widefield getting better images than spinning disk

[Nico Stuurman-3]

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Dear Andrea,

> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes (fill factor 8.2%).

Could you please explain what that number means, or better, how it is
measured?  The way it is written, I have the impression that when a very
thin film of fluorescent material is excited by the same amount of light
you get 5-6% intensity in confocal mode and 100% in wide-field mode.  
Obviously, that would be pretty bad (and according to the discussion on
this list, you should not lose more than a few percent of the
in-focus-signal through the pinholes), so I assume that you mean
something else.

Best,

Nico

[Michael Giacomelli]

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Hi Rafael,

With any confocal system you'll get most of the light from a very thin
section of the sample (on the order of a few microns), and very little
light from elsewhere.  If your specimen is 30 microns thick, and your
confocal section is 3 microns thick, then you'll get about 10% of the
light in the confocal case (ignoring losses due to the disk, etc) as
compared to widefield.  So yes, it'll be a lot darker just because you
are imaging so much less material per pixel.

Using spinning disk rather than ordinary confocal further means that
the illumination is greatly attenuated, but you can compensate for
that just by turning up the laser power.

Mike

On Mon, May 23, 2016 at 10:34 AM, 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 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.
>>

[Tim Feinstein]

Hi Rafael,

I would break down how the two technologies work in very small time fragments.  In a 10 ms exposure the spinning disc is still adding together a huge number of small point exposures.  In fact in that time a specific fluorescent molecule will get exited a few times at best for maybe a couple microseconds at a time.  To counteract the inevitable noise problem from such short exposures you do a ton of averaging, in other words exposing for long enough for the disc to spin many times.  The excitation power at each point is somewhat brighter than widefield because of the focused laser, but it cannot be orders of magnitude brighter or you will rapidly destroy your sample by causing a ton of triplet state excitation.  Resonant point-scanning confocals have a similar limitation, so people generally use averaging in resonant mode unless speed is of utmost importance.  
       
In a widefield, if you expose the image for 10 ms then the detector sees every fluorescent molecule for the full 10 ms.  That means each pixel has had a chance to see a lot more emitted photons than a pixel in a spinning disc that has been integrating over the same period of time.  

Best,


Tim
               
Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Dept. of Developmental Biology



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rafael Jaimes III
Sent: Monday, May 23, 2016 10:34 AM
To: [hidden email]
Subject: Re: widefield getting better images than spinning disk

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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-he
> uristic-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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*****

dear Nico,
the spinning disk  itself acts as an optical filter on thick samples.
Spinning Disk fill factor means 'Open Area Ratio' - holes area / blocked area.
by inserting the disk within the optical path the theoretical throughput
collection value would be 8.2% with respect to widefield (both excitation
and emission pass through spinning disk of course), on 4 um beads, GFP
excitation channel.

the value 5-6% on 4um beads (and other values I'm reporting), has been
measured by using "Measuring and interpreting point spread functions to
determine confocal microscope resolution and ensure quality control Richard
W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume 6, Pages:
1929–1941(2011)" method.

briefly (Macro is running in NIS Element SW):
"

1.) Sample: subresolution beads prepared according to „Measuring and
interpreting point spread functions to determine confocal microscope
resolution and ensure quality control
Richard W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume
6, Pages: 1929–1941(2011)
2.) Technical setup of Microscope:
a. The microscope is set up   for a typical point spread function
acquisition to characterize the resolution/imaging quality using a high-end
 100x oil objective.
b. A regular fluorescence light source is used for all acquisitions in
combination with a filter cube.  This is essential and the bottom principle
of the test. The same illumination with the same sample is used to compare
wide field intensity with the confocal intensity of bead images. This test
assesses only the emission light transmission of the spinning disc device.    
i. Option one preferred: left and right port of the microscope is used so
that one port holds the spinning disc device with camera and the other port
an identical second camera in simple wide field mode.
ii. Alternatively one can run the acquisition of test data first with the
spinning disc  +camera and then remove the scan head and use the same camera
alone on the same port where the spinning disc scan head was mounted.
iii. Toggle between wide field mode and spinning disc mode if the to be
tested scan head provides such an option and (important!) if  the wide field
mode does not have more than a single mirror in the light path  (otherwise
the transmission loss of the additional optical elements in the wide field
mode of the scan head results in a lower intensity measurements . This in
turn would artificially “improve” the result so that one would think the
ratio wide field intensity / confocal intensity  is higher and therefore
pretending to be the better instrument)  

3.) Procedure
Time required:  about 1 -2 hours
Overview of work flow: acquisition of image z-stacks of beads at 3 different
exposure times in each mode (confocal and wide field) while illumination
remains constant.
Prepare collapsed stack images (max projection)
rolling ball background reduction
find beads and set ROIs
calculate mean or median intensity (in our hands it does not make a difference)
plot intenstity vs. exposure time for both modes
determine slope
ratio of slopes = result

In detail:
a.) Set up beads in focus with the scanning device. The signal should be
chosen at an intensity (illumination light intensity ) , so that with the
same illumination wide field images are possible (sufficiently short shutter
time should be available)
b.) Stacks of images are taken at  3 different exposure times of different
view fields to avoid the issue of bleaching,
c.) Repeat the steps a) and b) in wide field mode  
d.) Run macro in ImageJ (enclosed) to analyze bead intensity

Find different ROIs in each maximum projection :

run("Z Project...", "projection=[Max Intensity]");
run("Subtract Background...", "rolling=5");
run("Threshold...");
 title = "WaitForUserDemo";
  msg = "If necessary, use the \"Threshold\" tool to\nadjust the threshold,
then click \"OK\".";
  waitForUser(title, msg);
getThreshold(lower, upper)
run("Analyze Particles...", "size=1-4 exclude summarize");


or:   you run the macro above on the  stack with the lowest exposure time
and then – to keep the ROIs the same for all stacks- yu run the macro below
on the remaining stacks

run("Z Project...", "projection=[Max Intensity]");
run("Subtract Background...", "rolling=5");
roiManager("measure")


e.) Prepare plots of the so calculated intensities vs the exposure time
f.) Calculate slope
g.) Ratio the slope.

"


regards.

Andrea

Andrea Latini
President
CrestOptics Srl


On Mon, 23 May 2016 08:42:07 -0700, Nico Stuurman <[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 Andrea,
>
>> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
(fill factor 8.2%).

>
>Could you please explain what that number means, or better, how it is
>measured?  The way it is written, I have the impression that when a very
>thin film of fluorescent material is excited by the same amount of light
>you get 5-6% intensity in confocal mode and 100% in wide-field mode.
>Obviously, that would be pretty bad (and according to the discussion on
>this list, you should not lose more than a few percent of the
>in-focus-signal through the pinholes), so I assume that you mean
>something else.
>
>Best,
>
>Nico

[Zdenek Svindrych-2]

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*****

Dear Andrea,
quite off the original topic, but, whatever...

As already mentioned, it's very important to separate the overall efficiency
into excitation efficiency and collection efficiency.

5% excitation efficiency would be tolerable, e.g. with a spinning disk
without microlenses...

But in detection path "every photon counts", 80% efficiency should be
possible with > 1AU pinholes.

What's missing in the protocol is "measure the illumination intensity in the
BFP (or image plane) of the objective in both cases and do the math"...

And what would be the useful piece of information for the customer? Perhaps
something like "increase tour illumination power 27.35 times (don't quote me
here :-) to get the same average illumination intensity if you want to
compare spinning disk to widefield..."

Best, zdenek






---------- Původní zpráva ----------
Od: Andrea Latini <[hidden email]>
Komu: [hidden email]
Datum: 23. 5. 2016 14:40:42
Předmět: Re: [QUAR] 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.
*****

dear Nico,
the spinning disk itself acts as an optical filter on thick samples.
Spinning Disk fill factor means 'Open Area Ratio' - holes area / blocked
area.
by inserting the disk within the optical path the theoretical throughput
collection value would be 8.2% with respect to widefield (both excitation
and emission pass through spinning disk of course), on 4 um beads, GFP
excitation channel.

the value 5-6% on 4um beads (and other values I'm reporting), has been
measured by using "Measuring and interpreting point spread functions to
determine confocal microscope resolution and ensure quality control Richard
W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume 6, Pages:
1929–1941(2011)" method.

briefly (Macro is running in NIS Element SW):
"

1.) Sample: subresolution beads prepared according to „Measuring and
interpreting point spread functions to determine confocal microscope
resolution and ensure quality control
Richard W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume
6, Pages: 1929–1941(2011)
2.) Technical setup of Microscope:
a. The microscope is set up for a typical point spread function
acquisition to characterize the resolution/imaging quality using a high-end
100x oil objective.
b. A regular fluorescence light source is used for all acquisitions in
combination with a filter cube. This is essential and the bottom principle
of the test. The same illumination with the same sample is used to compare
wide field intensity with the confocal intensity of bead images. This test
assesses only the emission light transmission of the spinning disc device.
i. Option one preferred: left and right port of the microscope is used so
that one port holds the spinning disc device with camera and the other port
an identical second camera in simple wide field mode.
ii. Alternatively one can run the acquisition of test data first with the
spinning disc +camera and then remove the scan head and use the same camera
alone on the same port where the spinning disc scan head was mounted.
iii. Toggle between wide field mode and spinning disc mode if the to be
tested scan head provides such an option and (important!) if the wide field
mode does not have more than a single mirror in the light path (otherwise
the transmission loss of the additional optical elements in the wide field
mode of the scan head results in a lower intensity measurements . This in
turn would artificially “improve” the result so that one would think the
ratio wide field intensity / confocal intensity is higher and therefore
pretending to be the better instrument)

3.) Procedure
Time required: about 1 -2 hours
Overview of work flow: acquisition of image z-stacks of beads at 3 different
exposure times in each mode (confocal and wide field) while illumination
remains constant.
Prepare collapsed stack images (max projection)
rolling ball background reduction
find beads and set ROIs
calculate mean or median intensity (in our hands it does not make a
difference)
plot intenstity vs. exposure time for both modes
determine slope
ratio of slopes = result

In detail:
a.) Set up beads in focus with the scanning device. The signal should be
chosen at an intensity (illumination light intensity ) , so that with the
same illumination wide field images are possible (sufficiently short shutter
time should be available)
b.) Stacks of images are taken at 3 different exposure times of different
view fields to avoid the issue of bleaching,
c.) Repeat the steps a) and b) in wide field mode
d.) Run macro in ImageJ (enclosed) to analyze bead intensity

Find different ROIs in each maximum projection :

run("Z Project...", "projection=[Max Intensity]");
run("Subtract Background...", "rolling=5");
run("Threshold...");
title = "WaitForUserDemo";
msg = "If necessary, use the \"Threshold\" tool to\nadjust the threshold,
then click \"OK\".";
waitForUser(title, msg);
getThreshold(lower, upper)
run("Analyze Particles...", "size=1-4 exclude summarize");


or: you run the macro above on the stack with the lowest exposure time
and then – to keep the ROIs the same for all stacks- yu run the macro below
on the remaining stacks

run("Z Project...", "projection=[Max Intensity]");
run("Subtract Background...", "rolling=5");
roiManager("measure")


e.) Prepare plots of the so calculated intensities vs the exposure time
f.) Calculate slope
g.) Ratio the slope.

"


regards.

Andrea

Andrea Latini
President
CrestOptics Srl


On Mon, 23 May 2016 08:42:07 -0700, Nico Stuurman <[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 Andrea,
>
>> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
(fill factor 8.2%).

>
>Could you please explain what that number means, or better, how it is
>measured? The way it is written, I have the impression that when a very
>thin film of fluorescent material is excited by the same amount of light
>you get 5-6% intensity in confocal mode and 100% in wide-field mode.
>Obviously, that would be pretty bad (and according to the discussion on
>this list, you should not lose more than a few percent of the
>in-focus-signal through the pinholes), so I assume that you mean
>something else.
>
>Best,
>
>Nico"

[Andrea Latini]

*****
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 Zdenek,
I guess this comparison, we've got from enduser, would be interesting for
others.
using same protocol.

"..
- Throughput confocal performance ratio (CF/WF %), taking into account both
excitation and collection efficiency:

o Visitech 1% efficiency
o Yoko CSU X1  2.8% efficiency
o Crest X-Light 5.5%-6.3% efficiency

Note: we have been using 200nm beads, 100X 1,49 obj and SpectraX LED system
(not even lasers), Evolve CCD
.."

regards.

Andrea Latini
President
CrestOptics Srl



On Mon, 23 May 2016 21:21:14 +0200, Zdenek Svindrych <[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 Andrea,
quite off the original topic, but, whatever...

As already mentioned, it's very important to separate the overall efficiency
into excitation efficiency and collection efficiency.

5% excitation efficiency would be tolerable, e.g. with a spinning disk
without microlenses...

But in detection path "every photon counts", 80% efficiency should be
possible with > 1AU pinholes.

What's missing in the protocol is "measure the illumination intensity in the
BFP (or image plane) of the objective in both cases and do the math"...

And what would be the useful piece of information for the customer? Perhaps
something like "increase tour illumination power 27.35 times (don't quote me
here :-) to get the same average illumination intensity if you want to
compare spinning disk to widefield..."

Best, zdenek






---------- Původní zpráva ----------
Od: Andrea Latini <[hidden email]>
Komu: [hidden email]
Datum: 23. 5. 2016 14:40:42
Předmět: Re: [QUAR] 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.
*****

dear Nico,
the spinning disk itself acts as an optical filter on thick samples.
Spinning Disk fill factor means 'Open Area Ratio' - holes area / blocked
area.
by inserting the disk within the optical path the theoretical throughput
collection value would be 8.2% with respect to widefield (both excitation
and emission pass through spinning disk of course), on 4 um beads, GFP
excitation channel.

the value 5-6% on 4um beads (and other values I'm reporting), has been
measured by using "Measuring and interpreting point spread functions to
determine confocal microscope resolution and ensure quality control Richard
W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume 6, Pages:
1929–1941(2011)" method.

briefly (Macro is running in NIS Element SW):
"

1.) Sample: subresolution beads prepared according to „Measuring and
interpreting point spread functions to determine confocal microscope
resolution and ensure quality control
Richard W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume
6, Pages: 1929–1941(2011)
2.) Technical setup of Microscope:
a. The microscope is set up for a typical point spread function
acquisition to characterize the resolution/imaging quality using a high-end
100x oil objective.
b. A regular fluorescence light source is used for all acquisitions in
combination with a filter cube. This is essential and the bottom principle
of the test. The same illumination with the same sample is used to compare
wide field intensity with the confocal intensity of bead images. This test
assesses only the emission light transmission of the spinning disc device.
i. Option one preferred: left and right port of the microscope is used so
that one port holds the spinning disc device with camera and the other port
an identical second camera in simple wide field mode.
ii. Alternatively one can run the acquisition of test data first with the
spinning disc +camera and then remove the scan head and use the same camera
alone on the same port where the spinning disc scan head was mounted.
iii. Toggle between wide field mode and spinning disc mode if the to be
tested scan head provides such an option and (important!) if the wide field
mode does not have more than a single mirror in the light path (otherwise
the transmission loss of the additional optical elements in the wide field
mode of the scan head results in a lower intensity measurements . This in
turn would artificially “improve� the result so that one would think the
ratio wide field intensity / confocal intensity is higher and therefore
pretending to be the better instrument)

3.) Procedure
Time required: about 1 -2 hours
Overview of work flow: acquisition of image z-stacks of beads at 3 different
exposure times in each mode (confocal and wide field) while illumination
remains constant.
Prepare collapsed stack images (max projection)
rolling ball background reduction
find beads and set ROIs
calculate mean or median intensity (in our hands it does not make a
difference)
plot intenstity vs. exposure time for both modes
determine slope
ratio of slopes = result

In detail:
a.) Set up beads in focus with the scanning device. The signal should be
chosen at an intensity (illumination light intensity ) , so that with the
same illumination wide field images are possible (sufficiently short shutter
time should be available)
b.) Stacks of images are taken at 3 different exposure times of different
view fields to avoid the issue of bleaching,
c.) Repeat the steps a) and b) in wide field mode
d.) Run macro in ImageJ (enclosed) to analyze bead intensity

Find different ROIs in each maximum projection :

run("Z Project...", "projection=[Max Intensity]");
run("Subtract Background...", "rolling=5");
run("Threshold...");
title = "WaitForUserDemo";
msg = "If necessary, use the \"Threshold\" tool to\nadjust the threshold,
then click \"OK\".";
waitForUser(title, msg);
getThreshold(lower, upper)
run("Analyze Particles...", "size=1-4 exclude summarize");


or: you run the macro above on the stack with the lowest exposure time
and then – to keep the ROIs the same for all stacks- yu run the macro below
on the remaining stacks

run("Z Project...", "projection=[Max Intensity]");
run("Subtract Background...", "rolling=5");
roiManager("measure")


e.) Prepare plots of the so calculated intensities vs the exposure time
f.) Calculate slope
g.) Ratio the slope.

"


regards.

Andrea

Andrea Latini
President
CrestOptics Srl


On Mon, 23 May 2016 08:42:07 -0700, Nico Stuurman <[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 Andrea,
>
>> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
(fill factor 8.2%).

>
>Could you please explain what that number means, or better, how it is
>measured? The way it is written, I have the impression that when a very
>thin film of fluorescent material is excited by the same amount of light
>you get 5-6% intensity in confocal mode and 100% in wide-field mode.
>Obviously, that would be pretty bad (and according to the discussion on
>this list, you should not lose more than a few percent of the
>in-focus-signal through the pinholes), so I assume that you mean
>something else.
>
>Best,
>
>Nico"

[Andrea Latini]

*****
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.
*****

With full respect for the below named Vendors please allow me to specify
that my below reported measurements were not carried out at our labs nor
confirmed by us (we did confirm only our system final results, of course).
Enduser carried them out in specific experimental conditions that were not
under our control.
Reason for posting them was in trying to answer Zdenek's question about
'what is useful piece of information for the customer' in using such
measurement protocols: is some specific case they 'could' be used for system
comparison and performance evaluation under experimental condition of own
interest.

Hence, they must not be intended as different Vendor's Confocal System
'overall and general' performance comparison.

regards.


Andrea Latini
President
CrestOptics Srl


On Mon, 23 May 2016 15:13:13 -0500, 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 Zdenek,
>I guess this comparison, we've got from enduser, would be interesting for
>others.
>using same protocol.
>
>"..
>- Throughput confocal performance ratio (CF/WF %), taking into account both
>excitation and collection efficiency:
>
>o Visitech 1% efficiency
>o Yoko CSU X1  2.8% efficiency
>o Crest X-Light 5.5%-6.3% efficiency
>
>Note: we have been using 200nm beads, 100X 1,49 obj and SpectraX LED system
>(not even lasers), Evolve CCD
>.."
>
>regards.
>
>Andrea Latini
>President
>CrestOptics Srl
>
>
>
>On Mon, 23 May 2016 21:21:14 +0200, Zdenek Svindrych <[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 Andrea,
>quite off the original topic, but, whatever...
>
>As already mentioned, it's very important to separate the overall efficiency
>into excitation efficiency and collection efficiency.
>
>5% excitation efficiency would be tolerable, e.g. with a spinning disk
>without microlenses...
>
>But in detection path "every photon counts", 80% efficiency should be
>possible with > 1AU pinholes.
>
>What's missing in the protocol is "measure the illumination intensity in the
>BFP (or image plane) of the objective in both cases and do the math"...
>
>And what would be the useful piece of information for the customer? Perhaps
>something like "increase tour illumination power 27.35 times (don't quote me
>here :-) to get the same average illumination intensity if you want to
>compare spinning disk to widefield..."
>
>Best, zdenek
>
>
>
>
>
>
>---------- Původní zpráva ----------
>Od: Andrea Latini <[hidden email]>
>Komu: [hidden email]
>Datum: 23. 5. 2016 14:40:42
>Předmět: Re: [QUAR] 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.
>*****
>
>dear Nico,
>the spinning disk itself acts as an optical filter on thick samples.
>Spinning Disk fill factor means 'Open Area Ratio' - holes area / blocked
>area.
>by inserting the disk within the optical path the theoretical throughput
>collection value would be 8.2% with respect to widefield (both excitation
>and emission pass through spinning disk of course), on 4 um beads, GFP
>excitation channel.
>
>the value 5-6% on 4um beads (and other values I'm reporting), has been
>measured by using "Measuring and interpreting point spread functions to
>determine confocal microscope resolution and ensure quality control Richard
>W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume 6, Pages:
>1929–1941(2011)" method.
>
>briefly (Macro is running in NIS Element SW):
>"
>
>1.) Sample: subresolution beads prepared according to „Measuring and
>interpreting point spread functions to determine confocal microscope
>resolution and ensure quality control
>Richard W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume
>6, Pages: 1929–1941(2011)
>2.) Technical setup of Microscope:
>a. The microscope is set up for a typical point spread function
>acquisition to characterize the resolution/imaging quality using a high-end
>100x oil objective.
>b. A regular fluorescence light source is used for all acquisitions in
>combination with a filter cube. This is essential and the bottom principle
>of the test. The same illumination with the same sample is used to compare
>wide field intensity with the confocal intensity of bead images. This test
>assesses only the emission light transmission of the spinning disc device.
>i. Option one preferred: left and right port of the microscope is used so
>that one port holds the spinning disc device with camera and the other port
>an identical second camera in simple wide field mode.
>ii. Alternatively one can run the acquisition of test data first with the
>spinning disc +camera and then remove the scan head and use the same camera
>alone on the same port where the spinning disc scan head was mounted.
>iii. Toggle between wide field mode and spinning disc mode if the to be
>tested scan head provides such an option and (important!) if the wide field
>mode does not have more than a single mirror in the light path (otherwise
>the transmission loss of the additional optical elements in the wide field
>mode of the scan head results in a lower intensity measurements . This in
>turn would artificially “improve� the result so that one would think the
>ratio wide field intensity / confocal intensity is higher and therefore
>pretending to be the better instrument)
>
>3.) Procedure
>Time required: about 1 -2 hours
>Overview of work flow: acquisition of image z-stacks of beads at 3 different
>exposure times in each mode (confocal and wide field) while illumination
>remains constant.
>Prepare collapsed stack images (max projection)
>rolling ball background reduction
>find beads and set ROIs
>calculate mean or median intensity (in our hands it does not make a
>difference)
>plot intenstity vs. exposure time for both modes
>determine slope
>ratio of slopes = result
>
>In detail:
>a.) Set up beads in focus with the scanning device. The signal should be
>chosen at an intensity (illumination light intensity ) , so that with the
>same illumination wide field images are possible (sufficiently short shutter
>time should be available)
>b.) Stacks of images are taken at 3 different exposure times of different
>view fields to avoid the issue of bleaching,
>c.) Repeat the steps a) and b) in wide field mode
>d.) Run macro in ImageJ (enclosed) to analyze bead intensity
>
>Find different ROIs in each maximum projection :
>
>run("Z Project...", "projection=[Max Intensity]");
>run("Subtract Background...", "rolling=5");
>run("Threshold...");
>title = "WaitForUserDemo";
>msg = "If necessary, use the \"Threshold\" tool to\nadjust the threshold,
>then click \"OK\".";
>waitForUser(title, msg);
>getThreshold(lower, upper)
>run("Analyze Particles...", "size=1-4 exclude summarize");
>
>
>or: you run the macro above on the stack with the lowest exposure time
>and then – to keep the ROIs the same for all stacks- yu run the macro below
>on the remaining stacks
>
>run("Z Project...", "projection=[Max Intensity]");
>run("Subtract Background...", "rolling=5");
>roiManager("measure")
>
>
>e.) Prepare plots of the so calculated intensities vs the exposure time
>f.) Calculate slope
>g.) Ratio the slope.
>
>"
>
>
>regards.
>
>Andrea
>
>Andrea Latini
>President
>CrestOptics Srl
>
>
>On Mon, 23 May 2016 08:42:07 -0700, Nico Stuurman <[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 Andrea,
>>
>>> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
>(fill factor 8.2%).
>>
>>Could you please explain what that number means, or better, how it is
>>measured? The way it is written, I have the impression that when a very
>>thin film of fluorescent material is excited by the same amount of light
>>you get 5-6% intensity in confocal mode and 100% in wide-field mode.
>>Obviously, that would be pretty bad (and according to the discussion on
>>this list, you should not lose more than a few percent of the
>>in-focus-signal through the pinholes), so I assume that you mean
>>something else.
>>
>>Best,
>>
>>Nico"

[samjlord]

*****
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 realize that for fast calcium imaging, my recommendation for a longer exposure
time may not actually help because the fluorescent bursts are so short. And Jeff
Reece's original response was a very good point: Unless your sample is thicker
than 20 um, there should be little change in the detection side using 40 um
pinholes and 10x/0.45NA objective.

Therefore, I suspect the major culprit is the significant reduction in excitation
intensity that you have not been able to compensate for by simply turning up the
illumination power. With a 40 um pinhole disk, I think the fill-factor of 4.2% means
that only 4.2% of the excitation light hits the sample (compared to wide field).
Unless you have very high-power lasers, or you were using them on very low
power for your wide field images, your excitation irradiance will be significantly
lower with the disk in, so your images will appear noisier. The up-side is that your
sample should bleach more slowly. :)

On Sun, 22 May 2016 05:55:14 +0000, Guy Cox <[hidden email]>
wrote:
>Repeat 100 times:
>
>CONFOCAL ONLY THROWS OUT
>OUT OF FOCUS LIGHT!  

My statement that "confocal is designed to throw out light in order to improve
optical slicing" is correct technically and in a practically sense. The pinholes both
block a significant portion of the excitation light and also reject out-of-focus
fluorescence. Both these facts mean that fewer photons hit the camera, giving the
low S/N result that the original poster observed.

I understand that a pinhole does not reject in-focus light, but that doesn't change
the fact that the excitation light is dimmer. And for thicker features in a sample,
rejecting the out-of-focus light actually makes each feature dimmer, because you
are throwing out fluorescence that originated from a different part of the same
feature.

Anyway, I was just trying to assist someone who has never used a spinning disk
before by offering some practical pointers.

-Sam

[Guy Cox-2]

*****
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.
*****

As I said, the light is sacrificed for speed, NOT confocality.  Single point scanning confocal wastes no excitation light.  Nipkow disc confocal wastes an awful lot of excitation light (as you say) but that sacrifice is entirely for speed, and no fluorescence is wasted.  Micro-lens spinning disk systems (Yokogawa, Perkin Elmer) salvage quite a lot of that excitation light and so come somewhere in between.

                                                                           Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Lord
Sent: Tuesday, 24 May 2016 9:43 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 realize that for fast calcium imaging, my recommendation for a longer exposure time may not actually help because the fluorescent bursts are so short. And Jeff Reece's original response was a very good point: Unless your sample is thicker than 20 um, there should be little change in the detection side using 40 um pinholes and 10x/0.45NA objective.

Therefore, I suspect the major culprit is the significant reduction in excitation intensity that you have not been able to compensate for by simply turning up the illumination power. With a 40 um pinhole disk, I think the fill-factor of 4.2% means that only 4.2% of the excitation light hits the sample (compared to wide field).
Unless you have very high-power lasers, or you were using them on very low power for your wide field images, your excitation irradiance will be significantly lower with the disk in, so your images will appear noisier. The up-side is that your sample should bleach more slowly. :)

On Sun, 22 May 2016 05:55:14 +0000, Guy Cox <[hidden email]>
wrote:
>Repeat 100 times:
>
>CONFOCAL ONLY THROWS OUT
>OUT OF FOCUS LIGHT!  

My statement that "confocal is designed to throw out light in order to improve optical slicing" is correct technically and in a practically sense. The pinholes both block a significant portion of the excitation light and also reject out-of-focus fluorescence. Both these facts mean that fewer photons hit the camera, giving the low S/N result that the original poster observed.

I understand that a pinhole does not reject in-focus light, but that doesn't change the fact that the excitation light is dimmer. And for thicker features in a sample, rejecting the out-of-focus light actually makes each feature dimmer, because you are throwing out fluorescence that originated from a different part of the same feature.

Anyway, I was just trying to assist someone who has never used a spinning disk before by offering some practical pointers.

-Sam

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

*****
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 for the double posting.  The listserv might be letting messages through whether they are approved or not.

Apologies to Sam for misreading your earlier post.  Don't stop contributing!

Cheers,
Jeff

-----Original Message-----
From: Reece, Jeff (NIH/NIDDK) [C]
Sent: Sunday, May 22, 2016 1:41 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.
*****

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

*****
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.

[Guy Cox-2]

*****
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.
*****

Look, it's not the light in the psf that contributes to resolution, it's the light in the Airy disk.  Letting the rings through would make the image worse, not better.  Actually that may be why people sometimes say they are getting improved resolution with a confocal when the pinhole, at one Airy unit, would not actually be delivering any confocal super-resolution.  

                        Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Reece, Jeff (NIH/NIDDK) [C]
Sent: Monday, 23 May 2016 3:41 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 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

*****
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.