Re: IMAGEJ Digest - 6 Jul 2010 to 7 Jul 2010 (#2010-15)

[Daniel James White]

Dear Slavena


On Jul 8, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:

>
> Date:    Wed, 7 Jul 2010 14:48:58 +0200
> From:    Slavena Trifonova <[hidden email]>
> Subject: Re: Colocalization plugins
>
> Dear All,
>
> Thank you very much for your replies,I am a beginner in this field. I =
> used
> JaCoP manual and tried it on images but still have some questions using =
> this
> colocalization measurement plugin. I would really appreciate any help. =
> To
> analyze the colocalization of the blue and the red signal which refer to =
> Cy3
> for Glucocorticoid receptor and Dapi for nucleus in lymphocytes I =
> focused on
> objects based analysis.I choose to work on centre to centre distances
> suspecting that the cells are not close to the optical resolution.

In that case an object based method will work ok, but the pixel intensity correlation method will also work quite well,
depending on the biology and the images.
BUT you have a big problem, in that there is a page shift in xy position between the two colour channels,
so any coloc meansurement you do will be way off until you correct that.

if you merege the images,
using
Image -c color - merge channels,
and then zoom in you can see that ovjects that clearly should overlp in fact do no,
and are off by several pixels.

these are stiched images... but not very well stitched perhaps,
because the blue and red channels images are no the same size...

a really good stiching plugin is in Fiji distro of ImageJ,
btu we need to get the zvi data into it somehow...
http://pacific.mpi-cbg.de/wiki/index.php/Stitch_and_Align_a_sequence_of_grid_images_Tutorial
http://pacific.mpi-cbg.de/wiki/index.php/Stitching_2D/3D



> I =
> don=92t
> know which picture format I have to use the original file-zvi or it can =
> be a
> tif file.

open the .zvi file using the bio-formats importer plugin so all the meta data is properly read. (eg pixel spacing)

(in Fiji its in the plugins - LOCI menu item already - nothing extra to install)


> I have also some difficulties in setting up the three tabs =
> that
> appear on the screen using JaCoP. First I don=92t know how to adjust the
> threshold of the two images.

you must first subtract  the background, if any, then use the auto threshold method in JACoP
Manual set threshpld as totally subjective and not reproducible... a bad idea.

> Second the xy and z (nm) calib -where shall =
> I
> take this numbers from and what NA means-

If you donr know what NA means, then you really need to talk to a local microscopist,
who can teach you the basics of optics and microscopy.

if you dont know the microscopy basics, then you will make terrible mistakes in the data analysis.

NA mean numerical aperture, and Abbe told us that it determined the lateral resolution of the objective lens - d

d = light wavelength / 2 x NA

> it is not pointed out anywhere.

you need to learn and understand about spatial resolution, opticval resolution and basic microscope optics.

see here:
https://ifn.mpi-cbg.de/wiki/ifn/images/6/69/2010-07-Basics_of_Imaging_Processing_Course-final2.pdf
which is part of the material at
https://ifn.mpi-cbg.de/wiki/ifn/index.php/Teaching_Material



> Another issue is how to measure the particle size with imageJ in order =
> to
> filter and select only the small lymphocytes excluding the big =
> granulocytes?

Analyze Particles function has a size filter in it.
see:
http://rsbweb.nih.gov/ij/docs/menus/analyze.html#ap

>
>
cheers

Dan




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)

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( white (at) mpi-cbg.de )