***Commercial response***
Johan, et al.,
We do something similar to what you describe in terms of labeling
the structure of interest and then using that image to determine an ROI
for assessing localization or another label to that structure. I
agree that the absolute error in doing this with a 2D projection of a
single cell may be high due to orientation and focus considerations.
However, it is not necessary to form a 3D image if you have the ability
to measure enough individual cells in 2D, whether using our system or
any other 2D imaging platform. Rather than relying on the
localization measurement of a single cell, if you assess localization by
measuring the distribution of measurements over many cells the
assessment
will be very robust. The error in each individual measurement will tend
to
broaden the distribution but since the odds of all the cumulative errors
being
in the "same direction" will be very low, the mean of the distribution
will
be very consistent.
Best Regards,
David
David Basiji, Ph.D.
President and CEO, Amnis Corporation
2505 Third Ave., Suite 210
Seattle, WA 98121
+1 206 374 7165 direct
+1 206 919 3342 mobile
+1 206 576 6895 fax
www.amnis.com
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Johan Henriksson
Sent: Monday, November 17, 2008 3:44 AM
To: [log in to unmask]
Subject: Re: image analysis-distribution of protein
Chris Tully wrote:
> Anchal,
>
> Regardless of which software you are using, you need some way of
> isolating the ER and Golgi from the rest of the cell. You may want to
> consider fixing the cells and using a membrane stain for isolate the
> ER and Golgi based on shape, or you will need to express a third FP in
> one of the proteins that populates the ER and Golgi membranes. With a
> seperate channel it should be relatively easy to derive ROIs from the
> membrane channel for use in measuring the Protein A distribution in
> the main image. Now, this technique is only partially accurate.
> Since both the ER and Golgi are relatively small bodies that do not
> fill the entire vertical space of the cell you really need to look at
> this problem in 3D. You will need a lot of slices because you want to
> get as close to a cubic voxel as possible.
>
> I am sure there are some 3D tools for Image J but I would recommend
> that you also check out commercial software such as 3D Constructor
> (for Image-Pro Plus; http://www.mediacy.com <http://www.mediacy.com>),
> Volocity (http://www.improvision.com/products/volocity/), and Imaris
> (http://www.bitplane.com/).
>
or the newer open source packages, Endrov, OME and Bioimagexd
/Johan
--
--
------------------------------------------------
Johan Henriksson
MSc Engineering
PhD student, Karolinska Institutet
http://mahogny.areta.org http://www.endrov.net
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