Robust and automatic brightness evaluation for fluorescent spots

[Kyle Michael Douglass]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello everyone,

Would anyone be able to point me towards some recommended papers or
open-source software for automatic and robust evaluation of the
fluorescence signal strength from fluorescence-labeled nuclear loci such
as one might observe in FISH experiments?

Automatic segmentation and detection of the spots is not a problem;
however, quantifying the fluorescence signal strength in our data is
tricky because

1) different nuclei have different background fluorescence levels,
2) different loci within the same nucleus sometimes lie within a few
pixels of one another, which confounds automated local background estimates,
3) the loci do not necessarily possess circular symmetries, and
4) we have several hundred images to process, so manual methods are not
feasible.

I appreciate the feedback. Cheers, and happy Friday.

Kyle

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

http://rajlab.seas.upenn.edu/StarSearch/launch.html

It helped Arjun make PopSci

http://www.popsci.com/science/article/2013-09/arjun-raj

Arjun's early papers - PubMed    raj a tyagi   -- showed that RNA in
mammalian cells are transcribed in bursts. See also iceFISHing

http://www.ncbi.nlm.nih.gov/pubmed/23416756

http://stellarisgallery.biosearchtech.com/IceFISH


What you want to do may be facilitated by his/his lab's work with Ed
Boyden's on Expansion Microscopy FISHing:

http://www.nature.com/nmeth/journal/v13/n8/full/nmeth.3899.html

//

By the way, if you need to go whole organism (mouse, rat, not adult
person or blue whale), deliberately shrinking with the optical clearing
solution is useful:

http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.3964.html

I can envision "whole mouse single molecule  RNA FISHing" ... with the
transcriptional burst sites being easier to image.


George

p.s. if you want multiplex with that, check out fast joint spatial
deconvolution and spatial unmixing (not just for FRET, more plex the
better),

http://www.ncbi.nlm.nih.gov/pubmed/27023704



On 8/26/2016 1:25 AM, Kyle Douglass wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello everyone,
>
> Would anyone be able to point me towards some recommended papers or
> open-source software for automatic and robust evaluation of the
> fluorescence signal strength from fluorescence-labeled nuclear loci
> such as one might observe in FISH experiments?
>
> Automatic segmentation and detection of the spots is not a problem;
> however, quantifying the fluorescence signal strength in our data is
> tricky because
>
> 1) different nuclei have different background fluorescence levels,
> 2) different loci within the same nucleus sometimes lie within a few
> pixels of one another, which confounds automated local background
> estimates,
> 3) the loci do not necessarily possess circular symmetries, and
> 4) we have several hundred images to process, so manual methods are
> not feasible.
>
> I appreciate the feedback. Cheers, and happy Friday.
>
> Kyle
>

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650