Question regarding fluorescence colocalization

[sivaram mylavarapu]

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Dear colleagues,

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

Thanks much!

Sivaram.  

--

[xavier Sanjuan]

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Hi Sivaram,

try this (sorry if a more updated one is available!): https://www.ncbi.nlm.nih.gov/pubmed/17210054

Best,

Xavi.

2016-11-02 15:12 GMT+01:00 sivaram mylavarapu <[hidden email]>:

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Dear colleagues,

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

Thanks much!

Sivaram.  

--

[Gary G. Li-3]

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A practical guide to evaluating colocalization in biological microscopy.

Dunn KW, Kamocka MM, McDonald JH.

Am J Physiol Cell Physiol. 2011 Apr;300(4):C723-42

https://www.ncbi.nlm.nih.gov/pubmed/21209361

On Wed, Nov 2, 2016 at 10:12 AM, sivaram mylavarapu <[hidden email]> wrote:

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Dear colleagues,

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

Thanks much!

Sivaram.  

--

[Steffen Dietzel]

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Pitfalls?

search for "The 39 Steps: A Cautionary Tale of Quantitative 3-D
Fluorescence Microscopy" by Jim Pawley with your favorite search engine.

Steffen

Am 02.11.2016 um 15:12 schrieb sivaram mylavarapu:

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Dear colleagues,

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

Thanks much!

Sivaram.  

--

[sivaram mylavarapu]

***** 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. *****

Thanks Steffen, will check it out.

On 2 Nov 2016 10:26 p.m., "Steffen Dietzel" <[hidden email]> wrote:

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Pitfalls?

search for "The 39 Steps: A Cautionary Tale of Quantitative 3-D
Fluorescence Microscopy" by Jim Pawley with your favorite search engine.

Steffen

Am 02.11.2016 um 15:12 schrieb sivaram mylavarapu:

***** 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. *****

Dear colleagues,

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

Thanks much!

Sivaram.  

--

[Pongor Csaba]

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Dear Sivaram!

I can recomend the following papers in addition:

1. Bolte S,Cordelières FP, (2006) A guided tour into subcellular colocalization analysis in light microscopy, Journal of Microscopy, Volume 224, Issue 3: 213-232.

2. Dunn KW, Kamocka MM, McDonald JH., (2011) A practical guide to evaluating colocalization in biological microscopy. American Journal of Physiology - Cell Physiology 300:C723-C7421

I hope it helps!

Yours,

Csaba Pongor

---

Feladó: "sivaram mylavarapu" <[hidden email]>
Címzett: [hidden email]
Elküldött üzenetek: Szerda, 2016. november 2. 15:12:41
Tárgy: Question regarding fluorescence colocalization

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Dear colleagues,

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

Thanks much!

Sivaram.  

--

[Jeremy Adler-5]

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Hej Sivaram,

 

Measuring colocalization is bit of a minefield – there are too many coefficients and different ways of applying them.

We have been pushing the idea that colocalization has two components, that reported together provide the clearest description.

of the relative distribution of two fluorophores.

1)    Co-occurrence, are the fluorophores in the same place – measured by the Manders M1 & M2 coefficients

And

2)    Correlation, is there a relationship between the intensities across a population of pixels. Pearson or the Spearman correlation coeffs

An important consideration in measuring correlation is which pixels to include – we strongly argue that only pixels with both fluorophores present should be included.

Another important consideration is the ROI (region of interest), the part of the image used to make the measurements and how this is defined/chosen.

 

Below is a review article and two papers focused on the details of making colocalization measurements

I would also recommend the Dunn review – mentioned in another post.

 

Quantifying colocalization: thresholding, void voxels and the H(coef).

Adler J, Parmryd I.

PLoS One. 2014 Nov 6;9(11):e111983. doi: 10.1371/journal.pone.0111983.

PMID:25375829

Free PMC Article

Colocalization analysis in fluorescence microscopy.

Adler J, Parmryd I.

Methods Mol Biol. 2013;931:97-109. doi: 10.1007/978-1-62703-056-4_5.

PMID:23026999

Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.

Adler J, Parmryd I.

Cytometry A. 2010 Aug;77(8):733-42. doi: 10.1002/cyto.a.20896.

PMID:20653013

Free Article

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of sivaram mylavarapu
Sent: den 2 november 2016 15:13
To: [hidden email]
Subject: Question regarding fluorescence colocalization

 

***** 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. *****

Dear colleagues,

 

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

 

Thanks much!

 

Sivaram.  

 

--

Sivaram Mylavarapu.
[hidden email]

[phil laissue-2]

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Hi Sivaram, 

colocalisation has got a bit of a disreputation because it's often done badly, but if done the right way, I think it's more robust than, say, intensity-based FRET. We developed a centroid/histogram-based approach. If you have punctate structures, this works well, even for sparse colocalisation.

https://www.researchgate.net/publication/235401654_A_novel_method_for_quantified_superresolved_three-dimensional_colocalisation_of_isotropic_fluorescent_particles

Just drop me a line if you'd like to use the Matlab GUI, and I'll send you the code. 

With kind regards, 

Philippe

On 3 November 2016 at 08:40, Jeremy Adler <[hidden email]> wrote:

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Hej Sivaram,

 

Measuring colocalization is bit of a minefield – there are too many coefficients and different ways of applying them.

We have been pushing the idea that colocalization has two components, that reported together provide the clearest description.

of the relative distribution of two fluorophores.

1)    Co-occurrence, are the fluorophores in the same place – measured by the Manders M1 & M2 coefficients

And

2)    Correlation, is there a relationship between the intensities across a population of pixels. Pearson or the Spearman correlation coeffs

An important consideration in measuring correlation is which pixels to include – we strongly argue that only pixels with both fluorophores present should be included.

Another important consideration is the ROI (region of interest), the part of the image used to make the measurements and how this is defined/chosen.

 

Below is a review article and two papers focused on the details of making colocalization measurements

I would also recommend the Dunn review – mentioned in another post.

 

Quantifying colocalization: thresholding, void voxels and the H(coef).

Adler J, Parmryd I.

PLoS One. 2014 Nov 6;9(11):e111983. doi: 10.1371/journal.pone.0111983.

PMID:25375829

Free PMC Article

Colocalization analysis in fluorescence microscopy.

Adler J, Parmryd I.

Methods Mol Biol. 2013;931:97-109. doi: 10.1007/978-1-62703-056-4_5.

PMID:23026999

Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.

Adler J, Parmryd I.

Cytometry A. 2010 Aug;77(8):733-42. doi: 10.1002/cyto.a.20896.

PMID:20653013

Free Article

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of sivaram mylavarapu
Sent: den 2 november 2016 15:13
To: [hidden email]
Subject: Question regarding fluorescence colocalization

 

***** 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. *****

Dear colleagues,

 

I am looking for an authoritative review(s) on the principles and best practices to be followed for measuring immunofluorescence co-localization, including obtaining quantitative information like the Pearson's coefficient. It would be very useful if the most common pitfalls to be avoided were also dwelt upon on in the articles. I would appreciate if you could point me to such literature.

 

Thanks much!

 

Sivaram.  

 

--

Sivaram Mylavarapu.
[hidden email]