imaging 100 μM neurospheres

[Bridget-Ann Kenny]

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Good evening,

I have been trying to image neurospheres (assembled from three separately
prepared primary cultures of cells, astrocytes, pericytes and brain
microvascular endothelial cells, so a bit unlike the neurospheres generally
found in the literature) but have been finding it very difficult.

I have been using a Zeiss LSM 700 and mounting entire neurospheres in
mowiol/dabco within a concave hollow of a glass slide so they are not
squashed. The neurospheres are about 100 μM in diameter. Starting with Z
stacks it appears as though I can take images for one half of a neurosphere
but then I get nothing. The stains I've used appear to work so I don't
think the issue is density for staining permeation.

I have since started trying the approach of cryosectioning them and
staining but it would be wonderful to be able to take a Z stack or
something a lot more straightforward as I would like to use this model
intensively and decreasing processing for imaging would be ideal.

If anyone can advise on improving my approach I would be very grateful.
Many thanks.

Best wishes,

Bridget-Ann

[Barbara Foster]

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Hi, Bridget,

This is just a shot in the dark (no pun
intended!), but I googled neurospheres and, at
the size range you are discussing, I think that
the CytoViva accessory would be ideal.  I worked
with Dr. Vodynoy the inventor) on the original
product launch and, although the CytoViva website
explains this technique as though it is
darkfield, there is actually a very different
imaging mechanism working.  The result  gives you
high contrast (a la darkfield) but also allows
you to optically section (very unlike darkfield,
which has an "infinitely deep" depth of field).
Also, while darkfield is a detection-limited
process, what is going on with CV has actual
resolution limits.  I tested them at about 90nm
and the younger, more clear-eyed apps specialist
at the time, at 82nm (Richardson test target).

Because your sample prep is so straight forward,
I would request that they loan you a system for a
short time, to test to see if this approach is
viable.  Imaging would be instantaneous (compared
to the  LSM), you should be able to optically
section then focus stack
(https://en.wikipedia.org/wiki/Focus_stacking).
The system works with fluorescence as well,
although if you use the original version, you
should not have to use fluorphores unless you
want to differentiate/tag specific structures. .

Their website is www.CytoViva.com and it also
lists their contact information.  Please feel free to use my name.

Caveat: No commercial interest.

I hope this was helpful.  Good hunting!

Best regards,
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education  ... "Education, not Training"
7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: 972-924-5310
www.MicroscopyEducation.com


NEW!   Getting involved in Raman or FTIR?
MME is now offering courses in these areas specifically for microscopists!
Now scheduling courses through the end of
2015.  We can customize a course on nearly any
topic, from fluorescence to confocal to image analysis to SEM/TEM.
Call today for a free training evaluation.





At 05:53 AM 9/26/2015, Bridget-Ann Kenny 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.
>*****
>
>Good evening,
>
>I have been trying to image neurospheres (assembled from three separately
>prepared primary cultures of cells, astrocytes, pericytes and brain
>microvascular endothelial cells, so a bit unlike the neurospheres generally
>found in the literature) but have been finding it very difficult.
>
>I have been using a Zeiss LSM 700 and mounting entire neurospheres in
>mowiol/dabco within a concave hollow of a glass slide so they are not
>squashed. The neurospheres are about 100 μM in diameter. Starting with Z
>stacks it appears as though I can take images for one half of a neurosphere
>but then I get nothing. The stains I've used appear to work so I don't
>think the issue is density for staining permeation.
>
>I have since started trying the approach of cryosectioning them and
>staining but it would be wonderful to be able to take a Z stack or
>something a lot more straightforward as I would like to use this model
>intensively and decreasing processing for imaging would be ideal.
>
>If anyone can advise on improving my approach I would be very grateful.
>Many thanks.
>
>Best wishes,
>
>Bridget-Ann

[mcammer]

Is the lens crashing the coverslip?

Is there too much light scattering?

Do you use the depth correction feature in the Z series window of Zen that can increase laser power and gain as you image deeper?


_________________________________________
Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy
http://microscopynotes.com/
Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Bridget-Ann Kenny [[hidden email]]
Sent: Saturday, September 26, 2015 12:28 PM
To: [hidden email]
Subject: imaging 100 μM neurospheres

*****
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*****

Good evening,

I have been trying to image neurospheres (assembled from three separately
prepared primary cultures of cells, astrocytes, pericytes and brain
microvascular endothelial cells, so a bit unlike the neurospheres generally
found in the literature) but have been finding it very difficult.

I have been using a Zeiss LSM 700 and mounting entire neurospheres in
mowiol/dabco within a concave hollow of a glass slide so they are not
squashed. The neurospheres are about 100 μM in diameter. Starting with Z
stacks it appears as though I can take images for one half of a neurosphere
but then I get nothing. The stains I've used appear to work so I don't
think the issue is density for staining permeation.

I have since started trying the approach of cryosectioning them and
staining but it would be wonderful to be able to take a Z stack or
something a lot more straightforward as I would like to use this model
intensively and decreasing processing for imaging would be ideal.

If anyone can advise on improving my approach I would be very grateful.
Many thanks.

Best wishes,

Bridget-Ann

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=================================

[Mark Cannell-2]

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*****

Hi

100 um is pretty thick. Scattering and self absorption could be problems. If the former can you clear the prep? 2 photon could really help if scattering and self absorption are problems.

My 2c

Cheers Mark


On 26/09/2015, at 5:28 pm, Bridget-Ann Kenny <[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.
> *****
>
> Good evening,
>
> I have been trying to image neurospheres (assembled from three separately
> prepared primary cultures of cells, astrocytes, pericytes and brain
> microvascular endothelial cells, so a bit unlike the neurospheres generally
> found in the literature) but have been finding it very difficult.
>
> I have been using a Zeiss LSM 700 and mounting entire neurospheres in
> mowiol/dabco within a concave hollow of a glass slide so they are not
> squashed. The neurospheres are about 100 μM in diameter. Starting with Z
> stacks it appears as though I can take images for one half of a neurosphere
> but then I get nothing. The stains I've used appear to work so I don't
> think the issue is density for staining permeation.
>
> I have since started trying the approach of cryosectioning them and
> staining but it would be wonderful to be able to take a Z stack or
> something a lot more straightforward as I would like to use this model
> intensively and decreasing processing for imaging would be ideal.
>
> If anyone can advise on improving my approach I would be very grateful.
> Many thanks.
>
> Best wishes,
>
> Bridget-Ann

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[George McNamara]

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*****

Hi Bridget-Ann,

Since your specimens are fixed, you can use optical clearing to make it
transparent. A couple of recent web links:

Costantini et al 2015
http://www.nature.com/articles/srep09808
http://arxiv.org/ftp/arxiv/papers/1504/1504.03855.pdf

and
http://blogs.zeiss.com/microscopy/news/en/references-for-clearing-protocols 
... updated for Miyawaki's et al latest ScaleS.
(Zeiss is not the only vendor with web pages on o.c.).

As noted in an earlier reply, working distance could be an issue. For
example, oil immersion objective lenses may not have the working
distance to image through a thick specimen, especially if it is not at
the coverglass. You can get a bit more working distance by using #0
coverglasses instead of the standard #1.5 (you did not specify if you
were imaging through the coverglass or slide - use the former). I
suggest testing the objectives you have on your microscope, starting low
magnification, say 10x, and working up -- in combinatin with the right
fluorophores:

A long wavelength excitation nuclear label, such as Propidium iodide
(PI), or To-Pro-3, or DRAQ5 (the latter also works in live cells), could
be used to label all the cell. If you cannot see nuclei all the way
through an optical cleared, far-red/near-infrared bright dye, you should
go find a better microscope.

Once you have optics and simple staining worked out, you can add more
markers. For example, Brainbow like labeling of the different cell
populations with fluorescent proteins (optionally cell type specifc
promoters for different colors ... and of course mulimerizing and
localizing is better ... see Brainbow 3.0 and my writings on Tattletales
and T-Bow), or much simpler, stain with DiO (green), DiI (orange-red),
DiD (far-red/near infrared) lipophilic dyes, before making the
neurospheres. However, FPs may lose intensity with optical clearing
agents (even if the papers say the signal is retained) and Di_'s may be
stripped by clearing methods.

Two more tips:

*. You are now required by law to use pinhole=1.0 Airy units. Once you
have some staining "at the far end", test whether larger pinhole
setting(s) get you more useful data.

* if you do have any signal, use quantitative deconvolution to make it
better. I recommend www.microvolution.com (not just because that is my
and colleagues image data on their home page). You can also test
AutoQuant and SVI (Huygens). It is possible that Zeiss now has a
deconvolution option in its software - don't assume it works as well as
Microvolution, AQ or Huygens. If you have an infinite amount of time
(processing) and patience (finding usable settings), you can try out the
free devonvolbers in Fiji ImageJ.

best wishes,

George
p.s. um, not uM.



On 9/26/2015 11:28 AM, Bridget-Ann Kenny 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.
> *****
>
> Good evening,
>
> I have been trying to image neurospheres (assembled from three separately
> prepared primary cultures of cells, astrocytes, pericytes and brain
> microvascular endothelial cells, so a bit unlike the neurospheres generally
> found in the literature) but have been finding it very difficult.
>
> I have been using a Zeiss LSM 700 and mounting entire neurospheres in
> mowiol/dabco within a concave hollow of a glass slide so they are not
> squashed. The neurospheres are about 100 μM in diameter. Starting with Z
> stacks it appears as though I can take images for one half of a neurosphere
> but then I get nothing. The stains I've used appear to work so I don't
> think the issue is density for staining permeation.
>
> I have since started trying the approach of cryosectioning them and
> staining but it would be wonderful to be able to take a Z stack or
> something a lot more straightforward as I would like to use this model
> intensively and decreasing processing for imaging would be ideal.
>
> If anyone can advise on improving my approach I would be very grateful.
> Many thanks.
>
> Best wishes,
>
> Bridget-Ann
>
>    

--



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

[Sylvie Le Guyader]

Hi Bridget-Ann

We have imaged a lot of embryos and spheroids without clearing them. We can image completely spheres of up to 200um diameter without clearing if the signal is good. I agree with several advices you already got from the list. Here are my own.

To be able to image your neurospheres you need to do the following :
- If you are using antibodies, optimize your staining to get the brightest signal and lowest background. Changing to a brighter fluorophore, using the longest wavelength for the dimest signal, using freshly fixed and stained samples, staining with low antibody concentration in the fridge for a longer time to increase stringency, not using DAPI... often goes a long way. If you work with proteins you can enhance the signal with eg an anti GFP antibody.
- reduce scattering as much as possible. This point is crucial so match your refractive indices: use an upright system, a water dipping objective eg zeiss 20x NA1.0 dipping. Mount your sample in 1% low melting point agarose in PBS, cover in PBS and image by dipping the objective in the PBS without coverslip between the sample and the objective. It is worth investing in an objective of you do not have a water objective.
- Use the z compensation to change the Imaging settings from low last power/low gain at the top of your stack to high at the bottom.
- Using 2 photon excitation and sensitive detectors like NDDs or GaAsP definitely helps but 100um is to my experience no problem with single photon and descanned PMTs.
- And of course make sure your emission filters are optimal for your fluorophores but I am sure that you have done that. If not, you are welcome to check the bleedthrough video on our website under teaching material. :-)

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD
Live Cell Imaging Unit Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


---- Bridget-Ann Kenny wrote ----

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Good evening,

I have been trying to image neurospheres (assembled from three separately
prepared primary cultures of cells, astrocytes, pericytes and brain
microvascular endothelial cells, so a bit unlike the neurospheres generally
found in the literature) but have been finding it very difficult.

I have been using a Zeiss LSM 700 and mounting entire neurospheres in
mowiol/dabco within a concave hollow of a glass slide so they are not
squashed. The neurospheres are about 100 μM in diameter. Starting with Z
stacks it appears as though I can take images for one half of a neurosphere
but then I get nothing. The stains I've used appear to work so I don't
think the issue is density for staining permeation.

I have since started trying the approach of cryosectioning them and
staining but it would be wonderful to be able to take a Z stack or
something a lot more straightforward as I would like to use this model
intensively and decreasing processing for imaging would be ideal.

If anyone can advise on improving my approach I would be very grateful.
Many thanks.

Best wishes,

Bridget-Ann