Live cell imaging using a inverted confocal microscope

[Faizan Malik]

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Hi Everyone,

I am currently working on optimizing a protocol for live cell imaging of
embryonic brains (mice) with a inverted confocal microscope.

Does anyone have a specific set up or protocol they use? Or a certain type
of perfusion chamber design? I have found a lot of literature regarding live
cell imaging with normal confocal microscopes, but there does not seem to be
much there on live imaging with inverted confocal microscopes. Is there any
specific way that you attach your samples to the chamber in a way that
prevents damage to morphology as well as prevent reduced image quality due
to the using substances like glue or collagen to hold the specimen in place
(a problem not seen with non-inverted confocal microscopes). We use a Zeiss
LSM 700 in our lab.

Your help is appreciated!

Cheers,
Faizan

[Craig Brideau]

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Inverted systems work best for live when you can pull the organ out of the
body partially and lay it next to the animal. Kidney etc. works well for
this. For brain I think the embryo would be tricky to position although
perfusion might be easier since the part you want to image would be deeper
in the chamber. You might want to look into 3d printing for solutions if
you need to build a custom chamber. I've done a fair bit of this for spinal
cord holders/supports and sciatic nerve and being able to create my own
shapes was very helpful!

Craig

On Wed, May 27, 2015 at 4:11 PM, Faizan Malik <[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 Everyone,
>
> I am currently working on optimizing a protocol for live cell imaging of
> embryonic brains (mice) with a inverted confocal microscope.
>
> Does anyone have a specific set up or protocol they use? Or a certain type
> of perfusion chamber design? I have found a lot of literature regarding
> live
> cell imaging with normal confocal microscopes, but there does not seem to
> be
> much there on live imaging with inverted confocal microscopes. Is there any
> specific way that you attach your samples to the chamber in a way that
> prevents damage to morphology as well as prevent reduced image quality due
> to the using substances like glue or collagen to hold the specimen in place
> (a problem not seen with non-inverted confocal microscopes). We use a Zeiss
> LSM 700 in our lab.
>
> Your help is appreciated!
>
> Cheers,
> Faizan
>

[Luis Farias]

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Hello Faizan,
Just to clarify my doubt, you don't want to use collagen with your cells?
Cause I'd succeeded in live imaging using an LSM 5 Pascal (inverted) using
Collagen I protocols. But there is other options without using Collagen,
you just need the suitable dish plates with cover slips on the base.


hope it helps,

regards


Luis Henrique Seabra de Farias*, Msc.*
E-mail: [hidden email] <[hidden email]>
Universidade Federal do Pará
Instituto de Ciências Biológicas
Laboratório de Parasitologia/Laboratório de Biologia Estrutural
Telefone: 913201-7927/913201-8232

2015-05-27 19:11 GMT-03:00 Faizan Malik <[hidden email]>:

> *****
> 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 Everyone,
>
> I am currently working on optimizing a protocol for live cell imaging of
> embryonic brains (mice) with a inverted confocal microscope.
>
> Does anyone have a specific set up or protocol they use? Or a certain type
> of perfusion chamber design? I have found a lot of literature regarding
> live
> cell imaging with normal confocal microscopes, but there does not seem to
> be
> much there on live imaging with inverted confocal microscopes. Is there any
> specific way that you attach your samples to the chamber in a way that
> prevents damage to morphology as well as prevent reduced image quality due
> to the using substances like glue or collagen to hold the specimen in place
> (a problem not seen with non-inverted confocal microscopes). We use a Zeiss
> LSM 700 in our lab.
>
> Your help is appreciated!
>
> Cheers,
> Faizan
>

[Richard Mort]

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Hi Faizan,

Its a challenge, especially if you require an air-liquid interface. We
solved this problem using lummox gas permeable membranes (from Greiner -
I have no association) and this works well for embryonic skin culture.
We also tried on embryonic brain slices recently and it looked
promising. The membranes can be cut out of the dishes to use for custom
apparatus.

The most recent of the following papers may be the most useful but if
you don't have a workshop you may be able to adapt a 50mm lummox dish if
you can find a way to immobilize your sample.

http://www.ncbi.nlm.nih.gov/pubmed/24894489
http://www.ncbi.nlm.nih.gov/pubmed/20067551

We found that if handled carefully the membranes were compatible with
Nikon PFS which was a great help.
Best wishes
Richard



On 27/05/15 23:11, Faizan Malik 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 Everyone,
>
> I am currently working on optimizing a protocol for live cell imaging of
> embryonic brains (mice) with a inverted confocal microscope.
>
> Does anyone have a specific set up or protocol they use? Or a certain type
> of perfusion chamber design? I have found a lot of literature regarding live
> cell imaging with normal confocal microscopes, but there does not seem to be
> much there on live imaging with inverted confocal microscopes. Is there any
> specific way that you attach your samples to the chamber in a way that
> prevents damage to morphology as well as prevent reduced image quality due
> to the using substances like glue or collagen to hold the specimen in place
> (a problem not seen with non-inverted confocal microscopes). We use a Zeiss
> LSM 700 in our lab.
>
> Your help is appreciated!
>
> Cheers,
> Faizan

--
Dr Richard Mort
Research Fellow
MRC Human Genetics Unit
Institute of Genetics and Molecular Medicine
University of Edinburgh
Western General Hospital
Crewe Road
Edinburgh
EH4 2XU

Tel: [phone]


The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

[George McNamara]

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Hi Richard,
Thanks for the tip on the Greiner Bio-One products - product line has
only one m -- Lumox. Product pdf is

http://greinerbioone.com/UserFiles/File/IVSSbrochure.pdf

Hi Fazian,
You may want to invest in an Objective Inverter from LSM Tech

http://www.lsmtech.com/product_objective_inverter.html

enables your specimen to be viewed at any angle. Depending on your
microscope layout, you might also want one of their XYZ Side Stages,

http://www.lsmtech.com/xyz_sidestage.html

though maybe a lab jack next to the microscope would be sufficient.

Enjoy,

George




On 5/28/2015 4:17 AM, Richard Mort 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 Faizan,
>
> Its a challenge, especially if you require an air-liquid interface. We
> solved this problem using lummox gas permeable membranes (from Greiner
> - I have no association) and this works well for embryonic skin
> culture. We also tried on embryonic brain slices recently and it
> looked promising. The membranes can be cut out of the dishes to use
> for custom apparatus.
>
> The most recent of the following papers may be the most useful but if
> you don't have a workshop you may be able to adapt a 50mm lummox dish
> if you can find a way to immobilize your sample.
>
> http://www.ncbi.nlm.nih.gov/pubmed/24894489
> http://www.ncbi.nlm.nih.gov/pubmed/20067551
>
> We found that if handled carefully the membranes were compatible with
> Nikon PFS which was a great help.
> Best wishes
> Richard
>
>
>
> On 27/05/15 23:11, Faizan Malik 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 Everyone,
>>
>> I am currently working on optimizing a protocol for live cell imaging of
>> embryonic brains (mice) with a inverted confocal microscope.
>>
>> Does anyone have a specific set up or protocol they use? Or a certain
>> type
>> of perfusion chamber design? I have found a lot of literature
>> regarding live
>> cell imaging with normal confocal microscopes, but there does not
>> seem to be
>> much there on live imaging with inverted confocal microscopes. Is
>> there any
>> specific way that you attach your samples to the chamber in a way that
>> prevents damage to morphology as well as prevent reduced image
>> quality due
>> to the using substances like glue or collagen to hold the specimen in
>> place
>> (a problem not seen with non-inverted confocal microscopes). We use a
>> Zeiss
>> LSM 700 in our lab.
>>
>> Your help is appreciated!
>>
>> Cheers,
>> Faizan
>

--



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

[Dan Focht]

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*****

Faizan


I just want to add to George’s suggestion of the LSM Tech inverter.
If you use one of these devices you may find it helpful to place your specimen on top of a Corning™ Snapwell membrane in a Bioptechs™ intrinsically temperature controlled Delta T dish.
You can then perfuse media under the membrane to nourish the specimen resting on top of the membrane and image directly from above.  
We have a perfusion system that will maintain this configuration without overflowing making it a convenient methodology.

Another approach is to use our Brain slice adapter in a Delta T dish micro-environmental system.
Link:
 
http://www.bioptechs.com/Products/Delta_T/Options/options.html#Brain%20A



Dan


On May 27, 2015, at 6:11 PM, Faizan Malik <[hidden email]> wrote:

*****
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*****

Hi Everyone,

I am currently working on optimizing a protocol for live cell imaging of
embryonic brains (mice) with a inverted confocal microscope.

Does anyone have a specific set up or protocol they use? Or a certain type
of perfusion chamber design? I have found a lot of literature regarding live
cell imaging with normal confocal microscopes, but there does not seem to be
much there on live imaging with inverted confocal microscopes. Is there any
specific way that you attach your samples to the chamber in a way that
prevents damage to morphology as well as prevent reduced image quality due
to the using substances like glue or collagen to hold the specimen in place
(a problem not seen with non-inverted confocal microscopes). We use a Zeiss
LSM 700 in our lab.

Your help is appreciated!

Cheers,
Faizan

Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler, PA 16002
724-282-7145
Toll Free 877-LIVE-CELL (548-3235)