Dyes for apoplastic pH in leaf tissue

[Ahmad Kabir]

Hi,
I would like to measure apoplastic pH in plant leaf by using confocal
microscope. I am wondering which dyes (pH sensitive and pH
insensitive) would be suitable to use?

[Teng-Leong Chew]

Hi all,

Pardon me for opening up a can of worms of colocalization issue.

I wonder if anyone has experience in thresholding a biological
object using a channel (say, green actin in a contractile ring)
and use only that selected ROI to quantify Pearson coeefficient
between green and a red-labeled protein? Bottom line question
is: how much of the red protein co-localizes to green within
the object and not the whole cell?

We have Volocity, MetaMorph and ImageJ.

Thanks in advance for your help.

Regards,
Leong


--
Teng-Leong Chew, Ph.D.
Director, Cell Imaging Facility & Nikon Imaging Center
Director of University Imaging Resources
Feinberg School of Medicine & Office of Research
Northwestern University
303 E. Chicago Avenue
Chicago, IL 60611
[phone]
[phone] (W, F)

[Armstrong, Louise]

*COMMERCIAL RESPONSE*

Dear Leong

From your email I see that you have our Volocity software and so I would
like to direct you to some of our new training videos and tutorials
which I hope will help with your question.

Please visit our website www.cellularimaging.com and look under the
tutorials section.

Alternatively you should be able to click on this link
http://www.cellularimaging.com/Tutorials/


On the tutorials page there are two videos about colocalization.  The
first by Dr Andrew Barlow deals with the theory of colocalization and
takes you through the interpretation of correlated co-efficients using
simulated and real cell data.  The second by Claire Stewart shows you
how to perform colocalization analyses in our latest version of Volocity
5.4, released last month.

If you don't have the latest version of the software then you can
download and try our new free evaluation/demo version and recreate the
steps yourself using some customer/demonstration data.  You will find
this here http://www.cellularimaging.com/products/volocity/demo/ 

Please feel free to contact me off-list if you would like to discuss
this any further.

Regards

Louise


Louise Armstrong-Denby PhD | Global Product Manager, Bio-discovery
PerkinElmer | For the Better

[hidden email]
www.perkinelmer.com

Please consider the environment before printing this e-mail.
This e-mail message and any attachments are confidential and proprietary
to PerkinElmer, Inc.  If you are not the intended recipient of this
message, please inform the sender by replying to this email or sending a
message to the sender and destroy the message and any attachments.
Thank you.




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Teng-Leong Chew
Sent: 07 September 2010 20:00
To: [hidden email]
Subject: Colocalization question

Hi all,

Pardon me for opening up a can of worms of colocalization issue.

I wonder if anyone has experience in thresholding a biological
object using a channel (say, green actin in a contractile ring)
and use only that selected ROI to quantify Pearson coeefficient
between green and a red-labeled protein? Bottom line question
is: how much of the red protein co-localizes to green within
the object and not the whole cell?

We have Volocity, MetaMorph and ImageJ.

Thanks in advance for your help.

Regards,
Leong


--
Teng-Leong Chew, Ph.D.
Director, Cell Imaging Facility & Nikon Imaging Center
Director of University Imaging Resources
Feinberg School of Medicine & Office of Research
Northwestern University
303 E. Chicago Avenue
Chicago, IL 60611
[phone]
[phone] (W, F)

[Gitta Hamel]

Commercial response

Hello Teng-Leong,

As I understand your question, you would like to calculate the Pearson coefficient on a predefined ROI which is based on a threshold of 1 channel. Our Huygens software contains an option "Colocalization Analyzer" which can calculate the Pearson coefficient after setting a (different) threshold on both channels and take only those voxels into account that are above these thresholds.

Something else that might be interesting for you is the Object Analyzer option. This module make it possible to create an ROI and clip your data to this ROI. You can than put this information in the Colocalization Analyzer which than computes the Pearson coefficient over your ROI only, which is, as I understood it, exactly what you want. Secondly you can apply object based colocalization in the Object Analyzer itself.

You can read more about this Colocalization Analyzer on http://www.svi.nl/CoLocalization.
More about this Object Analyzer is written on http://www.svi.nl/ObjectAnalyzer

If you would like to try out these modules or if you would like to talk more about this matter, please contact us for further information.

With best wishes,

Gitta Hamel

******************************
Gitta Hamel
Managing Director
Scientific Volume Imaging bv
Developers of the Huygens software
The Netherlands
www.svi.nl
[hidden email]
tel: ++ 31 35 6 42 16 26
******************************

Teng-Leong Chew wrote:

Hi all,

Pardon me for opening up a can of worms of colocalization issue.

I wonder if anyone has experience in thresholding a biological
object using a channel (say, green actin in a contractile ring)
and use only that selected ROI to quantify Pearson coeefficient
between green and a red-labeled protein? Bottom line question
is: how much of the red protein co-localizes to green within
the object and not the whole cell?

We have Volocity, MetaMorph and ImageJ.

Thanks in advance for your help.

Regards,
Leong


  

[Barlow, Andrew]

Hello Leong,
You can measure correlation coefficients based around a region of interest in Volocity by selecting a region of interest in the Colocalization view using any ROI tool, or you can make a measurement protocol in the measurements view to select either single or multiple ROIs. You can then either add a "Calculate object colocalisation" task (for per object measurements) or select the colocalization view.

In the colocalization view coefficients are always based on the ROI if one is present.

If you wish to answer your bottom line question, "how much of the red protein co-localizes to green within the object and not the whole cell?" you should study Manders' coefficients which provide a good measurement of co-localization.  Use Pearson's correlation coefficient to get a good measurement of the quality of the relationship.

If you require any assistance with this please do call our technical support team or send me a message off list.
Regards,
Andrew


Andrew Barlow PhD | Applications Specialist
PerkinElmer | For the Better
[hidden email]
Phone:  + 44 2476 692229|   Fax:  [phone]   |   Mobile:   [phone]
Millburn Hill Road, Coventry, CV4 7HS, UK
www.perkinelmer.com
Please consider the environment before printing this e-mail.
This e-mail message and any attachments are confidential and proprietary to PerkinElmer, Inc.  If you are not the intended recipient of this message, please inform the sender by replying to this email or sending a message to the sender and destroy the message and any attachments.  Thank you.

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Teng-Leong Chew
> Sent: 07 September 2010 20:00
> To: [hidden email]
> Subject: Colocalization question
>
> Hi all,
>
> Pardon me for opening up a can of worms of colocalization issue.
>
> I wonder if anyone has experience in thresholding a biological
> object using a channel (say, green actin in a contractile ring)
> and use only that selected ROI to quantify Pearson coeefficient
> between green and a red-labeled protein? Bottom line question
> is: how much of the red protein co-localizes to green within
> the object and not the whole cell?
>
> We have Volocity, MetaMorph and ImageJ.
>
> Thanks in advance for your help.
>
> Regards,
> Leong
>
>
> --
> Teng-Leong Chew, Ph.D.
> Director, Cell Imaging Facility & Nikon Imaging Center
> Director of University Imaging Resources
> Feinberg School of Medicine & Office of Research
> Northwestern University
> 303 E. Chicago Avenue
> Chicago, IL 60611
> [phone]
> [phone] (W, F)

[Daniel James White]

Hi Leong,


On Sep 8, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:

>
> Date:    Tue, 7 Sep 2010 13:59:37 -0500
> From:    Teng-Leong Chew <[hidden email]>
> Subject: Colocalization question
>
> Hi all,
>
> Pardon me for opening up a can of worms of colocalization issue.
>
no need to excuse yourself for asking a sensible question!

> I wonder if anyone has experience in thresholding a biological
> object using a channel (say, green actin in a contractile ring)
> and use only that selected ROI to quantify Pearson coeefficient
> between green and a red-labeled protein? Bottom line question
> is: how much of the red protein co-localizes to green within
> the object and not the whole cell?

Its a pretty sensible idea to do something like that.
Rarely should one analyze the whole of an image,
as the biology one is interested in is only contained in a certain region of interest.
Thus one should only analyse that region of interest.

So, you need a good way to make the region of interest.
One way is just to draw it (a bit subjective)
but another more objective way, as you suggest is to use a simple threshold or other clever way to segment one of the channels,
and use the result as the mask to do the colocalization measurement in.

I suggest  using the Costes methods for auto thresholding (in addition to the ROI or Mask)
and statistical significance
and to report both Pearsons coefficient for the masked/RIO area and Pearsons above the auto threshold levels,
and the auto thresholds,
and the Manders coefficients M1 and M2 and thresholded Manders tM1 and tM2
Manders coeff  tell you about coloc from the perspective of each channel,
and thats what you mentioned you want to know i believe.

Lots of details and pitfalls are explained at
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis

>
> We have Volocity, MetaMorph and ImageJ.
>

Fiji is just imageJ, but has the latest bug fixes
(by us Fiji-ers - including a bug spotted by the Volocity guys which eas recenbtly fixed)
of the ImageJ plugins:
Colocalization Threshold and Test
along with lots of other nice stuff added on top of imageJ.
(Fiji is becoming the de facto ImageJ distro of choice for cell biology etc.
and you wont find the latest bug fixes for colo in the other plugin distributions I expect.)

I don't know about metamorph coloc analysis - never saw it
but Volocity is very good and has been apparently improved recently on coloc analysis....
(but its not open source so you cant check the maths yourself unfortunately... its a very nice black box.)

> Thanks in advance for your help.

I'm always happy to help with Colocalization!!!

cheers

Dan

>
> Regards,
> Leong
>
>
> --
> Teng-Leong Chew, Ph.D.
> Director, Cell Imaging Facility & Nikon Imaging Center
> Director of University Imaging Resources
> Feinberg School of Medicine & Office of Research
> Northwestern University
> 303 E. Chicago Avenue
> Chicago, IL 60611
> [phone]
> [phone] (W, F)

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Jeremy Adler-3]

There are lots of ways to mess up colocalization measurements - the  
coefficients still have reassuring numbers of decimal places but their  
value shrinks.

ROIs and thresholding - we recommend both. The ROI should have  
biological relevance and thresholds should be set for both channels  
that exclude background pixels. This is illustrated in Fig 1 of our  
hot off the presses article (Cytometry August 2010, Adler & Parmryd).  
The article also shows that including background pixels can actually  
increase the measured correlation and also considers how the  
correlation measurement is confused when the there is more than 1  
relationship present (always look at the scattergram). We also argue  
strongly that the Manders Overlap Coefficient (not M1 and M2) is, at  
best, a highly confused measurement and should be abandoned - use the  
Pearson, Spearman or ICQ correlation coefficients instead.

Setting thresholds for both channels then measuring the colocalization  
between pixels that contain both fluorophores means that the remaining  
fluorescence is excluded from whichever correlation coefficient you  
use. Therefore we recommend qualifying the correlation with the M1 and  
M2 coefficients which give the fraction of the total fluorescence for  
each fluorophore to which the correlation applies. M1 and M2 are not  
measures of correlation but are measures co occurence. You might also  
report the fraction fraction of the pixels that contain both  
fluorophores in your chosen ROI.

Adler, J. and Parmryd, I. (2010), Quantifying colocalization by  
correlation: The Pearson correlation coefficient is superior to the  
Mander's overlap coefficient. Cytometry Part A, 77A: 733?742. doi:  
10.1002/cyto.a.20896

Jeremy Adler

> Hi all,
>
> Pardon me for opening up a can of worms of colocalization issue.
>
> I wonder if anyone has experience in thresholding a biological
> object using a channel (say, green actin in a contractile ring)
> and use only that selected ROI to quantify Pearson coeefficient
> between green and a red-labeled protein? Bottom line question
> is: how much of the red protein co-localizes to green within
> the object and not the whole cell?
>
> We have Volocity, MetaMorph and ImageJ.
>
> Thanks in advance for your help.
>
> Regards,
> Leong
>
>
> --
> Teng-Leong Chew, Ph.D.
> Director, Cell Imaging Facility & Nikon Imaging Center
> Director of University Imaging Resources
> Feinberg School of Medicine & Office of Research
> Northwestern University
> 303 E. Chicago Avenue
> Chicago, IL 60611
> [phone]
> [phone] (W, F)
>

Jeremy Adler
Genetics & Pathology
Rudbeck Labs
Uppsala U
Sweden

0046 (0)18 471 4607

[Teng-Leong Chew]

Dear list,

Thank you so much for all the suggestions and ideas, we will
probably need some time to explore the carious options that
have been suggested, but I am sure at least a few of these
should deliver what we need.

Thanks,
Leong

[Aryeh Weiss]

============================================================
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
============================================================

Not confocal, but I think that this group has the expertise...
I am interested in which digital SLR cameras (eg, Nikon D70 or D700) are
being used for digital microscopy, and with which software.
Are these cameras used in densitometry? How about fluorescence?

Thanks in advance
--aryeh
--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]