quantifying focal adhesions

[Knecht, David]

We are trying to come up with a way to quantify the difference in the  
number/size of focal adhesions in cells under different conditions.  
The images were taken on a widefield scope and so there is significant  
and variable cellular background fluorescence.  The focal adhesions  
themselves have pixel values above the local background that surrounds  
them, so they are easy to pick out by eye, but you can't threshold  
easily because there vary in intensity and there are other areas of  
the cell which have the same or higher and lower pixel values than the  
focal adhesions we want to target.  Is there a way of classifying/
thresholding that will compare structures to the local background  
surrounding it so as to allow it to be identified and quantified?  
Thanks- Dave

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Stoyan Pavlov]

Hello,
I have a similar probem.There are several ways to deal with it. You
can try the "Process=>Binary=>Find Maxima" command which finds the
local maxima in brightness within certain noise tollerance limits (say
for example 40 brightness
levels)(http://rsb.info.nih.gov/ij/docs/menus/process.html#binary) .
You can try also try either the build in "Subtract background" command
or the "Background subtractor" plugin (
http://www.mosaic.ethz.ch/Downloads/bgs ). The two latter produce not
a thresholded binary image but keep the format of the images instead,
however the filtered images are very easily thresholded.
If this still doesn't work either you may want to try some more
advanced form of morphological filtering (for example the
"GreyScaleTopHat"or "Find regional Maxima" macros from Landini's
Morphology collection of plugins is very useful"
Good luck!
--
 Stoyan P. Pavlov, MD
Departament of Anatomy, Histology and Embryology
Medical University "Prof. Dr. Paraskev Stoyanov", Varna
Prof. Marin Drinov Str.55
9002 Varna
 Bulgaria
Tel: +359 (0) 52 - 677 - 050 #2638
e-mail: [hidden email]

> We are trying to come up with a way to quantify the difference in the
> number/size of focal adhesions in cells under different conditions.  The
> images were taken on a widefield scope and so there is significant and
> variable cellular background fluorescence.  The focal adhesions themselves
> have pixel values above the local background that surrounds them, so they
> are easy to pick out by eye, but you can't threshold easily because there
> vary in intensity and there are other areas of the cell which have the same
> or higher and lower pixel values than the focal adhesions we want to target.
>  Is there a way of classifying/thresholding that will compare structures to
> the local background surrounding it so as to allow it to be identified and
> quantified?  Thanks- Dave
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>

[Stoyan Pavlov]

Hello,
I have a similar probem.There are several ways to deal with it. You
can try the "Process=>Binary=>Find Maxima" command which finds the
local maxima in brightness within certain noise tollerance limits (say
for example 40 brightness
levels)(http://rsb.info.nih.gov/ij/docs/menus/process.html#binary) .
You can try also try either the build in "Subtract background" command
or the "Background subtractor" plugin (
http://www.mosaic.ethz.ch/Downloads/bgs ). The two latter produce not
a thresholded binary image but keep the format of the images instead,
however the filtered images are very easily thresholded.
If this still doesn't work either you may want to try some more
advanced form of morphological filtering (for example the
"GreyScaleTopHat"or "Find regional Maxima" macros from Landini's
Morphology collection of plugins is very useful"
Good luck!
--
 Stoyan P. Pavlov, MD
Departament of Anatomy, Histology and Embryology
Medical University "Prof. Dr. Paraskev Stoyanov", Varna
Prof. Marin Drinov Str.55
9002 Varna
 Bulgaria
Tel: +359 (0) 52 - 677 - 050 #2638
e-mail: [hidden email]

On Thu, Nov 12, 2009 at 4:52 AM, David Knecht ATT
<[hidden email]> wrote:

> We are trying to come up with a way to quantify the difference in the
> number/size of focal adhesions in cells under different conditions.  The
> images were taken on a widefield scope and so there is significant and
> variable cellular background fluorescence.  The focal adhesions themselves
> have pixel values above the local background that surrounds them, so they
> are easy to pick out by eye, but you can't threshold easily because there
> vary in intensity and there are other areas of the cell which have the same
> or higher and lower pixel values than the focal adhesions we want to target.
>  Is there a way of classifying/thresholding that will compare structures to
> the local background surrounding it so as to allow it to be identified and
> quantified?  Thanks- Dave
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>

--
Dr. Stoyan P. Pavlov, MD
Departament of Anatomy, Histology and Embryology
Medical University "Prof. Dr. Paraskev Stoyanov", Varna
Prof. Marin Drinov Str.55
9002 Varna
 Bulgaria
Tel: +359 (0) 52 - 677 - 050 #2638
e-mail: [hidden email]

[Joel Sheffield]

Hi,

One more possibility is to try to normalize the background by using
the FFT bandpass filter.  You may be able to filter out the
background variation (essentialy low frequency components, while
retaining the small points (the high frequency components.)
Joel

> Hello,
> I have a similar probem.There are several ways to deal with it.
> You
> can try the "Process=>Binary=>Find Maxima" command which finds the
> local maxima in brightness within certain noise tollerance limits
> (say
> for example 40 brightness
> levels)(http://rsb.info.nih.gov/ij/docs/menus/process.html#binary)
> .
> You can try also try either the build in "Subtract background"
> command
> or the "Background subtractor" plugin (
> http://www.mosaic.ethz.ch/Downloads/bgs ). The two latter produce
> not
> a thresholded binary image but keep the format of the images
> instead,
> however the filtered images are very easily thresholded.
> If this still doesn't work either you may want to try some more
> advanced form of morphological filtering (for example the
> "GreyScaleTopHat"or "Find regional Maxima" macros from Landini's
> Morphology collection of plugins is very useful"
> Good luck!
> --
>  Stoyan P. Pavlov, MD
> Departament of Anatomy, Histology and Embryology
> Medical University "Prof. Dr. Paraskev Stoyanov", Varna
> Prof. Marin Drinov Str.55
> 9002 Varna
>  Bulgaria
> Tel: +359 (0) 52 - 677 - 050 #2638
> e-mail: [hidden email]
>
> On Thu, Nov 12, 2009 at 4:52 AM, David Knecht ATT
> <[hidden email]> wrote:
> > We are trying to come up with a way to quantify the difference in
> the
> > number/size of focal adhesions in cells under different
> conditions.  The
> > images were taken on a widefield scope and so there is significant
> and
> > variable cellular background fluorescence.  The focal adhesions
> themselves
> > have pixel values above the local background that surrounds them,
> so they
> > are easy to pick out by eye, but you can't threshold easily
> because there
> > vary in intensity and there are other areas of the cell which have
> the same
> > or higher and lower pixel values than the focal adhesions we want
> to target.
> >  Is there a way of classifying/thresholding that will compare
> structures to
> > the local background surrounding it so as to allow it to be
> identified and
> > quantified?  Thanks- Dave
> >
> > Dr. David Knecht
> > Department of Molecular and Cell Biology
> > Co-head Flow Cytometry and Confocal Microscopy Facility
> > U-3125
> > 91 N. Eagleville Rd.
> > University of Connecticut
> > Storrs, CT 06269
> > 860-486-2200
> > 860-486-4331 (fax)
> >
>
>
>
> --
> Dr. Stoyan P. Pavlov, MD
> Departament of Anatomy, Histology and Embryology
> Medical University "Prof. Dr. Paraskev Stoyanov", Varna
> Prof. Marin Drinov Str.55
> 9002 Varna
>  Bulgaria
> Tel: +359 (0) 52 - 677 - 050 #2638
> e-mail: [hidden email]

--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]
[phone], fax [phone]
http://astro.temple.edu/~jbs