Re: How can we Distinguish In/Out of focus cells in confocal microscope?

Posted by Richard Mort-2 on May 26, 2014; 9:59am
URL: http://imagej.273.s1.nabble.com/How-can-we-Distinguish-In-Out-of-focus-cells-in-confocal-microscope-tp5007911p5007920.html

It might be better to ensure all cells are in focus if possible so you
don't waste data. If you were using widefield I guess you could use
deconvolution to make sure your entire field is in focus. I find that
using confocal you often just want the best z-position for each cell and
you don't need the rest of the data. If all cells are in the same
z-position you can take a confocal stack and then use the normalized
variance to judge the most "in-focus" slice, here is a method:

http://imagejdocu.tudor.lu/doku.php?id=macro:normalized_variance

You can do this on a time lapse sequence using:

http://imagejdocu.tudor.lu/doku.php?id=macro:autofocus_hyperstack

If you have variation is the best z-position across your field you can
break the stack down into tiles and chose the best z-position for each
tile then re-assemble into a composite image. The following code works
on time-lapse so you may need to adapt it to work on a single stack. You
can chose the number of tiles so you could use a size thats roughly a
cell diameter and see if that helps:

http://imagejdocu.tudor.lu/doku.php?id=macro:tiled_autofocus_hyperstack

Best
R


On 25/05/14 19:16, JOEL B. SHEFFIELD wrote:

> Hi Mohamed,
>
> It would help if you could post an example image so that we could see what
> you are dealing with.
>
> 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 1:00 PM, Mohamed Tleis <[hidden email]> wrote:
>
>> 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.
>>>>
>>>> --
>>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>>>
>>>>   --
>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
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>>>
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--
Dr Richard Mort
MRC Human Genetics Unit
MRC IGMM
University of Edinburgh
Western General Hospital
Crewe Road
Edinburgh.
EH4 2XU, UK

Tel: [phone]
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