Stereologically count triple labelled nuclei in 3D in ImageJ

[L'assegnista]

Dear list,
I would like to count nuclei labelled with three markers in a
histological section acquired with the confocal microscope. In this
analysis I would like to obtain the total number of objectes labelled
for each marker and of those that are co-labelled. In addition I would
like to exclude  all the nuclei that contact the upper (or lower)
confocal plane in order to have a stereological estimate of the
particles in the analyzed volume.

Any suggestion to perform an automatic analysis (maybe with the 3D
object counter and Coloc plugins?) will be greately appreciated!

Kinds regards

Federico Luzzati

--
Federico Luzzati, PhD
Researcher
Dept. Life Sciences and Systems Biology
University of Turin
Via Accademia Albertina, 13
10123 Torino
Neuroscience Institute Cavalieri Ottolenghi (NICO)
Regione Gonzole, 10
10043 Orbassano (Torino)
ITALY
tel. [phone]/ -6631

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Jeremy Adler]

I am not sure that rejecting nuclei that touch either the upper or lower planes is the correct selection criteria.
This produces a bias against large nuclei or elongated nuclei.
It is better to exclude nuclei that cut one of the outer planes, but include those that cut the other plane.

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Federico Luzzati
Sent: den 6 mars 2014 14:38
To: [hidden email]
Subject: Stereologically count triple labelled nuclei in 3D in ImageJ

Dear list,
I would like to count nuclei labelled with three markers in a histological section acquired with the confocal microscope. In this analysis I would like to obtain the total number of objectes labelled for each marker and of those that are co-labelled. In addition I would like to exclude  all the nuclei that contact the upper (or lower) confocal plane in order to have a stereological estimate of the particles in the analyzed volume.

Any suggestion to perform an automatic analysis (maybe with the 3D object counter and Coloc plugins?) will be greately appreciated!

Kinds regards

Federico Luzzati

--
Federico Luzzati, PhD
Researcher
Dept. Life Sciences and Systems Biology
University of Turin
Via Accademia Albertina, 13
10123 Torino
Neuroscience Institute Cavalieri Ottolenghi (NICO) Regione Gonzole, 10
10043 Orbassano (Torino)
ITALY
tel. [phone]/ -6631

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[L'assegnista]

I'm working with 50 microns thick sections, thus there is no risk to
have nuclei that crosses both upper and lower surfaces of the sections.
In any case, the exclusion of objects that touches one particular side
of the section is a basic rule of stereology. The idea is to count the
number of nuclei tips in a specific direction(and thus exclude those
that have their tips in another section), in order to obtain an estimate
number that is independent on the object's shape and dimensions.

best regards

Federico Luzzati

Il 07.03.2014 13:36 Jeremy Adler ha scritto:

> I am not sure that rejecting nuclei that touch either the upper or
> lower planes is the correct selection criteria.
> This produces a bias against large nuclei or elongated nuclei.
> It is better to exclude nuclei that cut one of the outer planes, but
> include those that cut the other plane.
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Federico Luzzati
> Sent: den 6 mars 2014 14:38
> To: [hidden email]
> Subject: Stereologically count triple labelled nuclei in 3D in ImageJ
>
> Dear list,
> I would like to count nuclei labelled with three markers in a
> histological section acquired with the confocal microscope. In this
> analysis I would like to obtain the total number of objectes labelled
> for each marker and of those that are co-labelled. In addition I
> would
> like to exclude  all the nuclei that contact the upper (or lower)
> confocal plane in order to have a stereological estimate of the
> particles in the analyzed volume.
>
> Any suggestion to perform an automatic analysis (maybe with the 3D
> object counter and Coloc plugins?) will be greately appreciated!
>
> Kinds regards
>
> Federico Luzzati
>
> --
> Federico Luzzati, PhD
> Researcher
> Dept. Life Sciences and Systems Biology
> University of Turin
> Via Accademia Albertina, 13
> 10123 Torino
> Neuroscience Institute Cavalieri Ottolenghi (NICO) Regione Gonzole,
> 10
> 10043 Orbassano (Torino)
> ITALY
> tel. [phone]/ -6631
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
Federico Luzzati, PhD
Researcher
Dept. Life Sciences and Systems Biology
University of Turin
Via Accademia Albertina, 13
10123 Torino
Neuroscience Institute Cavalieri Ottolenghi (NICO)
Regione Gonzole, 10
10043 Orbassano (Torino)
ITALY
tel. [phone]/ -6631

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Gabriel Landini]

On Friday 07 Mar 2014 14:43:39 Federico Luzzati wrote:
> I'm working with 50 microns thick sections, thus there is no risk to
> have nuclei that crosses both upper and lower surfaces of the sections.
> In any case, the exclusion of objects that touches one particular side
> of the section is a basic rule of stereology.

My understanding is that for non-square windows, you do not count objects in
two *adjacent* sides of the image frame (i.e. top and left,  or top and right,
etc.).

Cheers

Gabriel

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[Jeremy Adler]

There are two issues
1) how to count the number of nuclei per unit volume
The protocol of counting new tips as you move through the stack of images is correct - tips only occur once per nuclei.

2) I presume the exclusion of nuclei that cut the upper or lower planes is to ensure that whole nuclei are available for the colabelling study.
But using both planes for exclusion will selectively exclude large or elongated nuclei.
A workable protocol for say 50x 1 um sections and a nuclei with a length of say 10 ums
Include all nuclei whose tips start in the first 40ums - the 10um at the bottom ensures that all nuclei will be complete in Z.
You might also want to limit the XY origins of tips by excluding those that are within a few ums of the sides - this ensures that a large fraction of each nuclei must be within the image volume even for nuclei that are at an angle to the vertical- which should be enough to answer the colabelling question.

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Federico Luzzati
Sent: den 7 mars 2014 14:44
To: [hidden email]
Subject: Re: Stereologically count triple labelled nuclei in 3D in ImageJ

I'm working with 50 microns thick sections, thus there is no risk to have nuclei that crosses both upper and lower surfaces of the sections.
In any case, the exclusion of objects that touches one particular side of the section is a basic rule of stereology. The idea is to count the number of nuclei tips in a specific direction(and thus exclude those that have their tips in another section), in order to obtain an estimate number that is independent on the object's shape and dimensions.

best regards

Federico Luzzati

Il 07.03.2014 13:36 Jeremy Adler ha scritto:

> I am not sure that rejecting nuclei that touch either the upper or
> lower planes is the correct selection criteria.
> This produces a bias against large nuclei or elongated nuclei.
> It is better to exclude nuclei that cut one of the outer planes, but
> include those that cut the other plane.
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Federico Luzzati
> Sent: den 6 mars 2014 14:38
> To: [hidden email]
> Subject: Stereologically count triple labelled nuclei in 3D in ImageJ
>
> Dear list,
> I would like to count nuclei labelled with three markers in a
> histological section acquired with the confocal microscope. In this
> analysis I would like to obtain the total number of objectes labelled
> for each marker and of those that are co-labelled. In addition I would
> like to exclude  all the nuclei that contact the upper (or lower)
> confocal plane in order to have a stereological estimate of the
> particles in the analyzed volume.
>
> Any suggestion to perform an automatic analysis (maybe with the 3D
> object counter and Coloc plugins?) will be greately appreciated!
>
> Kinds regards
>
> Federico Luzzati
>
> --
> Federico Luzzati, PhD
> Researcher
> Dept. Life Sciences and Systems Biology University of Turin Via
> Accademia Albertina, 13
> 10123 Torino
> Neuroscience Institute Cavalieri Ottolenghi (NICO) Regione Gonzole,
> 10
> 10043 Orbassano (Torino)
> ITALY
> tel. [phone]/ -6631
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
Federico Luzzati, PhD
Researcher
Dept. Life Sciences and Systems Biology
University of Turin
Via Accademia Albertina, 13
10123 Torino
Neuroscience Institute Cavalieri Ottolenghi (NICO) Regione Gonzole, 10
10043 Orbassano (Torino)
ITALY
tel. [phone]/ -6631

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html