Colocalization and Hybrid Detectors

[Neil Anthony]

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Hi all, I hope the scopes are treating you well.

I wonder if anybody has any thoughts on using Leica HyD detectors for colocalization.  If one was forced to colocalize a HyD channel to a PMT channel, how would this affect the results when compared to a PMT-PMT coloc?  I'm assuming here that both channels would have the same bit-depth.

Thanks in advance for your time.

Neil


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[Avi Jacob]

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I have 2 of each on my SP8 and I don't see any problem with this. I will
usually use both HyDs for the critical fluorophores anyway, but I see no
fundamental reason why there should be any problem coloc'ing signal from
HyD with PPT. At the end of the day, you get value at a specific
coordinate. Does not matter how it was acquired.

I would be happy to hear if any of the gurus here disagree, would be good
to know.
Avi

--
Avi Jacob, Ph.D.
Head of Light Microscopy
The Mina & Everard Goodman Faculty of Life Sciences
Bar-Ilan University, Ramat-Gan 5290002, Israel
972-3-5317647
http://tinyurl.com/BIU-Microscopy




On Wed, Nov 11, 2015 at 3:59 AM, Anthony, Neil <[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 hope the scopes are treating you well.
>
> I wonder if anybody has any thoughts on using Leica HyD detectors for
> colocalization.  If one was forced to colocalize a HyD channel to a PMT
> channel, how would this affect the results when compared to a PMT-PMT
> coloc?  I'm assuming here that both channels would have the same bit-depth.
>
> Thanks in advance for your time.
>
> Neil
>
>
> ________________________________
>
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[Arne Seitz]

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Colocalization is an artefact caused by the low pass filtering of the microscopes. So at best you can obtain a relative measure. In order to interpret your obtained parameter correctly you need to compare it against control samples. These controls should/must have been recorded on the exactly same system with exactly the same settings.
Once you follow this approach it should to my understanding not matter whether you use classical PMTs or HyD detectors. The latter ones might have a higher noise level at least if operated in photon counting mode. This might influence the dynamic range of your analysis.

Cheers
Arne  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Anthony, Neil
Sent: mercredi 11 novembre 2015 02:59
To: [hidden email]
Subject: Colocalization and Hybrid Detectors

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Hi all, I hope the scopes are treating you well.

I wonder if anybody has any thoughts on using Leica HyD detectors for colocalization.  If one was forced to colocalize a HyD channel to a PMT channel, how would this affect the results when compared to a PMT-PMT coloc?  I'm assuming here that both channels would have the same bit-depth.

Thanks in advance for your time.

Neil


________________________________

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[George McNamara]

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Hi Neil,

do the experiment all four ways and publish.

PMT #1 - PMT #2
PMT #1 - HyD #2
HyD #1 - PMT #2
HyD#1 - HyD #2

I disagree with the previous comment about HyD being noisier in photon
counting mode than a PMT. Acquiring with a PMT without frame averaging
for fair comparison.

George


On 11/10/2015 7:59 PM, Anthony, Neil 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 hope the scopes are treating you well.
>
> I wonder if anybody has any thoughts on using Leica HyD detectors for colocalization.  If one was forced to colocalize a HyD channel to a PMT channel, how would this affect the results when compared to a PMT-PMT coloc?  I'm assuming here that both channels would have the same bit-depth.
>
> Thanks in advance for your time.
>
> Neil
>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination, distribution
> or copying of this message (including any attachments) is strictly
> prohibited.
>
> If you have received this message in error, please contact
> the sender by reply e-mail message and destroy all copies of the
> original message (including attachments).
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
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[Massimiliano Garre']

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Hi Neil,

in my hands and on the SP5 we have in our facility I always had some doubts
using the HyD for colocalization: when I analyze the scatter plot in the
colocalization analisys of the Leica software, the pixels of the HyD channel
do not have an even distribution; they are concentrated only in certain
values of the full bit range. I don't know if this is due to the different
gain of the Hyd (used in standard mode) but I feel this effect could affect
the standard colocalization factors that you calculate (Perasons Overlap, ...).
Anyway I never tried a PMT vs HyD direct comparison.

My two cents

Max

On Wed, 11 Nov 2015 01:59:29 +0000, Anthony, Neil <[hidden email]> wrote:

>*****
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>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 hope the scopes are treating you well.
>
>I wonder if anybody has any thoughts on using Leica HyD detectors for
colocalization.  If one was forced to colocalize a HyD channel to a PMT
channel, how would this affect the results when compared to a PMT-PMT coloc?
 I'm assuming here that both channels would have the same bit-depth.

>
>Thanks in advance for your time.
>
>Neil
>
>
>________________________________
>
>This e-mail message (including any attachments) is for the sole use of
>the intended recipient(s) and may contain confidential and privileged
>information. If the reader of this message is not the intended
>recipient, you are hereby notified that any dissemination, distribution
>or copying of this message (including any attachments) is strictly
>prohibited.
>
>If you have received this message in error, please contact
>the sender by reply e-mail message and destroy all copies of the
>original message (including attachments).

[Zdenek Svindrych-2]

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Hi Max,
The HyDs are fast photon counting detectors. If you use them in "Photon
Counting" mode, your histogram will be perfectly smooth with no missing
values. But in "Standard" mode the photon counts are multiplied by some
constant (e.g. about 100x at 12-bit, 400Hz, 100% "gain") so they appear
brighter. Then your histogram has large gaps (a hundred of zero values
between two big numbers in your histogram, assuming bin width is 1). You may
also note that the signal saturates (intensity value of 4095 for 12-bit
range) at about 40 photons per pixel (at the above conditions). That's why
the "Photon Counting" images are so dim... The reason behind that high
multiplication factor is to ensure the detectors' behavior is linear (Leica
claims the detectors are linear up to 6*10^7 photons/s and in "Standard"
mode up to 3*10^8 photons/s - there is be some correction, although I haven'
t been able to seen it in the histograms of my images)...

In normal PMTs (in "analog integrating" mode), the sharp peaks corresponding
to 1 photon/pixel, 2 photons/pixel, etc., are smeared by multiplication
noise, amplifier noise and slow amplifier response so you cannot distinguish
them.

I can't see any reason why HyD images should be less suitable for
colocalization analysis.

Best, zdenek


---------- Původní zpráva ----------
Od: Max Garre' <[hidden email]>
Komu: [hidden email]
Datum: 12. 11. 2015 3:10:29
Předmět: Re: Colocalization and Hybrid Detectors

"*****
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.
*****

Hi Neil,

in my hands and on the SP5 we have in our facility I always had some doubts
using the HyD for colocalization: when I analyze the scatter plot in the
colocalization analisys of the Leica software, the pixels of the HyD channel
do not have an even distribution; they are concentrated only in certain
values of the full bit range. I don't know if this is due to the different
gain of the Hyd (used in standard mode) but I feel this effect could affect
the standard colocalization factors that you calculate (Perasons Overlap, ..
.).
Anyway I never tried a PMT vs HyD direct comparison.

My two cents

Max

On Wed, 11 Nov 2015 01:59:29 +0000, Anthony, Neil <[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 hope the scopes are treating you well.
>
>I wonder if anybody has any thoughts on using Leica HyD detectors for
colocalization. If one was forced to colocalize a HyD channel to a PMT
channel, how would this affect the results when compared to a PMT-PMT coloc?
I'm assuming here that both channels would have the same bit-depth.

>
>Thanks in advance for your time.
>
>Neil
>
>
>________________________________
>
>This e-mail message (including any attachments) is for the sole use of
>the intended recipient(s) and may contain confidential and privileged
>information. If the reader of this message is not the intended
>recipient, you are hereby notified that any dissemination, distribution
>or copying of this message (including any attachments) is strictly
>prohibited.
>
>If you have received this message in error, please contact
>the sender by reply e-mail message and destroy all copies of the
>original message (including attachments)."

[Massimiliano Garre']

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Hi Zdenek,

thank you for your detailed reply. I will try using the HyD in photon
counting mode then in case images have to be processed for colocalization or
other quantification analysis.

Best regards

Max

On Thu, 12 Nov 2015 16:53:47 +0100, Zdenek Svindrych <[hidden email]> wrote:

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Hi Max,
The HyDs are fast photon counting detectors. If you use them in "Photon
Counting" mode, your histogram will be perfectly smooth with no missing
values. But in "Standard" mode the photon counts are multiplied by some
constant (e.g. about 100x at 12-bit, 400Hz, 100% "gain") so they appear
brighter. Then your histogram has large gaps (a hundred of zero values
between two big numbers in your histogram, assuming bin width is 1). You may
also note that the signal saturates (intensity value of 4095 for 12-bit
range) at about 40 photons per pixel (at the above conditions). That's why
the "Photon Counting" images are so dim... The reason behind that high
multiplication factor is to ensure the detectors' behavior is linear (Leica
claims the detectors are linear up to 6*10^7 photons/s and in "Standard"
mode up to 3*10^8 photons/s - there is be some correction, although I haven'
t been able to seen it in the histograms of my images)...

In normal PMTs (in "analog integrating" mode), the sharp peaks corresponding
to 1 photon/pixel, 2 photons/pixel, etc., are smeared by multiplication
noise, amplifier noise and slow amplifier response so you cannot distinguish
them.

I can't see any reason why HyD images should be less suitable for
colocalization analysis.

Best, zdenek


---------- Původní zpráva ----------
Od: Max Garre' <[hidden email]>
Komu: [hidden email]
Datum: 12. 11. 2015 3:10:29
Předmět: Re: Colocalization and Hybrid Detectors

"*****
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 Neil,

in my hands and on the SP5 we have in our facility I always had some doubts
using the HyD for colocalization: when I analyze the scatter plot in the
colocalization analisys of the Leica software, the pixels of the HyD channel
do not have an even distribution; they are concentrated only in certain
values of the full bit range. I don't know if this is due to the different
gain of the Hyd (used in standard mode) but I feel this effect could affect
the standard colocalization factors that you calculate (Perasons Overlap, ..
.).
Anyway I never tried a PMT vs HyD direct comparison.

My two cents

Max

On Wed, 11 Nov 2015 01:59:29 +0000, Anthony, Neil <[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 hope the scopes are treating you well.
>
>I wonder if anybody has any thoughts on using Leica HyD detectors for
colocalization. If one was forced to colocalize a HyD channel to a PMT
channel, how would this affect the results when compared to a PMT-PMT coloc?
I'm assuming here that both channels would have the same bit-depth.

>
>Thanks in advance for your time.
>
>Neil
>
>
>________________________________
>
>This e-mail message (including any attachments) is for the sole use of
>the intended recipient(s) and may contain confidential and privileged
>information. If the reader of this message is not the intended
>recipient, you are hereby notified that any dissemination, distribution
>or copying of this message (including any attachments) is strictly
>prohibited.
>
>If you have received this message in error, please contact
>the sender by reply e-mail message and destroy all copies of the
>original message (including attachments)."

[Neil Anthony]

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Thanks for all your detailed replies.  I love the confocal listserv!

Good to know that using HyD's mixed with PMTs is not a faux pas, so to speak.  I guess as long as your thresholding and ROIs make sense, and you're comparing apples-to-apples, it should work well.

George, I like your idea.  I'll get some tetraspec bead data on different detector combinations and report how it goes.

Thanks
Neil

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Max Garre'
Sent: Friday, November 13, 2015 3:12 AM
To: [hidden email]
Subject: Re: Colocalization and Hybrid Detectors

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*****

Hi Zdenek,

thank you for your detailed reply. I will try using the HyD in photon counting mode then in case images have to be processed for colocalization or other quantification analysis.

Best regards

Max

On Thu, 12 Nov 2015 16:53:47 +0100, 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.
>*****
>
>Hi Max,
The HyDs are fast photon counting detectors. If you use them in "Photon Counting" mode, your histogram will be perfectly smooth with no missing values. But in "Standard" mode the photon counts are multiplied by some constant (e.g. about 100x at 12-bit, 400Hz, 100% "gain") so they appear brighter. Then your histogram has large gaps (a hundred of zero values between two big numbers in your histogram, assuming bin width is 1). You may also note that the signal saturates (intensity value of 4095 for 12-bit
range) at about 40 photons per pixel (at the above conditions). That's why the "Photon Counting" images are so dim... The reason behind that high multiplication factor is to ensure the detectors' behavior is linear (Leica claims the detectors are linear up to 6*10^7 photons/s and in "Standard"
mode up to 3*10^8 photons/s - there is be some correction, although I haven'
t been able to seen it in the histograms of my images)...

In normal PMTs (in "analog integrating" mode), the sharp peaks corresponding to 1 photon/pixel, 2 photons/pixel, etc., are smeared by multiplication noise, amplifier noise and slow amplifier response so you cannot distinguish them.

I can't see any reason why HyD images should be less suitable for colocalization analysis.

Best, zdenek


---------- Původní zpráva ----------
Od: Max Garre' <[hidden email]>
Komu: [hidden email]
Datum: 12. 11. 2015 3:10:29
PÅTedmÄ>t: Re: Colocalization and Hybrid Detectors

"*****
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 Neil,

in my hands and on the SP5 we have in our facility I always had some doubts using the HyD for colocalization: when I analyze the scatter plot in the colocalization analisys of the Leica software, the pixels of the HyD channel do not have an even distribution; they are concentrated only in certain values of the full bit range. I don't know if this is due to the different gain of the Hyd (used in standard mode) but I feel this effect could affect the standard colocalization factors that you calculate (Perasons Overlap, ..
.).
Anyway I never tried a PMT vs HyD direct comparison.

My two cents

Max

On Wed, 11 Nov 2015 01:59:29 +0000, Anthony, Neil <[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 hope the scopes are treating you well.
>
>I wonder if anybody has any thoughts on using Leica HyD detectors for
colocalization. If one was forced to colocalize a HyD channel to a PMT channel, how would this affect the results when compared to a PMT-PMT coloc?
I'm assuming here that both channels would have the same bit-depth.

>
>Thanks in advance for your time.
>
>Neil
>
>
>________________________________
>
>This e-mail message (including any attachments) is for the sole use of
>the intended recipient(s) and may contain confidential and privileged
>information. If the reader of this message is not the intended
>recipient, you are hereby notified that any dissemination, distribution
>or copying of this message (including any attachments) is strictly
>prohibited.
>
>If you have received this message in error, please contact the sender
>by reply e-mail message and destroy all copies of the original message
>(including attachments)."