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Podosome lifetime - which scope and software?

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Seb Stephens

Podosome lifetime - which scope and software?

Hi all, I am studying structures that are similar to focal adhesions called
podosomes. They are rich in actin and make an actin core of about 1 micron in
diameter.

These podosomes have a lifetime about about 2 mins (that is, it takes 2 mins
for a "dot" in the cell to become visible and dissappear again).

At this stage I use a CSU-X on an Andor system (63x, binned 2x2 Ocra ER)
but I find it very hard to resolve because there is an "actin cloud" that is
found within the podosome belt (which you see surrounds the gigantic
osteoclast cell). I will be trying TIRF soon.

I also do not have any software for analyzing this (apart from going frame by
frame in image j).

As such, would you have any suggestions as to the best way of acquiring the
images (CSU, point scanner etc) and software for this work?

I would also be interested in communicating with anyone who does this sort of
particle tracking.

Regards Sebastien

RICHARD BURRY

Re: Podosome lifetime - which scope and software?

Sebastien

We are using a Visitech Infinity3 2D array confocal with a Hamamatsu C9100 EM-13 camera (not binned) and see the podosome rings in images taken from z stacks. Image time is 150 to 300 msec.

Dick

----- Original Message -----
From: Sebastien Stephens <[hidden email]>
Date: Monday, July 6, 2009 4:00 pm
Subject: Podosome lifetime - which scope and software?
To: [hidden email]

> Hi all, I am studying structures that are similar to focal
> adhesions called
> podosomes. They are rich in actin and make an actin core of
> about 1 micron in
> diameter.
>
> These podosomes have a lifetime about about 2 mins (that is, it
> takes 2 mins
> for a "dot" in the cell to become visible and dissappear
> again).
>
> At this stage I use a CSU-X on an Andor system (63x, binned 2x2
> Ocra ER)
> but I find it very hard to resolve because there is an "actin
> cloud" that is
> found within the podosome belt (which you see surrounds the
> gigantic
> osteoclast cell). I will be trying TIRF soon.
>
> I also do not have any software for analyzing this (apart from
> going frame by
> frame in image j).
>
> As such, would you have any suggestions as to the best way of
> acquiring the
> images (CSU, point scanner etc) and software for this work?
>
> I would also be interested in communicating with anyone who does
> this sort of
> particle tracking.
>
> Regards Sebastien
>
>
> --
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Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814 Cell 614.638.3345 Fax 614.247.8849

Rietdorf, Jens

Re: Podosome lifetime - which scope and software?

In reply to this post by Seb Stephens

Sebastien,

I have been using TIR to image hemidesmosomes (which partly resemble
podosomes), they are very close to the cover glass and TIR gives an
excellent contrast, you can nicely follow the structures if actin is
labeled.
Automatic tracking should be easy because the structures do not move
fast, if at all.

Cheers, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Sebastien Stephens
Sent: Monday, July 06, 2009 9:49 PM
To: [hidden email]
Subject: Podosome lifetime - which scope and software?

Hi all, I am studying structures that are similar to focal adhesions
called
podosomes. They are rich in actin and make an actin core of about 1
micron in
diameter.

These podosomes have a lifetime about about 2 mins (that is, it takes 2
mins
for a "dot" in the cell to become visible and dissappear again).

At this stage I use a CSU-X on an Andor system (63x, binned 2x2 Ocra ER)

but I find it very hard to resolve because there is an "actin cloud"
that is
found within the podosome belt (which you see surrounds the gigantic
osteoclast cell). I will be trying TIRF soon.

I also do not have any software for analyzing this (apart from going
frame by
frame in image j).

As such, would you have any suggestions as to the best way of acquiring
the
images (CSU, point scanner etc) and software for this work?

I would also be interested in communicating with anyone who does this
sort of
particle tracking.

Regards Sebastien

Watkins, Simon C

Re: Podosome lifetime - which scope and software?

In reply to this post by RICHARD BURRY

I have been imaging podosomes in dendrititic cells for a good while now and TIRF is really the only way to go IMHO
Simon

Sent via BlackBerry by AT&T

From: RICHARD BURRY
Date: Mon, 6 Jul 2009 16:37:56 -0400
To: [hidden email]<[hidden email]>
Subject: Re: Podosome lifetime - which scope and software?

Seb Stephens

Re: Podosome lifetime - which scope and software?

In reply to this post by Seb Stephens

Hi Richard,

The system I use actually does have an ANDOR camera very similar to the one
you mentioned. I will indeed test this sometime and discuss it further with my
core manager.

Regards

Sebastien

Seb Stephens

Re: Podosome lifetime - which scope and software?

In reply to this post by Seb Stephens

Hi Jens,

Thanks for your reply.

I am using GFP-actin to track these podosomes. Im not sure whether I should
use a labelling kit or a commercially available labelled actin which which is far
smaller than GFP! I will be trying this.

Im wondering what software you use for auto tracking?

Furthermore, would you be able to send one of your videos? Id like to see
what your structures look like on TIRF. Of course, I understand that you may
not be able to share your videos which is fine!

regards

Seb Stephens

Re: Podosome lifetime - which scope and software?

In reply to this post by Seb Stephens

Simon,

What software do you use to track your pdosomes?

Would you be able to send me one of your videos so I can what you are
seeing and compare to what Im seeing?

Where are you located?

Seb

On Mon, 6 Jul 2009 18:41:38 -0400, Watkins, Simon C <[hidden email]>
wrote:

>I have been imaging podosomes in dendrititic cells for a good while now and
TIRF is really the only way to go IMHO
>Simon
>
>Sent via BlackBerry by AT&T