the excitation side is only 1.6%, compared to widefield illumination (disk pulled out).  So if you increase the camera exposure time by a factor of ~70 on the confocal image, the signal levels of the in-focus portion should be similar to the widefield image, if everything is aligned properly.  Theoretically.

Hope that helps.
Cheers,
Jeff


-----Original Message-----
From: Guy Cox [mailto:[hidden email]]
Sent: Sunday, May 22, 2016 1:55 AM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Repeat 100 times:

CONFOCAL ONLY THROWS OUT
OUT OF FOCUS LIGHT!  

The reason spinning disk systems lose light is to attain speed, and has nothing to do with the confocal principle.  

                                   Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis, Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Lord
Sent: Sunday, 22 May 2016 10:45 AM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

I would recommmend setting the exposure time to 100 ms or however long an exposure it takes to get a good image. If the image gets bright but the contrast still looks worse than wide field, then maybe there's an alignment issue. But I suspect your sample is just too dim to image with confocal at 10 ms per frame. Not many samples are bright enough for that.
Remember that confocal is designed to throw out light in order to improve optical slicing.

[Andrea Latini]

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dear Jeff,
collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
(fill factor 8.2%). 4.6-4.8% with 60um (fill factor 5.8%). about 3-3.5% with
40um pinholes (fill factor 4.2%). values measured at 500nm.

hence, my proposal to double check for proper system alignment (pinholes to
detector focusing and excitation).

regards
Andrea

Andrea Latini
President
CrestOptics Srl


On Sun, 22 May 2016 18:03:48 +0000, Reece, Jeff (NIH/NIDDK) [C]
<[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.
>*****
>
>I was wondering how long it would take before Guy responded.  There have
been discussions on the listserv in years past about signal as a function of
confocal pinhole, and Guy has always stressed this basic concept.  I can't
find those previous discussions, but there is a nice graph here that
illustrates the concept (scroll down to Figure 6, the graph on the right):
>http://www.leica-microsystems.com/science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/
(disclaimer: this is the only place I could find it on the web; thanks
Leica) which makes sense if you look at Figure 3 on the same page, just
visually estimating how much of the total light from the psf must be inside
the Airy Disk.
>So, another way to state the same concept: only 16% of in-focus light is
thrown away when the pinhole is ~1 AU (i.e. "confocal" in the general sense).  
>If anyone has the original references that show the graph of integrated
intensity vs AU, or another place it might be on the web for free, I would
be interested.  Perhaps those links are with the previous discussion on the
listserv that I can't find.
>
>With the 10x/0.45 lens, and the pinhole at ~3 AU, you are collecting more
like 94% of the in-focus light.
>And since the FWHM z-resolution is ~20 microns for that pinhole and lens
(assuming 500nm as the emission wavelength), then you are collecting ~47% of
the out-of-focus light that originates from 10 microns away from the focal
plane.  
>
>My understanding of the Crest Disk, from the info I see on their web site,
is that throughput on the excitation side is only 1.6%, compared to
widefield illumination (disk pulled out).  So if you increase the camera
exposure time by a factor of ~70 on the confocal image, the signal levels of
the in-focus portion should be similar to the widefield image, if everything
is aligned properly.  Theoretically.

>
>Hope that helps.
>Cheers,
>Jeff
>
>
>-----Original Message-----
>From: Guy Cox [mailto:[hidden email]]
>Sent: Sunday, May 22, 2016 1:55 AM
>To: [hidden email]
>Subject: Re: widefield getting better images than spinning disk
>
>*****
>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.
>*****
>
>Repeat 100 times:
>
>CONFOCAL ONLY THROWS OUT
>OUT OF FOCUS LIGHT!  
>
>The reason spinning disk systems lose light is to attain speed, and has

nothing to do with the confocal principle.  
>
>                                   Guy
>
>Guy Cox, Honorary Associate Professor
>School of Medical Sciences
>
>Australian Centre for Microscopy and Microanalysis, Madsen, F09, University
of Sydney, NSW 2006
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Sam Lord

>Sent: Sunday, 22 May 2016 10:45 AM
>To: [hidden email]
>Subject: Re: widefield getting better images than spinning disk
>
>*****
>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.
>*****
>
>I would recommmend setting the exposure time to 100 ms or however long an

exposure it takes to get a good image. If the image gets bright but the
contrast still looks worse than wide field, then maybe there's an alignment
issue. But I suspect your sample is just too dim to image with confocal at
10 ms per frame. Not many samples are bright enough for that.
>Remember that confocal is designed to throw out light in order to improve
optical slicing.

[RJ3]

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Hi all,

I'm still trying to wrap my head around the light loss situation. So I
should expect to collect the majority of in-focus light, but overall only
~5% light is collected compared to widefield? For excitation, only 1.6% of
light is transmitted through the disk to the specimen?

​ I am fairly certain the camera is focused correctly on the pinholes. But,
I am not sure about the other knob on the top of the Crest, that is one of
the things I am looking into now.

Thanks,
Rafael

On Sun, May 22, 2016 at 3:26 PM, Andrea Latini <[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.
> *****
>
> dear Jeff,
> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
> (fill factor 8.2%). 4.6-4.8% with 60um (fill factor 5.8%). about 3-3.5%
> with
> 40um pinholes (fill factor 4.2%). values measured at 500nm.
>
> hence, my proposal to double check for proper system alignment (pinholes to
> detector focusing and excitation).
>
> regards
> Andrea
>
> Andrea Latini
> President
> CrestOptics Srl
>
>
> On Sun, 22 May 2016 18:03:48 +0000, Reece, Jeff (NIH/NIDDK) [C]
> <[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.
> >*****
> >
> >I was wondering how long it would take before Guy responded.  There have
> been discussions on the listserv in years past about signal as a function
> of
> confocal pinhole, and Guy has always stressed this basic concept.  I can't
> find those previous discussions, but there is a nice graph here that
> illustrates the concept (scroll down to Figure 6, the graph on the right):
> >
> http://www.leica-microsystems.com/science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/
> (disclaimer: this is the only place I could find it on the web; thanks
> Leica) which makes sense if you look at Figure 3 on the same page, just
> visually estimating how much of the total light from the psf must be inside
> the Airy Disk.
> >So, another way to state the same concept: only 16% of in-focus light is
> thrown away when the pinhole is ~1 AU (i.e. "confocal" in the general
> sense).
> >If anyone has the original references that show the graph of integrated
> intensity vs AU, or another place it might be on the web for free, I would
> be interested.  Perhaps those links are with the previous discussion on the
> listserv that I can't find.
> >
> >With the 10x/0.45 lens, and the pinhole at ~3 AU, you are collecting more
> like 94% of the in-focus light.
> >And since the FWHM z-resolution is ~20 microns for that pinhole and lens
> (assuming 500nm as the emission wavelength), then you are collecting ~47%
> of
> the out-of-focus light that originates from 10 microns away from the focal
> plane.
> >
> >My understanding of the Crest Disk, from the info I see on their web site,
> is that throughput on the excitation side is only 1.6%, compared to
> widefield illumination (disk pulled out).  So if you increase the camera
> exposure time by a factor of ~70 on the confocal image, the signal levels
> of
> the in-focus portion should be similar to the widefield image, if
> everything
> is aligned properly.  Theoretically.
> >
> >Hope that helps.
> >Cheers,
> >Jeff
> >
> >
> >-----Original Message-----
> >From: Guy Cox [mailto:[hidden email]]
> >Sent: Sunday, May 22, 2016 1:55 AM
> >To: [hidden email]
> >Subject: Re: widefield getting better images than spinning disk
> >
> >*****
> >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.
> >*****
> >
> >Repeat 100 times:
> >
> >CONFOCAL ONLY THROWS OUT
> >OUT OF FOCUS LIGHT!
> >
> >The reason spinning disk systems lose light is to attain speed, and has
> nothing to do with the confocal principle.
> >
> >                                   Guy
> >
> >Guy Cox, Honorary Associate Professor
> >School of Medical Sciences
> >
> >Australian Centre for Microscopy and Microanalysis, Madsen, F09,
> University
> of Sydney, NSW 2006
> >
> >-----Original Message-----
> >From: Confocal Microscopy List [mailto:[hidden email]]
> On
> Behalf Of Sam Lord
> >Sent: Sunday, 22 May 2016 10:45 AM
> >To: [hidden email]
> >Subject: Re: widefield getting better images than spinning disk
> >
> >*****
> >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.
> >*****
> >
> >I would recommmend setting the exposure time to 100 ms or however long an
> exposure it takes to get a good image. If the image gets bright but the
> contrast still looks worse than wide field, then maybe there's an alignment
> issue. But I suspect your sample is just too dim to image with confocal at
> 10 ms per frame. Not many samples are bright enough for that.
> >Remember that confocal is designed to throw out light in order to improve
> optical slicing.
>

[Andrea Latini]

*****
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*****

Rafael,
please connect with me at [hidden email], our engineers will setup a remote session.
thank you.


Andrea Latini
President
CrestOptics. Srl