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Organ movement

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Michael Herron

Organ movement

I am trying to image intracellular bacteria in living tissue explants
and the tissue moves (sort of writhes) in a manner that moves the
bacteria out of the planes of my Z collections.
I cannot cool the specimen, but I was wondering if a "specific"
channel or second messenger inhibitor might reduce this movement.
This must be a common problem in Drosophila or zebra fish imaging?

Any suggestions?

Michael J. Herron, U of MN, Dept. of Entomology
[hidden email]
612-624-3688 (office) 612-625-5299 (FAX)

Stephen Cody-2

Re: Organ movement

G'day Michael,

We successfully held an excised rat heart still during imaging. We used a dipping lens on an inverted microscope, and a modified version of the "condom bath". We applied a mild vacuum to the condom using a venturi which sucked the heart down onto a small o-ring, which we imaged through. This part of the heart was held still while the rest of the heart was able to beat freely.

This may help with your tissue writhing problem.

See: http://ajpheart.physiology.org/cgi/content/abstract/272/1/H236

I just read in the bottom of your post that you are an entomologist. We also looked at malphigian tubules (Cooper, Cody and Williams, unpublished), from memory we stuck them down with cell-tack. The sucking bath may be too brutal for this type of tissue. There are a lot of recipes out there to make your coverslip sticky, the most common being Poly-L-Lysine.

Cheers,

Stephen Cody

Consulting Microscopist

2008/12/17 Michael Herron <[hidden email]>

I am trying to image intracellular bacteria in living tissue explants and the tissue moves (sort of writhes) in a manner that moves the bacteria out of the planes of my Z collections.
I cannot cool the specimen, but I was wondering if a "specific" channel or second messenger inhibitor might reduce this movement. This must be a common problem in Drosophila or zebra fish imaging?

Any suggestions?

Michael J. Herron, U of MN, Dept. of Entomology
[hidden email]
612-624-3688 (office) 612-625-5299 (FAX)

Steve Hunter-2

Re: Organ movement

In reply to this post by Michael Herron

Hi Michael,

You have not described what equipment you are using to collect your z-stacks. One solution may be to collect the stacks very quickly. I have had experience with spinning disc confocals and these are quite capable of obtaining a 10-20 slice z-stack in 1 second and because each slice is captured as a single image rather than collected point by point as is the case with the traditional confocals you have less registration issues. If you use a back-illuminated emccd camera then they are also far more sensitive than the traditional confocals.

I hope this helps. Incidentally I have no commercial interest with these systems.

Steve Hunter
Regional Sales Manager - Australasia & SE Asia
Phone: +61 3 9538-3364
Mob: +61 417 501460
http://www.optiscan.com.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Herron
Sent: Wednesday, 17 December 2008 6:27 AM
To: [hidden email]
Subject: Organ movement

I am trying to image intracellular bacteria in living tissue explants
and the tissue moves (sort of writhes) in a manner that moves the
bacteria out of the planes of my Z collections.
I cannot cool the specimen, but I was wondering if a "specific"
channel or second messenger inhibitor might reduce this movement.
This must be a common problem in Drosophila or zebra fish imaging?

Any suggestions?

Michael J. Herron, U of MN, Dept. of Entomology
[hidden email]
612-624-3688 (office) 612-625-5299 (FAX)

Paul Cooper-2-3

Re: Organ movement

In reply to this post by Stephen Cody-2

G'day Michael,

I can confirm that we were able to image Malpighian tubules as Steve suggested, although we weren't attempting to see bacteria, so the focal place was not as precise. We did use cell-tack, although I have used other compounds as well.

Paul Cooper

Australian National University

On 17/12/2008, at 10:11 AM, Stephen Cody wrote:

G'day Michael,

We successfully held an excised rat heart still during imaging. We used a dipping lens on an inverted microscope, and a modified version of the "condom bath". We applied a mild vacuum to the condom using a venturi which sucked the heart down onto a small o-ring, which we imaged through. This part of the heart was held still while the rest of the heart was able to beat freely.

This may help with your tissue writhing problem.

See: http://ajpheart.physiology.org/cgi/content/abstract/272/1/H236

I just read in the bottom of your post that you are an entomologist. We also looked at malphigian tubules (Cooper, Cody and Williams, unpublished), from memory we stuck them down with cell-tack. The sucking bath may be too brutal for this type of tissue. There are a lot of recipes out there to make your coverslip sticky, the most common being Poly-L-Lysine.

Cheers,

Stephen Cody

Consulting Microscopist

2008/12/17 Michael Herron <[hidden email]>

I am trying to image intracellular bacteria in living tissue explants and the tissue moves (sort of writhes) in a manner that moves the bacteria out of the planes of my Z collections.
I cannot cool the specimen, but I was wondering if a "specific" channel or second messenger inhibitor might reduce this movement. This must be a common problem in Drosophila or zebra fish imaging?

Any suggestions?

Michael J. Herron, U of MN, Dept. of Entomology
[hidden email]
612-624-3688 (office) 612-625-5299 (FAX)

Paul Cooper

School of Botany & Zoology

Australian National University

Canberra, ACT 0200

Australia

[hidden email]

Telephone: 61-2-61253069

FAX: 61-2-61255573

http://www.anu.edu.au/BoZo/

http://online.anu.edu.au/EES_BOZO/

CRICOS Provider #00120C

Marc M Takeno

Re: Organ movement

In reply to this post by Michael Herron

Hello Michael,

A group at MIT (Mehmet Yanik) has constructed a nice system to immobilize C. elegans while imaging and performing microsurgery. I don't know how involved the associated hardware and control might be, but a similar idea might be adapted for your lab.

http://web.mit.edu/newsoffice/2008/lab-on-chip-0410.html

The original PNAS paper:

http://www.pnas.org/content/104/35/13891.full

On Tue, 16 Dec 2008, Michael Herron wrote:

> I am trying to image intracellular bacteria in living tissue explants and the
> tissue moves (sort of writhes) in a manner that moves the bacteria out of the
> planes of my Z collections.
> I cannot cool the specimen, but I was wondering if a "specific" channel or
> second messenger inhibitor might reduce this movement. This must be a common
> problem in Drosophila or zebra fish imaging?
>
> Any suggestions?
>
>
> Michael J. Herron, U of MN, Dept. of Entomology
> [hidden email]
> 612-624-3688 (office) 612-625-5299 (FAX)
>

William Hatton

Re: Organ movement

Hi Michael,
Apart from physical restraint of the heart you could also try
nicardipine or nifedipine (10-2M), both are Ca2+ channel blockers and
will inhibit/stop contraction.
Good luck,
Will

William J. Hatton, PhD
Res. Asst. Professor
Director of Imaging and Morphology Core,
NIH Center of Biomedical Research Excellence (COBRE)
Department of Pharmacology/318
University of Nevada School of Medicine
Reno, NV 89557-0046 USA

Office phone: 775-784-6289
Lab phone: 775-784-4769
fax: 775-784-1620
e-mail: [hidden email]
http://www.medicine.nevada.edu/dept/Pharmacology/faculty/hatton.html

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Marc M Takeno
Sent: Wednesday, December 17, 2008 10:08 AM
To: [hidden email]
Subject: Re: Organ movement

Hello Michael,

A group at MIT (Mehmet Yanik) has constructed a nice system to
immobilize C. elegans while imaging and performing microsurgery. I don't
know how involved the associated hardware and control might be, but a
similar idea might be adapted for your lab.

http://web.mit.edu/newsoffice/2008/lab-on-chip-0410.html

The original PNAS paper:

http://www.pnas.org/content/104/35/13891.full

On Tue, 16 Dec 2008, Michael Herron wrote:

> I am trying to image intracellular bacteria in living tissue explants
and the
> tissue moves (sort of writhes) in a manner that moves the bacteria out
of the
> planes of my Z collections.
> I cannot cool the specimen, but I was wondering if a "specific"
channel or
> second messenger inhibitor might reduce this movement. This must be a
common
> problem in Drosophila or zebra fish imaging?
>
> Any suggestions?
>
>
> Michael J. Herron, U of MN, Dept. of Entomology
> [hidden email]
> 612-624-3688 (office) 612-625-5299 (FAX)
>

--
Marc Takeno, Ph.D. | Research Scientist | University of Washington
Department of Bioengineering | N330B Foege | Box 355061
(206) 543-4290 | [hidden email]