How to count FISH signals automatically?

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How to count FISH signals automatically?

Zofia
Hello,
I made fluorescent in situ hybridization and now I have to count all green
and red signals on chromosomes (blue). Can I count them automatically in
ImageJ? Can the program recognize what is the right signal and what is the
artifact? How to do it?

Thanks in advance!

<http://imagej.1557.x6.nabble.com/file/t382450/fish_rgb.png>



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Re: How to count FISH signals automatically?

G. Esteban Fernandez
Hi Zofia,

To quantify little dots from FISH staining, this function in ImageJ works
well for me:

Process > Find Maxima

Increase the Prominence to remove background and count only real signal.
Also, check the box to “Preview point selection” to help you get the right
settings. Good luck!

-Esteban


On Sat, Oct 12, 2019 at 11:35 AM Zofia <[hidden email]> wrote:

> Hello,
> I made fluorescent in situ hybridization and now I have to count all green
> and red signals on chromosomes (blue). Can I count them automatically in
> ImageJ? Can the program recognize what is the right signal and what is the
> artifact? How to do it?
>
> Thanks in advance!
>
> <http://imagej.1557.x6.nabble.com/file/t382450/fish_rgb.png>
>
>
>
> --
> Sent from: http://imagej.1557.x6.nabble.com/
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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Re: How to count FISH signals automatically?

Cammer, Michael-2
In reply to this post by Zofia
I looked at the image you sent.  What lens did you use (magnification and numerical aperture) and what is the size of each pixel?

Regardless, the dots are saturated which makes them appear much larger than they really are.  Also, saturation makes neighboring discrete spots merge together.  It would be far preferable to collect images that are not saturated.  Also, if the camera is 12 or 14 or 16 bits, keep the channels at their original bit depth.

Yes, ImageJ can select the large blue dots and then tell you whether there is green in them.  But there are a lot of green dots not associated with the blue chromosomes.  Is this ok?




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

[hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/

Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567



________________________________

Sent: Saturday, October 12, 2019 2:23 PM
Subject: How to count FISH signals automatically?

Hello,
I made fluorescent in situ hybridization and now I have to count all green
and red signals on chromosomes (blue). Can I count them automatically in
ImageJ? Can the program recognize what is the right signal and what is the
artifact? How to do it?

Thanks in advance!



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Re: How to count FISH signals automatically?

G. Esteban Fernandez
Good points Michael. I didn’t see that there was an image link so my
comment was for spot counting in general.

-Esteban


On Sat, Oct 12, 2019 at 8:44 PM Cammer, Michael <
[hidden email]> wrote:

> I looked at the image you sent.  What lens did you use (magnification and
> numerical aperture) and what is the size of each pixel?
>
> Regardless, the dots are saturated which makes them appear much larger
> than they really are.  Also, saturation makes neighboring discrete spots
> merge together.  It would be far preferable to collect images that are not
> saturated.  Also, if the camera is 12 or 14 or 16 bits, keep the channels
> at their original bit depth.
>
> Yes, ImageJ can select the large blue dots and then tell you whether there
> is green in them.  But there are a lot of green dots not associated with
> the blue chromosomes.  Is this ok?
>
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue
> <https://www.google.com/maps/search/540+First+Avenue?entry=gmail&source=g>,
> SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
>
> ________________________________
>
> Sent: Saturday, October 12, 2019 2:23 PM
> Subject: How to count FISH signals automatically?
>
> Hello,
> I made fluorescent in situ hybridization and now I have to count all green
> and red signals on chromosomes (blue). Can I count them automatically in
> ImageJ? Can the program recognize what is the right signal and what is the
> artifact? How to do it?
>
> Thanks in advance!
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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Re: How to count FISH signals automatically?

Zofia
Thank you for all the answers!

I know the pictures are too saturated, but this is the only way I can catch
smaller red signals (5S rDNA) with fewer repetitions…

So I came to the conclusion that automatic signal counting is applicable
only to very good preparations from large chromosomes and the number of
sequence repetitions that are mapped within one signal is relatively
constant. In my case, the chromosomes are small, there is a lot of cytoplasm
on the preparations, and the 5S (red) and 35S rDNA (green) sequences appear
in many copies.

So I decided to count the signals manually, because I'm afraid that the
machine will count non-specific signals (trash, glow, artifacts etc.), and
sometimes only „by eye” I can say with 100% certainty that we are dealing
with the right signal, because we see it at the same height in two
chromatids.

Thanks again for your attention!




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