Posted by
Mike Esterman on
May 26, 2014; 1:41am
URL: http://imagej.273.s1.nabble.com/How-can-we-Distinguish-In-Out-of-focus-cells-in-confocal-microscope-tp5007911p5007928.html
You should not have any out of focus cells in your image if you have set the pin hole correctly. That is the function of Confocal to reject out of focus light. If you set the pinhole wide open then you are just acquiring a wide field image. You in fact may have cells that are not as bright because the dye is not taken up as well in some cells as with others.
Mike Esterman, Secretary
Indiana Microscopy Society
-----Original Message-----
From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of Mohamed Tleis
Sent: Sunday, May 25, 2014 1:00 PM
To:
[hidden email]
Subject: Re: How can we Distinguish In/Out of focus cells in confocal microscope?
Dear Joel,
Thank you for your reply, My Cells are simple yeast cells. I do Image Analysis for some images acquired by Biologists. they try to change the focal level to the most optimal one then they acquire their images.
However, In the analysis part we need to be aware of out of focus cells, because they can wrongly affect the final measurement results. So my question is: is there any means to deal with such issues?
Best Regards,
M. Tleis
Phd. Candidate
LIACS, Leiden University
The Netherlands.
On 05/25/2014 06:36 PM, JOEL B. SHEFFIELD wrote:
> Hi Mohamed,
>
> You haven't specified the tissue, but I'll assume that both cells are
> within the z range of the instrument. If it were simply a focus
> issue, you should be able to change the focal level (i.e. do a z scan)
> and see if you get any changes.
>
> Joel
>
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail:
[hidden email]
> URL:
http://astro.temple.edu/~jbs>
>
> On Sun, May 25, 2014 at 9:54 AM, Mohamed Tleis <
[hidden email]> wrote:
>
>> Dear ImageJ members,
>>
>> We have two channel images one of bright-field and one of Green
>> Fluoresence Protein, acquired by a confocal microscope. Recently we
>> are concerned about some cells having less fluoresent due to the fact
>> that it is out of focus. In the bright-field channel it is a bit hard
>> to distinguish between the in and out of focus cells. I wonder
>> whether any of you came across a similar problem or knows of some literature about it.
>>
>> Thank you in Advance for your answers.
>>
>>
>> Best Regards,
>> M. Tleis
>> Phd. Candidate
>> LIACS, Leiden University
>> The Netherlands.
>>
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>> ImageJ mailing list:
http://imagej.nih.gov/ij/list.html>>
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