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Fastest spectral acquisition/unmixing

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G. Esteban Fernandez

Fastest spectral acquisition/unmixing

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Hi all,

Does anyone have a sense for how different point-scanning confocals
compare in terms of speed of spectral acquisition for unmixing? I
know that Zeiss and Nikon both have 34 parallel spectral channels and
this seems like the fastest solution over a broad range (with Zeiss
using GaAsP detectors so probably faster), just wondering what others
have experienced.

Thanks!

-Esteban

Craig Brideau

Re: Fastest spectral acquisition/unmixing

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To join, leave or search the confocal microscopy listserv, go to:
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The Zeiss and Nikon systems are fastest because they acquire in parallel.
Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you
are splitting your signal up into a large number of channels, so you need
fairly bright signal. I suggest demoing both systems to see what works best
for you.
If you are really signal starved, the sequential spectral systems like
Leica and Olympus are more sensitive, but take longer to acquire and may
have photobleaching issues depending on the photostability of your
fluorophore.

Craig

On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez <
[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,
>
> Does anyone have a sense for how different point-scanning confocals
> compare in terms of speed of spectral acquisition for unmixing? I
> know that Zeiss and Nikon both have 34 parallel spectral channels and
> this seems like the fastest solution over a broad range (with Zeiss
> using GaAsP detectors so probably faster), just wondering what others
> have experienced.
>
> Thanks!
>
> -Esteban
>

Cammer, Michael

Re: Fastest spectral acquisition/unmixing

*****
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.
*****

We routinely use the Zeiss 880 for spectral detection. The 34 channel detector tops out at 690 or 700 nm but we can use an additional GaAsP detector to catch the light from 700 to 750 nm in one additional bin.
We also demoed the Nikon A1 and were happy with the spectral detector but forget the specific wavelengths.
Cheers-

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
C: 914-309-3270 [hidden email] http://nyulmc.org/micros http://microscopynotes.com/

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Craig Brideau
Sent: Tuesday, April 23, 2019 5:46 PM
To: [hidden email]
Subject: Re: Fastest spectral acquisition/unmixing

The Zeiss and Nikon systems are fastest because they acquire in parallel.
Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you are splitting your signal up into a large number of channels, so you need fairly bright signal. I suggest demoing both systems to see what works best for you.
If you are really signal starved, the sequential spectral systems like Leica and Olympus are more sensitive, but take longer to acquire and may have photobleaching issues depending on the photostability of your fluorophore.

Craig

On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez < [hidden email]> wrote:

>
> Hi all,
>
> Does anyone have a sense for how different point-scanning confocals
> compare in terms of speed of spectral acquisition for unmixing? I
> know that Zeiss and Nikon both have 34 parallel spectral channels and
> this seems like the fastest solution over a broad range (with Zeiss
> using GaAsP detectors so probably faster), just wondering what others
> have experienced.
>
> Thanks!
>
> -Esteban
>

------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

G. Esteban Fernandez

Re: Fastest spectral acquisition/unmixing

*****
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.
*****

Thank you Craig and Michael for your responses. The point about light
splitting and sensitivity is well taken. Our current Zeiss 710 is in
heavy demand so we're favoring parallel over sequential spectral for
speed, with GaAsP in the spectral detector for sensitivity. I think
Zeiss and Nikon have about the same range (400 - 700 or 750 nm).

-Esteban

On Tue, Apr 23, 2019 at 2:57 PM Cammer, Michael
<[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.
> *****
>
> We routinely use the Zeiss 880 for spectral detection. The 34 channel detector tops out at 690 or 700 nm but we can use an additional GaAsP detector to catch the light from 700 to 750 nm in one additional bin.
> We also demoed the Nikon A1 and were happy with the spectral detector but forget the specific wavelengths.
> Cheers-
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
> C: 914-309-3270 [hidden email] http://nyulmc.org/micros http://microscopynotes.com/
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Craig Brideau
> Sent: Tuesday, April 23, 2019 5:46 PM
> To: [hidden email]
> Subject: Re: Fastest spectral acquisition/unmixing
>
> The Zeiss and Nikon systems are fastest because they acquire in parallel.
> Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you are splitting your signal up into a large number of channels, so you need fairly bright signal. I suggest demoing both systems to see what works best for you.
> If you are really signal starved, the sequential spectral systems like Leica and Olympus are more sensitive, but take longer to acquire and may have photobleaching issues depending on the photostability of your fluorophore.
>
> Craig
>
> On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez < [hidden email]> wrote:
>
> >
> > Hi all,
> >
> > Does anyone have a sense for how different point-scanning confocals
> > compare in terms of speed of spectral acquisition for unmixing? I
> > know that Zeiss and Nikon both have 34 parallel spectral channels and
> > this seems like the fastest solution over a broad range (with Zeiss
> > using GaAsP detectors so probably faster), just wondering what others
> > have experienced.
> >
> > Thanks!
> >
> > -Esteban
> >
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================
>

Cole, Richard W (HEALTH)-2

Re: Fastest spectral acquisition/unmixing

In reply to this post by G. Esteban Fernandez

*****
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.
*****

We use the Leica; it can collect the images for spectral unmixing either parallel (5 channels) or serially (adjustable range and bin width). Depending on the sample and the dyes, I often find that the five channels are sufficient.

Happy unmixing
Rich

Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center

Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York

120 New Scotland Avenue, Albany N.Y. 12208
518-474-7048 Phone
518-408-1730 Fax

-----Original Message-----
From: G. Esteban Fernandez <[hidden email]>
Sent: Wednesday, April 24, 2019 11:02 PM
Subject: Re: Fastest spectral acquisition/unmixing

*****
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.
*****

Thank you Craig and Michael for your responses. The point about light splitting and sensitivity is well taken. Our current Zeiss 710 is in heavy demand so we're favoring parallel over sequential spectral for speed, with GaAsP in the spectral detector for sensitivity. I think Zeiss and Nikon have about the same range (400 - 700 or 750 nm).

-Esteban

On Tue, Apr 23, 2019 at 2:57 PM Cammer, Michael <[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.
> *****
>
> We routinely use the Zeiss 880 for spectral detection. The 34 channel detector tops out at 690 or 700 nm but we can use an additional GaAsP detector to catch the light from 700 to 750 nm in one additional bin.
> We also demoed the Nikon A1 and were happy with the spectral detector but forget the specific wavelengths.
> Cheers-
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> C: 914-309-3270 [hidden email] http://nyulmc.org/micros
> http://microscopynotes.com/
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Craig Brideau
> Sent: Tuesday, April 23, 2019 5:46 PM
> To: [hidden email]
> Subject: Re: Fastest spectral acquisition/unmixing
>
> The Zeiss and Nikon systems are fastest because they acquire in parallel.
> Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you are splitting your signal up into a large number of channels, so you need fairly bright signal. I suggest demoing both systems to see what works best for you.
> If you are really signal starved, the sequential spectral systems like Leica and Olympus are more sensitive, but take longer to acquire and may have photobleaching issues depending on the photostability of your fluorophore.
>
> Craig
>
> On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez < [hidden email]> wrote:
>
> >
> > Hi all,
> >
> > Does anyone have a sense for how different point-scanning confocals
> > compare in terms of speed of spectral acquisition for unmixing? I
> > know that Zeiss and Nikon both have 34 parallel spectral channels
> > and this seems like the fastest solution over a broad range (with
> > Zeiss using GaAsP detectors so probably faster), just wondering what
> > others have experienced.
> >
> > Thanks!
> >
> > -Esteban
> >
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================
>

G. Esteban Fernandez

Re: Fastest spectral acquisition/unmixing

*****
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 for chiming in Richard. I've never tried it but figured the
limited Leica parallelization was enough for something like GFP/YFP,
two "red" Alexa's, and maybe even broad autofluorescence. The
separate gains in the 5 bins must be nice :-) But today I imaged DAPI
- AF488 - AF546 - AF594 - AF647 fast in one scan. Everything was
bright so sensitivity and common gain were not an issue...this time.

-Esteban

On Thu, Apr 25, 2019 at 4:29 AM Cole, Richard W (HEALTH)
<[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.
> *****
>
> We use the Leica; it can collect the images for spectral unmixing either parallel (5 channels) or serially (adjustable range and bin width). Depending on the sample and the dyes, I often find that the five channels are sufficient.
>
> Happy unmixing
> Rich
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy & Image Analysis Core
> Wadsworth Center
>
> Research Assistant Professor
> Dept. of Biomedical Sciences
> School of Public Health State University of New York
>
> 120 New Scotland Avenue, Albany N.Y. 12208
> 518-474-7048 Phone
> 518-408-1730 Fax
>
>
>
>
> -----Original Message-----
> From: G. Esteban Fernandez <[hidden email]>
> Sent: Wednesday, April 24, 2019 11:02 PM
> Subject: Re: Fastest spectral acquisition/unmixing
>
> *****
> 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.
> *****
>
> Thank you Craig and Michael for your responses. The point about light splitting and sensitivity is well taken. Our current Zeiss 710 is in heavy demand so we're favoring parallel over sequential spectral for speed, with GaAsP in the spectral detector for sensitivity. I think Zeiss and Nikon have about the same range (400 - 700 or 750 nm).
>
> -Esteban
>
> On Tue, Apr 23, 2019 at 2:57 PM Cammer, Michael <[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.
> > *****
> >
> > We routinely use the Zeiss 880 for spectral detection. The 34 channel detector tops out at 690 or 700 nm but we can use an additional GaAsP detector to catch the light from 700 to 750 nm in one additional bin.
> > We also demoed the Nikon A1 and were happy with the spectral detector but forget the specific wavelengths.
> > Cheers-
> >
> > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> > Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> > 10016
> > C: 914-309-3270 [hidden email] http://nyulmc.org/micros
> > http://microscopynotes.com/
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]> On
> > Behalf Of Craig Brideau
> > Sent: Tuesday, April 23, 2019 5:46 PM
> > To: [hidden email]
> > Subject: Re: Fastest spectral acquisition/unmixing
> >
> > The Zeiss and Nikon systems are fastest because they acquire in parallel.
> > Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you are splitting your signal up into a large number of channels, so you need fairly bright signal. I suggest demoing both systems to see what works best for you.
> > If you are really signal starved, the sequential spectral systems like Leica and Olympus are more sensitive, but take longer to acquire and may have photobleaching issues depending on the photostability of your fluorophore.
> >
> > Craig
> >
> > On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez < [hidden email]> wrote:
> >
> > >
> > > Hi all,
> > >
> > > Does anyone have a sense for how different point-scanning confocals
> > > compare in terms of speed of spectral acquisition for unmixing? I
> > > know that Zeiss and Nikon both have 34 parallel spectral channels
> > > and this seems like the fastest solution over a broad range (with
> > > Zeiss using GaAsP detectors so probably faster), just wondering what
> > > others have experienced.
> > >
> > > Thanks!
> > >
> > > -Esteban
> > >
> >
> > ------------------------------------------------------------
> > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> > =================================
> >

Craig Brideau

Re: Fastest spectral acquisition/unmixing

*****
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 think Zeiss and Nikon also have sequential scanning options. Depending on
how elaborate a system you are willing to buy, you could have parallel and
sequential on the same scope...

Craig

On Fri, Apr 26, 2019, 12:22 AM G. Esteban Fernandez <
[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.
> *****
>
> Thanks for chiming in Richard. I've never tried it but figured the
> limited Leica parallelization was enough for something like GFP/YFP,
> two "red" Alexa's, and maybe even broad autofluorescence. The
> separate gains in the 5 bins must be nice :-) But today I imaged DAPI
> - AF488 - AF546 - AF594 - AF647 fast in one scan. Everything was
> bright so sensitivity and common gain were not an issue...this time.
>
> -Esteban
>
> On Thu, Apr 25, 2019 at 4:29 AM Cole, Richard W (HEALTH)
> <[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.
> > *****
> >
> > We use the Leica; it can collect the images for spectral unmixing either
> parallel (5 channels) or serially (adjustable range and bin width).
> Depending on the sample and the dyes, I often find that the five channels
> are sufficient.
> >
> > Happy unmixing
> > Rich
> >
> > Richard Cole
> > Research Scientist V
> > Director: Advanced Light Microscopy & Image Analysis Core
> > Wadsworth Center
> >
> > Research Assistant Professor
> > Dept. of Biomedical Sciences
> > School of Public Health State University of New York
> >
> > 120 New Scotland Avenue, Albany N.Y. 12208
> > 518-474-7048 Phone
> > 518-408-1730 Fax
> >
> >
> >
> >
> > -----Original Message-----
> > From: G. Esteban Fernandez <[hidden email]>
> > Sent: Wednesday, April 24, 2019 11:02 PM
> > Subject: Re: Fastest spectral acquisition/unmixing
> >
> > *****
> > 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.
> > *****
> >
> > Thank you Craig and Michael for your responses. The point about light
> splitting and sensitivity is well taken. Our current Zeiss 710 is in heavy
> demand so we're favoring parallel over sequential spectral for speed, with
> GaAsP in the spectral detector for sensitivity. I think Zeiss and Nikon
> have about the same range (400 - 700 or 750 nm).
> >
> > -Esteban
> >
> > On Tue, Apr 23, 2019 at 2:57 PM Cammer, Michael <
> [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.
> > > *****
> > >
> > > We routinely use the Zeiss 880 for spectral detection. The 34 channel
> detector tops out at 690 or 700 nm but we can use an additional GaAsP
> detector to catch the light from 700 to 750 nm in one additional bin.
> > > We also demoed the Nikon A1 and were happy with the spectral detector
> but forget the specific wavelengths.
> > > Cheers-
> > >
> > > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> > > Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> > > 10016
> > > C: 914-309-3270 [hidden email] http://nyulmc.org/micros
> > > http://microscopynotes.com/
> > >
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List <[hidden email]> On
> > > Behalf Of Craig Brideau
> > > Sent: Tuesday, April 23, 2019 5:46 PM
> > > To: [hidden email]
> > > Subject: Re: Fastest spectral acquisition/unmixing
> > >
> > > The Zeiss and Nikon systems are fastest because they acquire in
> parallel.
> > > Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind
> you are splitting your signal up into a large number of channels, so you
> need fairly bright signal. I suggest demoing both systems to see what works
> best for you.
> > > If you are really signal starved, the sequential spectral systems like
> Leica and Olympus are more sensitive, but take longer to acquire and may
> have photobleaching issues depending on the photostability of your
> fluorophore.
> > >
> > > Craig
> > >
> > > On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez <
> [hidden email]> wrote:
> > >
> > > >
> > > > Hi all,
> > > >
> > > > Does anyone have a sense for how different point-scanning confocals
> > > > compare in terms of speed of spectral acquisition for unmixing? I
> > > > know that Zeiss and Nikon both have 34 parallel spectral channels
> > > > and this seems like the fastest solution over a broad range (with
> > > > Zeiss using GaAsP detectors so probably faster), just wondering what
> > > > others have experienced.
> > > >
> > > > Thanks!
> > > >
> > > > -Esteban
> > > >
> > >
> > > ------------------------------------------------------------
> > > This email message, including any attachments, is for the sole use of
> the intended recipient(s) and may contain information that is proprietary,
> confidential, and exempt from disclosure under applicable law. Any
> unauthorized review, use, disclosure, or distribution is prohibited. If you
> have received this email in error please notify the sender by return email
> and delete the original message. Please note, the recipient should check
> this email and any attachments for the presence of viruses. The
> organization accepts no liability for any damage caused by any virus
> transmitted by this email.
> > > =================================
> > >
>

Steffen Dietzel

Re: Fastest spectral acquisition/unmixing

In reply to this post by G. Esteban Fernandez

> *****
> 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 for chiming in Richard. I've never tried it but figured the
> limited Leica parallelization was enough for something like GFP/YFP,
> two "red" Alexa's, and maybe even broad autofluorescence. The
> separate gains in the 5 bins must be nice :-) But today I imaged DAPI
> - AF488 - AF546 - AF594 - AF647 fast in one scan. Everything was
> bright so sensitivity and common gain were not an issue...this time.
>
> -Esteban
>
> On Thu, Apr 25, 2019 at 4:29 AM Cole, Richard W (HEALTH)
> <[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.
>> *****
>>
>> We use the Leica; it can collect the images for spectral unmixing either parallel (5 channels) or serially (adjustable range and bin width). Depending on the sample and the dyes, I often find that the five channels are sufficient.
>>
>> Happy unmixing
>> Rich
>>
>> Richard Cole
>> Research Scientist V
>> Director: Advanced Light Microscopy & Image Analysis Core
>> Wadsworth Center
>>
>> Research Assistant Professor
>> Dept. of Biomedical Sciences
>> School of Public Health State University of New York
>>
>> 120 New Scotland Avenue, Albany N.Y. 12208
>> 518-474-7048 Phone
>> 518-408-1730 Fax
>>
>>
>>
>>
>> -----Original Message-----
>> From: G. Esteban Fernandez <[hidden email]>
>> Sent: Wednesday, April 24, 2019 11:02 PM
>> Subject: Re: Fastest spectral acquisition/unmixing
>>
>> *****
>> 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.
>> *****
>>
>> Thank you Craig and Michael for your responses. The point about light splitting and sensitivity is well taken. Our current Zeiss 710 is in heavy demand so we're favoring parallel over sequential spectral for speed, with GaAsP in the spectral detector for sensitivity. I think Zeiss and Nikon have about the same range (400 - 700 or 750 nm).
>>
>> -Esteban
>>
>> On Tue, Apr 23, 2019 at 2:57 PM Cammer, Michael <[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.
>>> *****
>>>
>>> We routinely use the Zeiss 880 for spectral detection. The 34 channel detector tops out at 690 or 700 nm but we can use an additional GaAsP detector to catch the light from 700 to 750 nm in one additional bin.
>>> We also demoed the Nikon A1 and were happy with the spectral detector but forget the specific wavelengths.
>>> Cheers-
>>>
>>> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
>>> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
>>> 10016
>>> C: 914-309-3270 [hidden email] http://nyulmc.org/micros
>>> http://microscopynotes.com/
>>>
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List <[hidden email]> On
>>> Behalf Of Craig Brideau
>>> Sent: Tuesday, April 23, 2019 5:46 PM
>>> To: [hidden email]
>>> Subject: Re: Fastest spectral acquisition/unmixing
>>>
>>> The Zeiss and Nikon systems are fastest because they acquire in parallel.
>>> Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you are splitting your signal up into a large number of channels, so you need fairly bright signal. I suggest demoing both systems to see what works best for you.
>>> If you are really signal starved, the sequential spectral systems like Leica and Olympus are more sensitive, but take longer to acquire and may have photobleaching issues depending on the photostability of your fluorophore.
>>>
>>> Craig
>>>
>>> On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez < [hidden email]> wrote:
>>>
>>>> Hi all,
>>>>
>>>> Does anyone have a sense for how different point-scanning confocals
>>>> compare in terms of speed of spectral acquisition for unmixing? I
>>>> know that Zeiss and Nikon both have 34 parallel spectral channels
>>>> and this seems like the fastest solution over a broad range (with
>>>> Zeiss using GaAsP detectors so probably faster), just wondering what
>>>> others have experienced.
>>>>
>>>> Thanks!
>>>>
>>>> -Esteban
>>>>
>>> ------------------------------------------------------------
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--
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
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http://www.bioimaging.bmc.med.uni-muenchen.de

0000001ed7f52e4a-dmarc-request

Re: Fastest spectral acquisition/unmixing

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> that's 5 colors. I was wondering what you need the other 29 channels for on a 34 channel system?
To get the right intensities of the different fluorophores, you need to solve a system of linear equations. When the spectra are too close together, this gets more difficult and the error in the result increases. Having more data points can help, especially if you have good enough signal to noise. But then splitting the signal into more channels deceases the SNR. It would be interesting to see if there is an optimum number of channels.
As I remember, the Zeiss software can do the spectral unmixing on the fly and display the unmixed virtual channels during imaging, not sure about the others.
If your features do not overlap, e.g. separate cells and high enough resolution to resolve them, you actually can get away with just 2-3 channels and distinguish many colours, similar to a RGB computer monitor can display them, e.g. CFP, GFP,YFP and RFP with 3 fluorescent channels. By combination of membrane and nuclear staining 10 different cell types can be distinguished using these 3 fluorescence channels.www.sciencemag.org/cgi/content/full/science.aad3439/DC1
Best wishes
Andreas

-----Original Message-----
From: Steffen Dietzel <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Tue, 30 Apr 2019 17:05
Subject: Re: Fastest spectral acquisition/unmixing

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Hi Esteban,

that's 5 colors. I was wondering what you need the other 29 channels for
on a 34 channel system? We have done six colors on the same slide (on a
5 channel system without unmixing, by sequential scanning, pretty much
your channels plus Alexa 680). If you manage more than 10 colors with a
reasonable separation I am impressed. With 10 or less, two sweeps in a 5
channel system should be sufficient, no? You would have to go over it
twice but you also collect only 10 times noise, not 34 times. I don't
have much experience with computational unmixing, but it does not seem a
sure thing to me which way of scanning you would reach the same image
quality faster. Maybe I am missing the point.

Steffen

Am 26.04.2019 um 08:21 schrieb G. Esteban Fernandez:

> *****
> 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 for chiming in Richard. I've never tried it but figured the
> limited Leica parallelization was enough for something like GFP/YFP,
> two "red" Alexa's, and maybe even broad autofluorescence. The
> separate gains in the 5 bins must be nice :-) But today I imaged DAPI
> - AF488 - AF546 - AF594 - AF647 fast in one scan. Everything was
> bright so sensitivity and common gain were not an issue...this time.
>
> -Esteban
>
> On Thu, Apr 25, 2019 at 4:29 AM Cole, Richard W (HEALTH)
> <[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.
>> *****
>>
>> We use the Leica; it can collect the images for spectral unmixing either parallel (5 channels) or serially (adjustable range and bin width). Depending on the sample and the dyes, I often find that the five channels are sufficient.
>>
>> Happy unmixing
>> Rich
>>
>> Richard Cole
>> Research Scientist V
>> Director: Advanced Light Microscopy & Image Analysis Core
>> Wadsworth Center
>>
>> Research Assistant Professor
>> Dept. of Biomedical Sciences
>> School of Public Health State University of New York
>>
>> 120 New Scotland Avenue, Albany N.Y. 12208
>> 518-474-7048 Phone
>> 518-408-1730 Fax
>>
>>
>>
>>
>> -----Original Message-----
>> From: G. Esteban Fernandez <[hidden email]>
>> Sent: Wednesday, April 24, 2019 11:02 PM
>> Subject: Re: Fastest spectral acquisition/unmixing
>>
>> *****
>> 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.
>> *****
>>
>> Thank you Craig and Michael for your responses. The point about light splitting and sensitivity is well taken. Our current Zeiss 710 is in heavy demand so we're favoring parallel over sequential spectral for speed, with GaAsP in the spectral detector for sensitivity. I think Zeiss and Nikon have about the same range (400 - 700 or 750 nm).
>>
>> -Esteban
>>
>> On Tue, Apr 23, 2019 at 2:57 PM Cammer, Michael <[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.
>>> *****
>>>
>>> We routinely use the Zeiss 880 for spectral detection. The 34 channel detector tops out at 690 or 700 nm but we can use an additional GaAsP detector to catch the light from 700 to 750 nm in one additional bin.
>>> We also demoed the Nikon A1 and were happy with the spectral detector but forget the specific wavelengths.
>>> Cheers-
>>>
>>> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
>>> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
>>> 10016
>>> C: 914-309-3270 [hidden email] http://nyulmc.org/micros
>>> http://microscopynotes.com/
>>>
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List <[hidden email]> On
>>> Behalf Of Craig Brideau
>>> Sent: Tuesday, April 23, 2019 5:46 PM
>>> To: [hidden email]
>>> Subject: Re: Fastest spectral acquisition/unmixing
>>>
>>> The Zeiss and Nikon systems are fastest because they acquire in parallel.
>>> Our Nikon A1 can capture spectral in 1us per pixel. Just keep in mind you are splitting your signal up into a large number of channels, so you need fairly bright signal. I suggest demoing both systems to see what works best for you.
>>> If you are really signal starved, the sequential spectral systems like Leica and Olympus are more sensitive, but take longer to acquire and may have photobleaching issues depending on the photostability of your fluorophore.
>>>
>>> Craig
>>>
>>> On Tue, Apr 23, 2019 at 1:44 PM G. Esteban Fernandez < [hidden email]> wrote:
>>>
>>>> Hi all,
>>>>
>>>> Does anyone have a sense for how different point-scanning confocals
>>>> compare in terms of speed of spectral acquisition for unmixing? I
>>>> know that Zeiss and Nikon both have 34 parallel spectral channels
>>>> and this seems like the fastest solution over a broad range (with
>>>> Zeiss using GaAsP detectors so probably faster), just wondering what
>>>> others have experienced.
>>>>
>>>> Thanks!
>>>>
>>>> -Esteban
>>>>
>>> ------------------------------------------------------------
>>> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
>>> =================================
>>>