organoid imaging

[Mary Ellen Pease]

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Hi all,

I have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.

Best,

Mary Ellen Pease
Manager, Wilmer Microscopy and Imaging Core Facility (MICF)
Johns Hopkins SOM, Baltimore MD

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Hello Mary Ellen,

We had an interesting talk by Daniel Fulton from the University of Birmingham, UK (https://www.birmingham.ac.uk/staff/profiles/inflammation-ageing/fulton-daniel.aspx)

at our recent on-line conference, where he spoke about long term live imaging of oligodendrocyte myelination in organotypic brain slice cultures. Maybe some of his techniques might be useful?

His talk is at the end of the session located at this link:

https://www.youtube.com/watch?v=0L7tDQ2vDsM


Best regards

Phillipa

Dr Phillipa Timmins
Head of Sales

Aurox Ltd
Culham Science Centre
Abingdon
Oxfordshire
OX14 3DB

Tel: [phone]



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Mary Ellen Pease
Sent: 28 April 2020 15:22
To: [hidden email]
Subject: organoid imaging

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*****

Hi all,

I have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.

Best,

Mary Ellen Pease
Manager, Wilmer Microscopy and Imaging Core Facility (MICF) Johns Hopkins SOM, Baltimore MD

[Philipp Tripal]

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Dear Mary Ellen,

staining of organoids within matrigel is a challange and results in bad
signal to noise ratios... but if your organoids are expressing GFP or
even better long wavelength fluorophores, imaging can be performed
within the matrigel matrix. A dilution of matrigel with medium works
also well and dilution of up to 1:5 are applicable.

To increase the amount of organoids close to the coverslip, you can
place the dish on an ice pack while you seed the
matrigel-medium-organoid suspensions and keep it on ice for 5 minutes.
Within cold matrigel the organoids sink by gravity and you´ll have more
organoids within the working distance of the lens... I recommend
spinning disc microscopy, due to low photo-toxic stress. Matrigel and
2Photon might be challenging, due to the second harmonics of the
collagen matrix...

Best regards,

Philipp


On 28/04/2020 16:21, Mary Ellen Pease 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 have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.
>
> Best,
>
> Mary Ellen Pease
> Manager, Wilmer Microscopy and Imaging Core Facility (MICF)
> Johns Hopkins SOM, Baltimore MD

--
Philipp Tripal
Research Associate
Optical Imaging Centre Erlangen

Cauerstr. 3
91058 Erlangen, Germany
[phone] (Office)
[phone] (Secretary)
[phone] (Fax)

www.oice.uni-erlangen.de

[Michael Doube-2]

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Hi Mary Ellen,

Some time ago I was helping a user with their zebrafish embryos. The system was an LSM 780 upright with dipping lenses. The embryos were maintained in position by pressing small dimples into the top surface of (I think) agarose, then resting each embryo in a dimple, covered in water. It was sufficient in most cases to prevent drift.

Best regards,

Michael

On 28/04/2020 22:21, Mary Ellen Pease wrote:

*****
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*****

Hi all,

I have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.

Best,

Mary Ellen Pease
Manager, Wilmer Microscopy and Imaging Core Facility (MICF)
Johns Hopkins SOM, Baltimore MD


--
[Jockey Club College of Veterinary Medicine and                      Life Sciences - City University of Hong Kong]



Dr. Michael Doube
BVSc, BPhil, PhD, PGCert Vet Ed, MRCVS, FRMS

Associate Professor
Department of Infectious Diseases
and Public Health

5/F, Block 2, To Yuen Building
31 To Yuen Street
Kowloon, Hong Kong
Tel: [phone]
Fax: [phone]
Website: www.cityu.edu.hk/ph<http://www.cityu.edu.hk/ph>



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[George McNamara]

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Hi Mary Allen,

Jawad Allen -- now Dr. Jawad Allen -- in Cindy Sears lab did long term
(weekend) imaging on our Ross Bldg (9th floor)  Olympus FV3000RS
confocal inverted (IX83) microscope (OkoLab stage top + plexiglas shroud
incubator, 37 C, tage top humidifier & optional 5% CO2 ... 10x/0.40 WD
3.1 mm or 20x/0.75 WD 0.6 mm, if I recall correctly ... standard lenses,
so equivalent on your LSM710MP would work fine),

http://confocal.jhu.edu/current-equipment/fv3000/

(I believe) embedded in matrigel, similar to Philipp Tripal's reply. You
should be able to get full details from Cindy (i.e. manuscript in prep).

Yes, Jawad brought his SBS plates between buildings (in an appropriate
container). I strongly recommend the research lab have a quarantine
incubator for plates that leave the lab and return.

Your user is welcome to try out our FV3000RS for comparison - once JHU
SOM re-opens. Full set of laser lines from 405-730 nm. We'll probably be
busy all summer catching up, but should be able to squeeze your user in.

I encourage use of thin bottom SBS plates or 35 mm imaging dish. Mattek
(and some others) offer #0 coverglass bottom, which provides an extra
~70 um working distance compared to #1.5 coverglass (nominally 170 um;
ibidi's bottoms are typically their image quality plastic, ~180 um).

George

p.s. my JHU email handle is gmcnama2


On 4/28/2020 10:21 AM, Mary Ellen Pease 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 have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.
>
> Best,
>
> Mary Ellen Pease
> Manager, Wilmer Microscopy and Imaging Core Facility (MICF)
> Johns Hopkins SOM, Baltimore MD

[Zbigniew Mikulski]

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*****

Hello Mary,

Have a look at CyGel (you can get it from Biostatus, Abcam also distribute it in the US). It's a liquid at 4C and it gels around 22-27C. It is reversible once you lower the temperature. They claim it's non-toxic. We have not tried with 2P imaging or organoids but it worked quite well in our hands with cells and fluorescence microscopy.

All the best, Zbigniew

Zbigniew Mikulski, PhD 
Director, Microscopy Core Facility, Instructor | La Jolla Institute for Immunology | 9420 Athena Circle, La Jolla, CA 92037

[Cammer, Michael-2]

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We kept organoids alive and apparently happy for 12-18 hours on a microscope stage using embedding in Matrigel.  In addition to organoid imaging alone, cells can be added to Matrigel and watch how they interact with organoids.  Imaging in paper below was at low mag, but (not used in the paper below) I fixed and labeled the organoids after overnight imaging to see the same ones at high resolution with confocal, so confident this would have worked live at high res too.



https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716041/

Small intestinal crypts were isolated and cultured as described previously (Sato et al., 2009<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716041/#bib47>). In brief, crypts of proximal small intestine were counted and embedded in 30 µl of Matrigel (Corning) at 10,000 crypts/ml and cultured in DMEM/F-12 ...




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

[hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/

Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Zbigniew Mikulski <[hidden email]>
Sent: Wednesday, April 29, 2020 2:55:09 AM
To: [hidden email]
Subject: Re: organoid imaging

[EXTERNAL]

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=aJsJQjnrPG2aNLszj0ShP3D-zXkejuG6AfyJkKY4bHE&s=syIjA5SFNswCO2uYAgxKQzbmn4ebARMDj75hRb8bchs&e=  and include the link in your posting.
*****

Hello Mary,

Have a look at CyGel (you can get it from Biostatus, Abcam also distribute it in the US). It's a liquid at 4C and it gels around 22-27C. It is reversible once you lower the temperature. They claim it's non-toxic. We have not tried with 2P imaging or organoids but it worked quite well in our hands with cells and fluorescence microscopy.

All the best, Zbigniew

Zbigniew Mikulski, PhD
Director, Microscopy Core Facility, Instructor | La Jolla Institute for Immunology | 9420 Athena Circle, La Jolla, CA 92037

[Mary Ellen Pease]

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*****

thanks George - i was hoping an on campus expert would be listening! I will
look into the manuscript, etc. I'm home for how and I don't know if/when we
will be ready to try again since everyone on campus is in shutdown - i know
the 2 labs I was working with have put things to be for now. These are
interesting times we live in.

my jhu address is [hidden email].

Best,
*Mary Ellen*


On Tue, Apr 28, 2020 at 11:13 AM George McNamara <[hidden email]>
wrote:

> Hi Mary Allen,
>
> Jawad Allen -- now Dr. Jawad Allen -- in Cindy Sears lab did long term
> (weekend) imaging on our Ross Bldg (9th floor)  Olympus FV3000RS
> confocal inverted (IX83) microscope (OkoLab stage top + plexiglas shroud
> incubator, 37 C, tage top humidifier & optional 5% CO2 ... 10x/0.40 WD
> 3.1 mm or 20x/0.75 WD 0.6 mm, if I recall correctly ... standard lenses,
> so equivalent on your LSM710MP would work fine),
>
> http://confocal.jhu.edu/current-equipment/fv3000/
>
> (I believe) embedded in matrigel, similar to Philipp Tripal's reply. You
> should be able to get full details from Cindy (i.e. manuscript in prep).
>
> Yes, Jawad brought his SBS plates between buildings (in an appropriate
> container). I strongly recommend the research lab have a quarantine
> incubator for plates that leave the lab and return.
>
> Your user is welcome to try out our FV3000RS for comparison - once JHU
> SOM re-opens. Full set of laser lines from 405-730 nm. We'll probably be
> busy all summer catching up, but should be able to squeeze your user in.
>
> I encourage use of thin bottom SBS plates or 35 mm imaging dish. Mattek
> (and some others) offer #0 coverglass bottom, which provides an extra
> ~70 um working distance compared to #1.5 coverglass (nominally 170 um;
> ibidi's bottoms are typically their image quality plastic, ~180 um).
>
> George
>
> p.s. my JHU email handle is gmcnama2
>
>
> On 4/28/2020 10:21 AM, Mary Ellen Pease 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 have a user who would like to image live organoids via
> confocal/multiphoton without jeopardizing the sample. Has anyone figured
> out a material in which the organoid could be placed that would eliminate
> movement/drift during imaging, without damaging it so the sample could go
> back into culture afterwards? So far, the papers I've looked at on this
> topic cryoembed them at a certain time point for immunohistochemistry. My
> user's lab has samples which are expressing GFP and RFP, and would like to
> look at them in vivo. Someone in our department suggested putting them in a
> mix of matrigel and culture media, which could slow them down temporarily,
> but in my experience, matrigel attenuates the signal. We have a multiphoton
> system on an inverted Zeiss platform (LSM 710). we can image using an
> objective inverter and dipping lenses, or from the bottom through a
> coverglass dish. Since we are working remotely at this point in time, I was
> hoping to get some direction from this group.
> >
> > Best,
> >
> > Mary Ellen Pease
> > Manager, Wilmer Microscopy and Imaging Core Facility (MICF)
> > Johns Hopkins SOM, Baltimore MD
>

[Mary Ellen Pease]

*****
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 very much! I will look into this as well.
take care,

*Mary Ellen*

Mary Ellen Pease
410-292-2551 (cell)
[hidden email]


On Wed, Apr 29, 2020 at 9:53 AM 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 kept organoids alive and apparently happy for 12-18 hours on a
> microscope stage using embedding in Matrigel.  In addition to organoid
> imaging alone, cells can be added to Matrigel and watch how they interact
> with organoids.  Imaging in paper below was at low mag, but (not used in
> the paper below) I fixed and labeled the organoids after overnight imaging
> to see the same ones at high resolution with confocal, so confident this
> would have worked live at high res too.
>
>
>
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716041/
>
> Small intestinal crypts were isolated and cultured as described previously
> (Sato et al., 2009<
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716041/#bib47>). In brief,
> crypts of proximal small intestine were counted and embedded in 30 µl of
> Matrigel (Corning) at 10,000 crypts/ml and cultured in DMEM/F-12 ...
>
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
>
> [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Zbigniew Mikulski <[hidden email]>
> Sent: Wednesday, April 29, 2020 2:55:09 AM
> To: [hidden email]
> Subject: Re: organoid imaging
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=aJsJQjnrPG2aNLszj0ShP3D-zXkejuG6AfyJkKY4bHE&s=UFU-FEcZrMDYGpjyqJ2bfLWPcOVEp_sfqyigL_JPB3w&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=aJsJQjnrPG2aNLszj0ShP3D-zXkejuG6AfyJkKY4bHE&s=syIjA5SFNswCO2uYAgxKQzbmn4ebARMDj75hRb8bchs&e=
> and include the link in your posting.
> *****
>
> Hello Mary,
>
> Have a look at CyGel (you can get it from Biostatus, Abcam also distribute
> it in the US). It's a liquid at 4C and it gels around 22-27C. It is
> reversible once you lower the temperature. They claim it's non-toxic. We
> have not tried with 2P imaging or organoids but it worked quite well in our
> hands with cells and fluorescence microscopy.
>
> All the best, Zbigniew
>
> Zbigniew Mikulski, PhD
> Director, Microscopy Core Facility, Instructor | La Jolla Institute for
> Immunology | 9420 Athena Circle, La Jolla, CA 92037
>

[Mary Ellen Pease]

*****
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.
*****

thanks, I will look at this :)

*Mary Ellen*

Mary Ellen Pease
410-292-2551 (cell)
[hidden email]


On Tue, Apr 28, 2020 at 10:47 AM Phillipa Timmins <
[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.
> *****
>
> Hello Mary Ellen,
>
> We had an interesting talk by Daniel Fulton from the University of
> Birmingham, UK (
> https://www.birmingham.ac.uk/staff/profiles/inflammation-ageing/fulton-daniel.aspx
> )
>
> at our recent on-line conference, where he spoke about long term live
> imaging of oligodendrocyte myelination in organotypic brain slice cultures.
> Maybe some of his techniques might be useful?
>
> His talk is at the end of the session located at this link:
>
> https://www.youtube.com/watch?v=0L7tDQ2vDsM
>
>
> Best regards
>
> Phillipa
>
> Dr Phillipa Timmins
> Head of Sales
>
> Aurox Ltd
> Culham Science Centre
> Abingdon
> Oxfordshire
> OX14 3DB
>
> Tel: [phone]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Mary Ellen Pease
> Sent: 28 April 2020 15:22
> To: [hidden email]
> Subject: organoid imaging
>
> *****
> 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 have a user who would like to image live organoids via
> confocal/multiphoton without jeopardizing the sample. Has anyone figured
> out a material in which the organoid could be placed that would eliminate
> movement/drift during imaging, without damaging it so the sample could go
> back into culture afterwards? So far, the papers I've looked at on this
> topic cryoembed them at a certain time point for immunohistochemistry. My
> user's lab has samples which are expressing GFP and RFP, and would like to
> look at them in vivo. Someone in our department suggested putting them in a
> mix of matrigel and culture media, which could slow them down temporarily,
> but in my experience, matrigel attenuates the signal. We have a multiphoton
> system on an inverted Zeiss platform (LSM 710). we can image using an
> objective inverter and dipping lenses, or from the bottom through a
> coverglass dish. Since we are working remotely at this point in time, I was
> hoping to get some direction from this group.
>
> Best,
>
> Mary Ellen Pease
> Manager, Wilmer Microscopy and Imaging Core Facility (MICF) Johns Hopkins
> SOM, Baltimore MD
>

[Mary Ellen Pease]

*****
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 so much - great suggestions and info!

*Mary Ellen*

Mary Ellen Pease
410-292-2551 (cell)
[hidden email]


On Tue, Apr 28, 2020 at 10:50 AM Philipp Tripal <[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 Mary Ellen,
>
> staining of organoids within matrigel is a challange and results in bad
> signal to noise ratios... but if your organoids are expressing GFP or
> even better long wavelength fluorophores, imaging can be performed
> within the matrigel matrix. A dilution of matrigel with medium works
> also well and dilution of up to 1:5 are applicable.
>
> To increase the amount of organoids close to the coverslip, you can
> place the dish on an ice pack while you seed the
> matrigel-medium-organoid suspensions and keep it on ice for 5 minutes.
> Within cold matrigel the organoids sink by gravity and you´ll have more
> organoids within the working distance of the lens... I recommend
> spinning disc microscopy, due to low photo-toxic stress. Matrigel and
> 2Photon might be challenging, due to the second harmonics of the
> collagen matrix...
>
> Best regards,
>
> Philipp
>
>
> On 28/04/2020 16:21, Mary Ellen Pease 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 have a user who would like to image live organoids via
> confocal/multiphoton without jeopardizing the sample. Has anyone figured
> out a material in which the organoid could be placed that would eliminate
> movement/drift during imaging, without damaging it so the sample could go
> back into culture afterwards? So far, the papers I've looked at on this
> topic cryoembed them at a certain time point for immunohistochemistry. My
> user's lab has samples which are expressing GFP and RFP, and would like to
> look at them in vivo. Someone in our department suggested putting them in a
> mix of matrigel and culture media, which could slow them down temporarily,
> but in my experience, matrigel attenuates the signal. We have a multiphoton
> system on an inverted Zeiss platform (LSM 710). we can image using an
> objective inverter and dipping lenses, or from the bottom through a
> coverglass dish. Since we are working remotely at this point in time, I was
> hoping to get some direction from this group.
> >
> > Best,
> >
> > Mary Ellen Pease
> > Manager, Wilmer Microscopy and Imaging Core Facility (MICF)
> > Johns Hopkins SOM, Baltimore MD
>
> --
> Philipp Tripal
> Research Associate
> Optical Imaging Centre Erlangen
>
> Cauerstr. 3
> 91058 Erlangen, Germany
> [phone] (Office)
> [phone] (Secretary)
> [phone] (Fax)
>
> www.oice.uni-erlangen.de
>

[Richard Lisle]

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*****

Hi Mary Ellen

We regularly image organoids embedded in matrigel on an LSM710 (invert) for up to 4 days.

We've imaged GFP, Venus, mBanana, RFP and mCherry, using the standard single photon laser lines. The organoids have been embedded in a matrigel cylinder around 3mm in diameter x 5mm deep, attached to the bottom of a 24 well plate with media filling the well. This has kept multiple organoids per matrigel cylinder viable for the full timecourse. Running non-imaged controls alongside as not shown any noticeable phototoxicity either.

We've been taking Z stacks up to approx. 1.5mm from the plate bottom without any problems or needing to increase gain or laser power through the stack, so we're not finding the matrigel to be a problem.

The caveat here is that we've been able to get away with using a 10x/0.45 objective for our needs, but as long as you have the working distance in the objective you need, it should be worth a try.

Best wishes

Richard
Ludwig Institute for Cancer Research, University of Oxford, Nuffield Department of Medicine, Old Road Campus Research Building
Roosevelt Drive, Oxford

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Mary Ellen Pease
Sent: 28 April 2020 15:22
To: [hidden email]
Subject: organoid imaging

*****
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*****

Hi all,

I have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.

Best,

Mary Ellen Pease
Manager, Wilmer Microscopy and Imaging Core Facility (MICF) Johns Hopkins SOM, Baltimore MD