receptor number quantification

[Clarence Dunn]

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Hello,

I'm interested in quantifying the number/density of a particular IL-
receptor in two different adherent cell populations. Most of what I've
come across in the liturature uses Flow Cytometry and calibration beads
or Western Blots using a known quantity of a reference proptein.
However, I'd prefer to include some imaging data...has anyone on this
ListServ had success using a microscopy-based techniques to generate
a quantitative, or even semi-quantitative measure of receptor density?

Thanks very much!

Clarence Dunn, Ph.D., MPH

[Smith, Benjamin E.]

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There is a fairly easy technique called number and brightness or N&B:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2257897/

This technique allows you to infer the relative number of proteins in each pixel in an image.  The only special equipment needed is a photon counting detector, so either a hybrid detector or a SPAD will work.  You can use a PMT, but as you can see in the methods section, converting a PMT analog signal to a counting-like signal is non-trivial.   You then simply set the dwell time and fluorescence intensity to match those in the manuscript, and then do an XYT scan for about 5 minutes.  The paper outlines the normalization math, or you can use the SimFCS software package from Dr. Enrico Gratton's lab.

Cheers,
   Ben Smith



________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Clarence Dunn <[hidden email]>
Sent: Monday, February 29, 2016 6:31 PM
To: [hidden email]
Subject: receptor number quantification

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Hello,

I'm interested in quantifying the number/density of a particular IL-
receptor in two different adherent cell populations. Most of what I've
come across in the liturature uses Flow Cytometry and calibration beads
or Western Blots using a known quantity of a reference proptein.
However, I'd prefer to include some imaging data...has anyone on this
ListServ had success using a microscopy-based techniques to generate
a quantitative, or even semi-quantitative measure of receptor density?

Thanks very much!

Clarence Dunn, Ph.D., MPH

[Michelle Digman]

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Dear Clarence,

I'm happy to help you in calibrating your system if you would like to try
number and Molecular Brightness analysis.

If you have a standard confocal microscope you can actually use even if it
has analog detectors (which most systems have) to do N&B analysis. We just
need to check a few parameters to insure you don't have a lot of noise from
your detectors.

Here are some quick tips to see if it will work:

1) If you happen to have a fixed sample such a a chroma slide or fixed
beads you can take a 256x256 image at a pixel dwell time of ~6-12us/pixel
for a total ~20-50 frames.

2) Download Globals for Images · SimFCS 4 (32 and 64-bit)
simfcs4-setup.exe · 65.9 MB
http://www.lfd.uci.edu/globals/

3) then follow these instructions:
https://webfiles.uci.edu/xythoswfs/webui/_xy-10068155_1-t_YAv8Vs6Y

Let me know if you have any questions.
Best,
Michelle
*******************************


*Michelle A. Digman, Ph.D.Assistant Professor, Department of Biomedical
Engineering, UCIrvine*Joint Assistant Professor, Department of Chemical
Engineering and Material Sciences,UCIrvine
Joint Assistant Professor, Department of Developmental and Cell Biology,
UCIrvine
Adjunct Lecturer in the School of Science and Technology, University of New
England, Armidale, Australia
Co-Investigator, Laboratory for Fluorescence Dynamics (LFD)
Director, W.M. Keck Nanoimaging Lab
Research Associate, Developmental and Cell Biology
*3103 Natural Sciences II*
*University of California Irvine*
*Irvine, CA 92697-2715*
*Tel: [phone]*
*Email: [hidden email] <[hidden email]> *
http://www.lfd.uci.edu/
http://faculty.sites.uci.edu/digmanlab/

On Mon, Feb 29, 2016 at 5:50 PM, Smith, Benjamin E. <[hidden email]>
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.
> *****
>
> There is a fairly easy technique called number and brightness or N&B:
>
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2257897/
>
> This technique allows you to infer the relative number of proteins in each
> pixel in an image.  The only special equipment needed is a photon counting
> detector, so either a hybrid detector or a SPAD will work.  You can use a
> PMT, but as you can see in the methods section, converting a PMT analog
> signal to a counting-like signal is non-trivial.   You then simply set the
> dwell time and fluorescence intensity to match those in the manuscript, and
> then do an XYT scan for about 5 minutes.  The paper outlines the
> normalization math, or you can use the SimFCS software package from Dr.
> Enrico Gratton's lab.
>
> Cheers,
>    Ben Smith
>
>
>
> ________________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Clarence Dunn <[hidden email]>
> Sent: Monday, February 29, 2016 6:31 PM
> To: [hidden email]
> Subject: receptor number quantification
>
> *****
> 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,
>
> I'm interested in quantifying the number/density of a particular IL-
> receptor in two different adherent cell populations. Most of what I've
> come across in the liturature uses Flow Cytometry and calibration beads
> or Western Blots using a known quantity of a reference proptein.
> However, I'd prefer to include some imaging data...has anyone on this
> ListServ had success using a microscopy-based techniques to generate
> a quantitative, or even semi-quantitative measure of receptor density?
>
> Thanks very much!
>
> Clarence Dunn, Ph.D., MPH
>

[Andreas Bruckbauer]

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Dear Clarence,

knowing how many fluorophores are in the image is one thing, but relating this information to a number of receptors is more challenging. We did this using dStorm for the B cell recptor and coreceptors
http://www.cell.com/immunity/abstract/S1074-7613%2813%2900097-6
http://onlinelibrary.wiley.com/doi/10.15252/embj.201593027/abstract
(see also the supporting information in the first paper)
You need to figure out what percentage of the receptors you are actually labelling and recording in the image, we did this using the bead assays you mentioned. In some special cases, e.g. you know the receptor has a certain known number of subunits, you could also get this information out of the images.

best wishes

Andreas


 

 

-----Original Message-----
From: Clarence Dunn <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Tue, 1 Mar 2016 0:46
Subject: receptor number quantification

*****
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*****

Hello,

I'm interested in quantifying the number/density of a particular IL-
receptor in two different adherent cell populations. Most of what I've
come across in the liturature uses Flow Cytometry and calibration beads
or Western Blots using a known quantity of a reference proptein.
However, I'd prefer to include some imaging data...has anyone on this
ListServ had success using a microscopy-based techniques to generate
a quantitative, or even semi-quantitative measure of receptor density?

Thanks very much!

Clarence Dunn, Ph.D., MPH

[Mark Cannell-2]

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If the number is time invariant you could try something like this, using the measured average brightness of a known fluorochrome. But watch out for the geometry problem…
Soeller, C., Crossman, D., Gilbert, R., Cannell, M.B., 2007. Analysis of ryanodine receptor clusters in rat and human cardiac myocytes. PNAS 104, 14958–14963. doi:10.1073/pnas.0703016104
Cheers


On 1/03/2016, at 12:31 am, Clarence Dunn <[hidden email]> 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,
>
> I'm interested in quantifying the number/density of a particular IL-
> receptor in two different adherent cell populations. Most of what I've
> come across in the liturature uses Flow Cytometry and calibration beads
> or Western Blots using a known quantity of a reference proptein.
> However, I'd prefer to include some imaging data...has anyone on this
> ListServ had success using a microscopy-based techniques to generate
> a quantitative, or even semi-quantitative measure of receptor density?
>
> Thanks very much!
>
> Clarence Dunn, Ph.D., MPH

[Michael Model]

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We have done that some years ago: Model MA, Reese JL, Fraizer GC.
Measurement of wheat germ agglutinin binding with a fluorescence
microscope. Cytometry A 75A, 874-881 (2009)

Mike Model


On Mon, Feb 29, 2016 at 7:31 PM, Clarence Dunn <[hidden email]>
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,
>
> I'm interested in quantifying the number/density of a particular IL-
> receptor in two different adherent cell populations. Most of what I've
> come across in the liturature uses Flow Cytometry and calibration beads
> or Western Blots using a known quantity of a reference proptein.
> However, I'd prefer to include some imaging data...has anyone on this
> ListServ had success using a microscopy-based techniques to generate
> a quantitative, or even semi-quantitative measure of receptor density?
>
> Thanks very much!
>
> Clarence Dunn, Ph.D., MPH
>