Elyra 7 system for bioluminescence

[Michał Majkowski-2]

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Dear List Members,
I am just thinking about the use of our Elyra 7 system to visualise
bioluminescence.
Have any of you ever tried something similar? In systems dedicated for
bioluminescence signal is detected with the same or similar camera (Andor
iXon Ultra 897) though light path is less complex.
My idea is to take time series with longest possible exposure time (1 sec),
convert T->Z and then sum up all slices. Does it have any sense?
Thank you in advance
Michal Majkowski

Imaging Specialist
University of Wroclaw
Biotechnology Faculty
Joliot-Curie 14a
50-383 Wroclaw POLAND

[Cammer, Michael-2]

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The structure for SIM is provided by the excitation light source. Does the emission in your system rely on this?
Cheers-
Michael Cammer
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Michał Majkowski <[hidden email]>
Sent: Monday, March 8, 2021 8:49:11 AM
To: [hidden email]
Subject: Elyra 7 system for bioluminescence

[EXTERNAL]

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Dear List Members,
I am just thinking about the use of our Elyra 7 system to visualise
bioluminescence.
Have any of you ever tried something similar? In systems dedicated for
bioluminescence signal is detected with the same or similar camera (Andor
iXon Ultra 897) though light path is less complex.
My idea is to take time series with longest possible exposure time (1 sec),
convert T->Z and then sum up all slices. Does it have any sense?
Thank you in advance
Michal Majkowski

Imaging Specialist
University of Wroclaw
Biotechnology Faculty
Joliot-Curie 14a
50-383 Wroclaw POLAND

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[Michał Majkowski-2]

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Dear Michael,

I want to use Elyra Laser WF mode. In this mode structure for SIM is not
used (as far as I understand SIM Elyra set-up). So a given laser
illuminates a sample and photons are collected through an emission filter.
Of course I do not want to use laser as photons are generated by chemical
reaction. Is it an answer for your question?

pon., 8 mar 2021 o 15:07 Cammer, Michael <
[hidden email]> napisał(a):

> *****
> 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.
> *****
>
> The structure for SIM is provided by the excitation light source. Does the
> emission in your system rely on this?
> Cheers-
> Michael Cammer
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Michał Majkowski <[hidden email]>
> Sent: Monday, March 8, 2021 8:49:11 AM
> To: [hidden email]
> Subject: Elyra 7 system for bioluminescence
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!MXfaZl3l!I4RyDm5zPisDOptpEl9KRSqfw2h7EHy7uCEecufCwes5tYMrt4oBsZbkdLKJgnwG5V0EZNQ$
> Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!I4RyDm5zPisDOptpEl9KRSqfw2h7EHy7uCEecufCwes5tYMrt4oBsZbkdLKJgnwGcs_TwAk$
> and include the link in your posting.
> *****
>
> Dear List Members,
> I am just thinking about the use of our Elyra 7 system to visualise
> bioluminescence.
> Have any of you ever tried something similar? In systems dedicated for
> bioluminescence signal is detected with the same or similar camera (Andor
> iXon Ultra 897) though light path is less complex.
> My idea is to take time series with longest possible exposure time (1 sec),
> convert T->Z and then sum up all slices. Does it have any sense?
> Thank you in advance
> Michal Majkowski
>
> Imaging Specialist
> University of Wroclaw
> Biotechnology Faculty
> Joliot-Curie 14a
> 50-383 Wroclaw POLAND
>
> ------------------------------------------------------------
> 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.
> =================================
>

[Zdenek Svindrych-2]

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Hi Michal,
you may want to use much longer exposure times (30 seconds for CMOS
cameras, hours for CCDs) depending how weak your luminescence is. With
EMCCDs, since the readout noise is negligible, you should get the same
results by summing shorter exposure images, like you suggested. You may
also bin the pixels for more SNR.
For optimization and troubleshooting you can measure the luminescence from
your cells with a suitable plate reader, they are much more sensitive.
I haven't tried this with the Zeiss Axio stand, but one issue I found with
the Nikon Ti-E and TE2000 is the stray light coming from internal
components (probably the encoders). I had to power off the microscope body
completely to get low background (I think Ti2-E can power off the encoders
only, if needed).
Also, dedicated systems may have much better light throughput, especially
when you trade off resolution for collection efficiency (just compare the
back focal plane aperture size of your microscope objective lens with let's
say Nikkor 50 mm F/# 1.2.
Good luck!
Best, zdenek

On Mon, Mar 8, 2021 at 8:50 AM Michał Majkowski <
[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 List Members,
> I am just thinking about the use of our Elyra 7 system to visualise
> bioluminescence.
> Have any of you ever tried something similar? In systems dedicated for
> bioluminescence signal is detected with the same or similar camera (Andor
> iXon Ultra 897) though light path is less complex.
> My idea is to take time series with longest possible exposure time (1 sec),
> convert T->Z and then sum up all slices. Does it have any sense?
> Thank you in advance
> Michal Majkowski
>
> Imaging Specialist
> University of Wroclaw
> Biotechnology Faculty
> Joliot-Curie 14a
> 50-383 Wroclaw POLAND
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Cammer, Michael-2]

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We use the system to image high speed transmission images, so for a sample that gives off its own light, I would expect you would turn off the light and use longer exposure times.
I would think you could put a sample on the stage, set the widefield transmission mode (no filters) and not turn the halogen lamp on, and collect with TV1 or TV2 depending which wavelengths you need.  Or you could put in filters if you want.  
Cheers-
Michael

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Michal Majkowski
Sent: Monday, March 8, 2021 9:32 AM
To: [hidden email]
Subject: Re: Elyra 7 system for bioluminescence

[EXTERNAL]


Dear Michael,

I want to use Elyra Laser WF mode. In this mode structure for SIM is not used (as far as I understand SIM Elyra set-up). So a given laser illuminates a sample and photons are collected through an emission filter.
Of course I do not want to use laser as photons are generated by chemical reaction. Is it an answer for your question?

pon., 8 mar 2021 o 15:07 Cammer, Michael < [hidden email]> napisał(a):

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confoca
> lmicroscopy__;!!MXfaZl3l!IaBvO0l0sTaSZWiFwkck7RQTKgvCkZTmNZLHYi8G8yWnn
> 1cltv_vM_tXBqihk8IHcd_YFyI$ Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!IaBvO0l0sTaSZWiFwkck7RQTKgvCkZTmNZLHYi8G8yWnn1cltv_vM_tXBqihk8IHCINck-c$  and include the link in your posting.
> *****
>
> The structure for SIM is provided by the excitation light source. Does
> the emission in your system rely on this?
> Cheers-
> Michael Cammer
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Michał Majkowski <[hidden email]>
> Sent: Monday, March 8, 2021 8:49:11 AM
> To: [hidden email]
> Subject: Elyra 7 system for bioluminescence
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confoca
> lmicroscopy__;!!MXfaZl3l!I4RyDm5zPisDOptpEl9KRSqfw2h7EHy7uCEecufCwes5t
> YMrt4oBsZbkdLKJgnwG5V0EZNQ$
> Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!I4RyDm5z
> PisDOptpEl9KRSqfw2h7EHy7uCEecufCwes5tYMrt4oBsZbkdLKJgnwGcs_TwAk$
> and include the link in your posting.
> *****
>
> Dear List Members,
> I am just thinking about the use of our Elyra 7 system to visualise
> bioluminescence.
> Have any of you ever tried something similar? In systems dedicated for
> bioluminescence signal is detected with the same or similar camera
> (Andor iXon Ultra 897) though light path is less complex.
> My idea is to take time series with longest possible exposure time (1
> sec), convert T->Z and then sum up all slices. Does it have any sense?
> Thank you in advance
> Michal Majkowski
>
> Imaging Specialist
> University of Wroclaw
> Biotechnology Faculty
> Joliot-Curie 14a
> 50-383 Wroclaw POLAND
>
> ------------------------------------------------------------
> 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.
> =================================
>

[Michał Majkowski-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Thank you for sharing your experience and opinion.
Best
Michal

pon., 8 mar 2021 o 16:08 Zdenek Svindrych <[hidden email]> napisał(a):

> *****
> 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 Michal,
> you may want to use much longer exposure times (30 seconds for CMOS
> cameras, hours for CCDs) depending how weak your luminescence is. With
> EMCCDs, since the readout noise is negligible, you should get the same
> results by summing shorter exposure images, like you suggested. You may
> also bin the pixels for more SNR.
> For optimization and troubleshooting you can measure the luminescence from
> your cells with a suitable plate reader, they are much more sensitive.
> I haven't tried this with the Zeiss Axio stand, but one issue I found with
> the Nikon Ti-E and TE2000 is the stray light coming from internal
> components (probably the encoders). I had to power off the microscope body
> completely to get low background (I think Ti2-E can power off the encoders
> only, if needed).
> Also, dedicated systems may have much better light throughput, especially
> when you trade off resolution for collection efficiency (just compare the
> back focal plane aperture size of your microscope objective lens with let's
> say Nikkor 50 mm F/# 1.2.
> Good luck!
> Best, zdenek
>
> On Mon, Mar 8, 2021 at 8:50 AM Michał Majkowski <
> [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 List Members,
> > I am just thinking about the use of our Elyra 7 system to visualise
> > bioluminescence.
> > Have any of you ever tried something similar? In systems dedicated for
> > bioluminescence signal is detected with the same or similar camera (Andor
> > iXon Ultra 897) though light path is less complex.
> > My idea is to take time series with longest possible exposure time (1
> sec),
> > convert T->Z and then sum up all slices. Does it have any sense?
> > Thank you in advance
> > Michal Majkowski
> >
> > Imaging Specialist
> > University of Wroclaw
> > Biotechnology Faculty
> > Joliot-Curie 14a
> > 50-383 Wroclaw POLAND
> >
>
>
> --
> --
> Zdenek Svindrych, Ph.D.
> Research Scientist - Microscopy Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>