Keeping zebrafish embryos in the field of view but letting them grow.

[Shalin Mehta]

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Hello listers,
We are imaging zebrafish embryos with 10x and the embryos just about
fit the field of view at the development stage of interest
(4hpf-30hpf).

Are there tried and tested methods for giving the embryos room to
grow, while keeping them within the field of view?

Embryos are mounted in agarose and we can make space in agarose pad
near the tail when the embryos are older. But, identifying the growth
axis at young stages (~4hpf) is difficult.

All inputs appreciated,
Thanks

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.

[jerie]

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*****

Dear Shalin,

have you seen *BMC Biotechnology* 2014, *14*:36.
doi:10.1186/1472-6750-14-36?

Kind regards, jens


On Wed, Jun 4, 2014 at 5:15 PM, Shalin Mehta <[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 listers,
> We are imaging zebrafish embryos with 10x and the embryos just about
> fit the field of view at the development stage of interest
> (4hpf-30hpf).
>
> Are there tried and tested methods for giving the embryos room to
> grow, while keeping them within the field of view?
>
> Embryos are mounted in agarose and we can make space in agarose pad
> near the tail when the embryos are older. But, identifying the growth
> axis at young stages (~4hpf) is difficult.
>
> All inputs appreciated,
> Thanks
>
> Assistant Research Scientist,
> Marine Biological Laboratory,
> 7 MBL Street, Woods Hole MA 02543, USA
>
> website: http://mshalin.com
> (office) Lillie 110, (ph) 508-289-7374.
>

--
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype Jens.Rietdorf

[Guy Cox-2]

Shalin,

                I know they do this sort of thing in the Monash Micro-Imaging Live Cell course so maybe one of the Steves will reply.  My own suggestion would to punch a hole just smaller than the FOV in your agarose, so the embryo can grow in any direction without you losing track of it.  A cow-sized hypodermic needle would probably do it.  (I've got several but I think it would be easier if you sourced them locally).  

                                Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shalin Mehta
Sent: Thursday, 5 June 2014 6:15 AM
To: [hidden email]
Subject: Keeping zebrafish embryos in the field of view but letting them grow.

*****
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*****

Hello listers,
We are imaging zebrafish embryos with 10x and the embryos just about fit the field of view at the development stage of interest (4hpf-30hpf).

Are there tried and tested methods for giving the embryos room to grow, while keeping them within the field of view?

Embryos are mounted in agarose and we can make space in agarose pad near the tail when the embryos are older. But, identifying the growth axis at young stages (~4hpf) is difficult.

All inputs appreciated,
Thanks

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.

[Cameron Nowell-3]

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*****

Hi Shalin,

Like Guy said we have done this at the Monash Live Cell Course. Normally we have used younger embryos but for older ones we have had good luck with CyGel. In the past i have done other imaging with older (96hpf) embryos and have done it just with low melt temp agarose. Its good for about 24 hours of imaging. If you need more then you have to chop away part around the tail end to allow the embryo to grow.

I also know of a group here in Melbourne that are building lab on a chip type microfuidic devices to hold embryos (2-30hpf) in place. They are really amazing little things and let you load up a heap of embroys with each one locking into place using micro fluidics. These can then be imaged without a problem

Drop me a message off list and i can put you in touch with them.

Cheers

Cam



Cameron J. Nowell
Research Facilities Manager

Monash Institute of Pharmaceutical Sciences
Monash University
399 Royal Parade
(Mail address: 381 Royal Parade)
Parkville, VIC, 3052
Australia

Email: [hidden email]
Mobile: [phone]
Office: [phone]

LinkedIn: Profile
Research Gate:  Profile


________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Guy Cox [[hidden email]]
Sent: Thursday, June 05, 2014 12:16 PM
To: [hidden email]
Subject: Re: Keeping zebrafish embryos in the field of view but letting them grow.

Shalin,

                I know they do this sort of thing in the Monash Micro-Imaging Live Cell course so maybe one of the Steves will reply.  My own suggestion would to punch a hole just smaller than the FOV in your agarose, so the embryo can grow in any direction without you losing track of it.  A cow-sized hypodermic needle would probably do it.  (I've got several but I think it would be easier if you sourced them locally).

                                Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shalin Mehta
Sent: Thursday, 5 June 2014 6:15 AM
To: [hidden email]
Subject: Keeping zebrafish embryos in the field of view but letting them grow.

*****
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 listers,
We are imaging zebrafish embryos with 10x and the embryos just about fit the field of view at the development stage of interest (4hpf-30hpf).

Are there tried and tested methods for giving the embryos room to grow, while keeping them within the field of view?

Embryos are mounted in agarose and we can make space in agarose pad near the tail when the embryos are older. But, identifying the growth axis at young stages (~4hpf) is difficult.

All inputs appreciated,
Thanks

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.

[Pamela Young]

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*****

I¹m looping my friend, Lauren, in on this conversation because I know her
lab at University of Wisconsin has developed microfluidic devices for this
purpose.  

Dr Pamela A. Young
 | Light and Optical Microscopist
Australian Centre for Microscopy & Microanalysis

THE UNIVERSITY OF SYDNEY
Rm 116A, Madsen Building F09 | The University of Sydney | NSW | 2006 |
Australia
T [phone] | F [phone]
E [hidden email] | W http://sydney.edu.au/acmm

Incorporating:
Australian Microscopy & Microanalysis Research Facility (AMMRF) | W
http://www.ammrf.org.au <http://www.ammrf.org.au/>
ARC Centre of Excellence for Design in Light Metals | W
http://www.arclightmetals.org.au <http://www.arclightmetals.org.au/>

CRICOS 00026A
This email plus any attachments to it are confidential. Any unauthorised
use is strictly prohibited. If you receive this email in error, please
delete it and any attachments.



On 5/06/2014 11:23 pm, "Cameron Nowell" <[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 Shalin,
>
>Like Guy said we have done this at the Monash Live Cell Course. Normally
>we have used younger embryos but for older ones we have had good luck
>with CyGel. In the past i have done other imaging with older (96hpf)
>embryos and have done it just with low melt temp agarose. Its good for
>about 24 hours of imaging. If you need more then you have to chop away
>part around the tail end to allow the embryo to grow.
>
>I also know of a group here in Melbourne that are building lab on a chip
>type microfuidic devices to hold embryos (2-30hpf) in place. They are
>really amazing little things and let you load up a heap of embroys with
>each one locking into place using micro fluidics. These can then be
>imaged without a problem
>
>Drop me a message off list and i can put you in touch with them.
>
>Cheers
>
>Cam
>
>
>
>Cameron J. Nowell
>Research Facilities Manager
>
>Monash Institute of Pharmaceutical Sciences
>Monash University
>399 Royal Parade
>(Mail address: 381 Royal Parade)
>Parkville, VIC, 3052
>Australia
>
>Email: [hidden email]
>Mobile: [phone]
>Office: [phone]
>
>LinkedIn: Profile
>Research Gate:  Profile
>
>
>________________________________________
>From: Confocal Microscopy List [[hidden email]] on
>behalf of Guy Cox [[hidden email]]
>Sent: Thursday, June 05, 2014 12:16 PM
>To: [hidden email]
>Subject: Re: Keeping zebrafish embryos in the field of view but letting
>them grow.
>
>Shalin,
>
>                I know they do this sort of thing in the Monash
>Micro-Imaging Live Cell course so maybe one of the Steves will reply.  My
>own suggestion would to punch a hole just smaller than the FOV in your
>agarose, so the embryo can grow in any direction without you losing track
>of it.  A cow-sized hypodermic needle would probably do it.  (I've got
>several but I think it would be easier if you sourced them locally).
>
>                                Guy
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Shalin Mehta
>Sent: Thursday, 5 June 2014 6:15 AM
>To: [hidden email]
>Subject: Keeping zebrafish embryos in the field of view but letting them
>grow.
>
>*****
>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 listers,
>We are imaging zebrafish embryos with 10x and the embryos just about fit
>the field of view at the development stage of interest (4hpf-30hpf).
>
>Are there tried and tested methods for giving the embryos room to grow,
>while keeping them within the field of view?
>
>Embryos are mounted in agarose and we can make space in agarose pad near
>the tail when the embryos are older. But, identifying the growth axis at
>young stages (~4hpf) is difficult.
>
>All inputs appreciated,
>Thanks
>
>Assistant Research Scientist,
>Marine Biological Laboratory,
>7 MBL Street, Woods Hole MA 02543, USA
>
>website: http://mshalin.com
>(office) Lillie 110, (ph) 508-289-7374.

[Dan Focht]

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*****

Shalin

Bioptechs manufactures glass culture cylinders that have been used for that purpose.
They are made of glass, reuseable, autoclavable, and don't require grease or wax to seal them.
link:  
http://www.bioptechs.com/Products/Delta_T/Options/options.html#Culture%20Cylinders

We have many sizes and can make custom geometries.  
They are an easy way to constrain specimens.
Ask, and I can send a sample.

Dan



On Jun 4, 2014, at 4:15 PM, Shalin Mehta wrote:

*****
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.
*****

Hello listers,
We are imaging zebrafish embryos with 10x and the embryos just about
fit the field of view at the development stage of interest
(4hpf-30hpf).

Are there tried and tested methods for giving the embryos room to
grow, while keeping them within the field of view?

Embryos are mounted in agarose and we can make space in agarose pad
near the tail when the embryos are older. But, identifying the growth
axis at young stages (~4hpf) is difficult.

All inputs appreciated,
Thanks

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler, PA 16002
www.bioptechs.com
P: [phone]
F: [phone]
[hidden email]

[Shalin Mehta]

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

Thanks everyone. We have started making the place holders in agarose
using 5ul pipette tip. I cut the tip's thinnest part and then fill it
with mounting medium. After the medium hardens, agarose can solidify
around this simple template attached to the cover of the petridish. I
hope to try some microfluidic approaches for achieving consisten
orientation of embryo from experiment to experiment.

Shalin
Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


On Thu, Jun 5, 2014 at 7:01 PM, Pamela Young <[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.
> *****
>
> I¹m looping my friend, Lauren, in on this conversation because I know her
> lab at University of Wisconsin has developed microfluidic devices for this
> purpose.
>
> Dr Pamela A. Young
>  | Light and Optical Microscopist
> Australian Centre for Microscopy & Microanalysis
>
> THE UNIVERSITY OF SYDNEY
> Rm 116A, Madsen Building F09 | The University of Sydney | NSW | 2006 |
> Australia
> T [phone] | F [phone]
> E [hidden email] | W http://sydney.edu.au/acmm
>
> Incorporating:
> Australian Microscopy & Microanalysis Research Facility (AMMRF) | W
> http://www.ammrf.org.au <http://www.ammrf.org.au/>
> ARC Centre of Excellence for Design in Light Metals | W
> http://www.arclightmetals.org.au <http://www.arclightmetals.org.au/>
>
> CRICOS 00026A
> This email plus any attachments to it are confidential. Any unauthorised
> use is strictly prohibited. If you receive this email in error, please
> delete it and any attachments.
>
>
>
> On 5/06/2014 11:23 pm, "Cameron Nowell" <[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 Shalin,
>>
>>Like Guy said we have done this at the Monash Live Cell Course. Normally
>>we have used younger embryos but for older ones we have had good luck
>>with CyGel. In the past i have done other imaging with older (96hpf)
>>embryos and have done it just with low melt temp agarose. Its good for
>>about 24 hours of imaging. If you need more then you have to chop away
>>part around the tail end to allow the embryo to grow.
>>
>>I also know of a group here in Melbourne that are building lab on a chip
>>type microfuidic devices to hold embryos (2-30hpf) in place. They are
>>really amazing little things and let you load up a heap of embroys with
>>each one locking into place using micro fluidics. These can then be
>>imaged without a problem
>>
>>Drop me a message off list and i can put you in touch with them.
>>
>>Cheers
>>
>>Cam
>>
>>
>>
>>Cameron J. Nowell
>>Research Facilities Manager
>>
>>Monash Institute of Pharmaceutical Sciences
>>Monash University
>>399 Royal Parade
>>(Mail address: 381 Royal Parade)
>>Parkville, VIC, 3052
>>Australia
>>
>>Email: [hidden email]
>>Mobile: [phone]
>>Office: [phone]
>>
>>LinkedIn: Profile
>>Research Gate:  Profile
>>
>>
>>________________________________________
>>From: Confocal Microscopy List [[hidden email]] on
>>behalf of Guy Cox [[hidden email]]
>>Sent: Thursday, June 05, 2014 12:16 PM
>>To: [hidden email]
>>Subject: Re: Keeping zebrafish embryos in the field of view but letting
>>them grow.
>>
>>Shalin,
>>
>>                I know they do this sort of thing in the Monash
>>Micro-Imaging Live Cell course so maybe one of the Steves will reply.  My
>>own suggestion would to punch a hole just smaller than the FOV in your
>>agarose, so the embryo can grow in any direction without you losing track
>>of it.  A cow-sized hypodermic needle would probably do it.  (I've got
>>several but I think it would be easier if you sourced them locally).
>>
>>                                Guy
>>
>>-----Original Message-----
>>From: Confocal Microscopy List [mailto:[hidden email]]
>>On Behalf Of Shalin Mehta
>>Sent: Thursday, 5 June 2014 6:15 AM
>>To: [hidden email]
>>Subject: Keeping zebrafish embryos in the field of view but letting them
>>grow.
>>
>>*****
>>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 listers,
>>We are imaging zebrafish embryos with 10x and the embryos just about fit
>>the field of view at the development stage of interest (4hpf-30hpf).
>>
>>Are there tried and tested methods for giving the embryos room to grow,
>>while keeping them within the field of view?
>>
>>Embryos are mounted in agarose and we can make space in agarose pad near
>>the tail when the embryos are older. But, identifying the growth axis at
>>young stages (~4hpf) is difficult.
>>
>>All inputs appreciated,
>>Thanks
>>
>>Assistant Research Scientist,
>>Marine Biological Laboratory,
>>7 MBL Street, Woods Hole MA 02543, USA
>>
>>website: http://mshalin.com
>>(office) Lillie 110, (ph) 508-289-7374.