Posted by
George McNamara on
Aug 26, 2016; 2:51pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Robust-and-automatic-brightness-evaluation-for-fluorescent-spots-tp7585583p7585588.html
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http://rajlab.seas.upenn.edu/StarSearch/launch.htmlIt helped Arjun make PopSci
http://www.popsci.com/science/article/2013-09/arjun-rajArjun'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/23416756http://stellarisgallery.biosearchtech.com/IceFISHWhat 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.htmlI 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/27023704On 8/26/2016 1:25 AM, Kyle Douglass wrote:
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>
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>
> 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/georgemcnamarahttps://works.bepress.com/gmcnamara/75/http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650