How can we Distinguish In/Out of focus cells in confocal microscope?
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How can we Distinguish In/Out of focus cells in confocal microscope?
 
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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 |
Re: How can we Distinguish In/Out of focus cells in confocal microscope?
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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 |
Re: How can we Distinguish In/Out of focus cells in confocal microscope?
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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 > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: How can we Distinguish In/Out of focus cells in confocal microscope?
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On 25/05/14 18:00, Mohamed Tleis 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. > > > Typically to deal with this sort of go/no-go situation, if your images have good contrast, I would try to score the focus of each cell based on it's rate of intensity change in an area with good contrast (eg edges or details in the cell). To get an idea for what I mean, find examples of well focused and poorly focused cells and draw a line selection through the cell, beginning and ending off the cell either side. Then plot a line profile (Ctrl+K I think) and study the 'shoulders' of the curve, the well focused cell should show much steeper rises at the edges than the poorly focused cell. It is possible to make use of this method automatically by writing a plugin/macro to suit your needs. Choosing the largest difference between two adjacent pixels on the edges of the curve as your sharpness score is often sufficient. Setting some threshold for this value gives an easy indication of go/no-go for focus dependent measurements. I hope that helps with the issue, please let me know if you need further assistance... Best regards, Ed -- [hidden email] www.esimagingsolutions.com -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: How can we Distinguish In/Out of focus cells in confocal microscope?
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In reply to this post by Mohamed Tleis
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 >> >> > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: How can we Distinguish In/Out of focus cells in confocal microscope?
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In reply to this post by Mohamed Tleis
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. >> >> -- >> 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 |
Re: How can we Distinguish In/Out of focus cells in confocal microscope?
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In reply to this post by Joel Sheffield
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 >>> >>> >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html >> > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > Dr Richard Mort MRC Human Genetics Unit MRC IGMM University of Edinburgh Western General Hospital Crewe Road Edinburgh. EH4 2XU, UK Tel: +44 (0)131 332 2471 Fax: +44 (0)131 467 8456 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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