How to calculate spot colocalization (content) in a 4 channel RNA-FSIH image with ImageJ?

[Wichgers Schreur, Paul]

Dear ImageJ users,

As a molecular virologist at the Wageningen University I am studying the genome content of individual bunyavirus virions using single molecule RNA fluorescence in situ hybridization in combination with immunofluorescence. The bunyavirus genome consists of 3 genome segments referred to as large (L), medium (M) and small (S).

After immobilization of the bunyavirus virions on coverslips I visualized the virions with an fluorescent antibody and evaluated the genome content of each virion with fluorescent probes specific for the L, M and S segment. So I obtained four channel spot images.

With the ComDet plugin of ImageJ I already successfully calculated colocalization of spots in two channels.
However, I would like to calculate colocalization of spots in the 4 channel image.
In other words: I would like to know how many virions contain 3, how many contain 2 and how many contain 1 or 0 genome segments.

I would be very grateful if anyone could be of some help!!

Thank you in advance!

Best wishes

Paul Wichgers Schreur


Dr. P.J. (Paul) Wichgers Schreur

Central Veterinary Institute of Wageningen UR
P.O. Box 65
8200 AB Lelystad
Visiting Address: Edelhertweg 15, 8219 PH Lelystad, The Netherlands
Tel. [phone]
Fax. [phone]
E-mail: [hidden email]<mailto:[hidden email]>
WWW: www.cvi.wur.nl<http://www.cvi.wur.nl/>
KvK: Stichting DLO, 09098104 - Arnhem

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[Gabriel Landini]

On Saturday 07 May 2016 20:45:14 Wichgers Schreur, Paul wrote:
> However, I would like to calculate
> colocalization of spots in the 4 channel image. In other words: I would
> like to know how many virions contain 3, how many contain 2 and how many
> contain 1 or 0 genome segments.

Isn't this the colocalisation between image pairs?
If you have the 3 types of spots in 3 channels, don't you already see what the
colours of the composite are? E.g:
0: nothing
1: red or green or blue
2: yellow, or cyan, or magenta
3: white
So, I do not understand why a 4th channel is needed, (most likely I might not
be understanding it correctly because I never used this kind of analysis).

Cheers

Gabriel

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[Jeremy Adler]

You have three fluorophores but mention 4 channels - what is the 4th image:  transmitted light ? and what use do you make of it ?

Gabriel  outline the answer, threshold each of the 3 fluorescent images and make an RGB image - look for the unique colour combinations

An alternative would be to take the 3 binary images, change the 255 values of one image to 1, the second to 2 and third to 4. Then sum the 3 images.
The sum of the three images produces a unique intensity for every possible combination and you can read the number of pixels of each type from a histogram.
There is a convenient macro command for changing the ImageJ intensities - changeValues(v1,v2,v3)

You also need to consider calculating the random associations between the 3 fluorophores e.g. if each two fluorophores each occupy 10% or your ROI then a 1% overlap would be expected with a random distribution.





-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Wichgers Schreur, Paul
Sent: den 7 maj 2016 22:45
To: [hidden email]
Subject: How to calculate spot colocalization (content) in a 4 channel RNA-FSIH image with ImageJ?

Dear ImageJ users,

As a molecular virologist at the Wageningen University I am studying the genome content of individual bunyavirus virions using single molecule RNA fluorescence in situ hybridization in combination with immunofluorescence. The bunyavirus genome consists of 3 genome segments referred to as large (L), medium (M) and small (S).

After immobilization of the bunyavirus virions on coverslips I visualized the virions with an fluorescent antibody and evaluated the genome content of each virion with fluorescent probes specific for the L, M and S segment. So I obtained four channel spot images.

With the ComDet plugin of ImageJ I already successfully calculated colocalization of spots in two channels.
However, I would like to calculate colocalization of spots in the 4 channel image.
In other words: I would like to know how many virions contain 3, how many contain 2 and how many contain 1 or 0 genome segments.

I would be very grateful if anyone could be of some help!!

Thank you in advance!

Best wishes

Paul Wichgers Schreur


Dr. P.J. (Paul) Wichgers Schreur

Central Veterinary Institute of Wageningen UR P.O. Box 65
8200 AB Lelystad
Visiting Address: Edelhertweg 15, 8219 PH Lelystad, The Netherlands Tel. [phone] Fax. [phone]
E-mail: [hidden email]<mailto:[hidden email]>
WWW: www.cvi.wur.nl<http://www.cvi.wur.nl/>
KvK: Stichting DLO, 09098104 - Arnhem

This message together with the attachment(s), if any, are confidential and may be privileged. Unauthorised use, dissemination or publishing (in whole or in part) by third parties is not allowed. If you received this message by mistake, please delete the message including any attachments and notify the sender immediately. This email message does not create any contractual obligations.


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