Red blood cells

[Petkov, Daniel]

Hello,
I am counting cells stained in green with the Cell counter. Red blood cells produce very strong green signal. How can I eliminate (reduce) the likelihood that I will count rbc instead of my target cells?
Thank you, Daniel

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[daschneider9]

Only count cells that have a nucleus?
Dave

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Petkov, Daniel
Sent: Thursday, September 17, 2015 9:42 AM
To: [hidden email]
Subject: Red blood cells

Hello,
I am counting cells stained in green with the Cell counter. Red blood cells produce very strong green signal. How can I eliminate (reduce) the likelihood that I will count rbc instead of my target cells?
Thank you, Daniel

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[Petkov, Daniel]

Yes but my macrophages in muscle are long and when tissue sections are prepared cells are cut unevenly and one cell may appear as rounded multiple objects (rbc) far from the nuclei.
Daniel

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Schneider, David A.
Sent: Thursday, September 17, 2015 1:10 PM
To: [hidden email]
Subject: Re: Red blood cells

Only count cells that have a nucleus?
Dave

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Petkov, Daniel
Sent: Thursday, September 17, 2015 9:42 AM
To: [hidden email]
Subject: Red blood cells

Hello,
I am counting cells stained in green with the Cell counter. Red blood cells produce very strong green signal. How can I eliminate (reduce) the likelihood that I will count rbc instead of my target cells?
Thank you, Daniel

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[Andrei Stefan]

Hi,

Can you post some images? It would help understand better.


Best regards,
Andrei

2015-09-17 19:20 GMT+01:00 Petkov, Daniel <[hidden email]>:

> Yes but my macrophages in muscle are long and when tissue sections are
> prepared cells are cut unevenly and one cell may appear as rounded multiple
> objects (rbc) far from the nuclei.
> Daniel
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Schneider, David A.
> Sent: Thursday, September 17, 2015 1:10 PM
> To: [hidden email]
> Subject: Re: Red blood cells
>
> Only count cells that have a nucleus?
> Dave
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Petkov, Daniel
> Sent: Thursday, September 17, 2015 9:42 AM
> To: [hidden email]
> Subject: Red blood cells
>
> Hello,
> I am counting cells stained in green with the Cell counter. Red blood
> cells produce very strong green signal. How can I eliminate (reduce) the
> likelihood that I will count rbc instead of my target cells?
> Thank you, Daniel
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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Andrei Cătălin ȘTEFAN, MRCVS, DVM, MVSc

E-mail: [hidden email]

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[daschneider9]

So by green do you mean trouble differentiating a fluorophore fluorescence in cells of interest from autofluorescence in RBCs? If so, then there are ways to reduce autofluorescence in RBCs that might help. If not, then more info needed.

Dave

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Petkov, Daniel
Sent: Thursday, September 17, 2015 11:21 AM
To: [hidden email]
Subject: Re: Red blood cells

Yes but my macrophages in muscle are long and when tissue sections are prepared cells are cut unevenly and one cell may appear as rounded multiple objects (rbc) far from the nuclei.
Daniel

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Schneider, David A.
Sent: Thursday, September 17, 2015 1:10 PM
To: [hidden email]
Subject: Re: Red blood cells

Only count cells that have a nucleus?
Dave

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Petkov, Daniel
Sent: Thursday, September 17, 2015 9:42 AM
To: [hidden email]
Subject: Red blood cells

Hello,
I am counting cells stained in green with the Cell counter. Red blood cells produce very strong green signal. How can I eliminate (reduce) the likelihood that I will count rbc instead of my target cells?
Thank you, Daniel

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[jerie]

Hi Daniel,

On Behalf Of Petkov, Daniel
Sent: Thursday, September 17, 2015 9:42 AM
Subject: Red blood cells
How can I eliminate (reduce) the likelihood that I will count rbc instead
of my target cells?

presuming the spectral excitation and emission properties of your stain are
not exactly identical to the fluorescence of the rbc's, you may want to
consider "spectral unmixing" to separate the signals; z.B.
http://rsb.info.nih.gov/ij/plugins/spectral-unmixing.html

Kind regards, Jens

Dr. Jens Rietdorf, visiting scientist @ center for technological
development in health CDTS, Rio de Janeiro, Brasil
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/


HRB 718417 ∙ AG Mannheim
Board of Managing Directors: Dr. Urban Liebel, Ralf Mulflur, Thomas Schurr
Supervisory Board: Rainer Baule (Deputy Chairman), Markus Hennig
(Chairman), Hans-Günther Hohmann

On Thu, Sep 17, 2015 at 3:41 PM, Schneider, David A. <[hidden email]>
wrote:

> So by green do you mean trouble differentiating a fluorophore fluorescence
> in cells of interest from autofluorescence in RBCs? If so, then there are
> ways to reduce autofluorescence in RBCs that might help. If not, then more
> info needed.
>
> Dave
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Petkov, Daniel
> Sent: Thursday, September 17, 2015 11:21 AM
> To: [hidden email]
> Subject: Re: Red blood cells
>
> Yes but my macrophages in muscle are long and when tissue sections are
> prepared cells are cut unevenly and one cell may appear as rounded multiple
> objects (rbc) far from the nuclei.
> Daniel
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Schneider, David A.
> Sent: Thursday, September 17, 2015 1:10 PM
> To: [hidden email]
> Subject: Re: Red blood cells
>
> Only count cells that have a nucleus?
> Dave
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Petkov, Daniel
> Sent: Thursday, September 17, 2015 9:42 AM
> To: [hidden email]
> Subject: Red blood cells
>
> Hello,
> I am counting cells stained in green with the Cell counter. Red blood
> cells produce very strong green signal. How can I eliminate (reduce) the
> likelihood that I will count rbc instead of my target cells?
> Thank you, Daniel
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[Petkov, Daniel]

Hi Jens,
Yes, I just compared numbers for the nuclei (DAPI) and targeted cells stained in green between unmixed images and images from ImageJ. While the numbers for the nuclei were very close there were substantial differences for the targeted cells.
Daniel  

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of jens rietdorf
Sent: Friday, September 18, 2015 8:30 AM
To: [hidden email]
Subject: Re: Red blood cells

Hi Daniel,

On Behalf Of Petkov, Daniel
Sent: Thursday, September 17, 2015 9:42 AM
Subject: Red blood cells
How can I eliminate (reduce) the likelihood that I will count rbc instead of my target cells?

presuming the spectral excitation and emission properties of your stain are not exactly identical to the fluorescence of the rbc's, you may want to consider "spectral unmixing" to separate the signals; z.B.
http://rsb.info.nih.gov/ij/plugins/spectral-unmixing.html

Kind regards, Jens

Dr. Jens Rietdorf, visiting scientist @ center for technological development in health CDTS, Rio de Janeiro, Brasil http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/


HRB 718417 ∙ AG Mannheim
Board of Managing Directors: Dr. Urban Liebel, Ralf Mulflur, Thomas Schurr Supervisory Board: Rainer Baule (Deputy Chairman), Markus Hennig (Chairman), Hans-Günther Hohmann

On Thu, Sep 17, 2015 at 3:41 PM, Schneider, David A. <[hidden email]>
wrote:

> So by green do you mean trouble differentiating a fluorophore
> fluorescence in cells of interest from autofluorescence in RBCs? If
> so, then there are ways to reduce autofluorescence in RBCs that might
> help. If not, then more info needed.
>
> Dave
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Petkov, Daniel
> Sent: Thursday, September 17, 2015 11:21 AM
> To: [hidden email]
> Subject: Re: Red blood cells
>
> Yes but my macrophages in muscle are long and when tissue sections are
> prepared cells are cut unevenly and one cell may appear as rounded
> multiple objects (rbc) far from the nuclei.
> Daniel
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Schneider, David A.
> Sent: Thursday, September 17, 2015 1:10 PM
> To: [hidden email]
> Subject: Re: Red blood cells
>
> Only count cells that have a nucleus?
> Dave
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Petkov, Daniel
> Sent: Thursday, September 17, 2015 9:42 AM
> To: [hidden email]
> Subject: Red blood cells
>
> Hello,
> I am counting cells stained in green with the Cell counter. Red blood
> cells produce very strong green signal. How can I eliminate (reduce)
> the likelihood that I will count rbc instead of my target cells?
> Thank you, Daniel
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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