who is interested in 10plex immunofluorescence imaging?

[George McNamara]

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Dear Confocal Listserv,

who is interested in doing 10plex immunofluorescence imaging?
(vendors - if you are offering or thinking of offering 10+plex imaging,
see bottom of message).

If you are, I encourage you to check out Dr. Dragan Maric's (10x1)
webinar on September 24 -

http://www.denovosoftware.com/site/UpcomingWebinars.shtml


      FCS Express 4 Image Cytometry Special Applications: Introduction
      to FCS Express and Application Examples from Dragan Maric, PhD.

We know that it can be difficult to try new software on your own, so we
have scheduled a live online demo with an application example from
Dragan Maric, Ph.D. NIH, to help you out. Agenda: De Novo Software:
Getting Started in FCS Express Image Cytometry. 30 minutes. Dragan
Maric, Ph.D., Staff Scientist, National Institutes of Health:
Quantitative in situ mapping of emerging neural cell phenotypes during
early rat brain development using FCS Express Image Cytometry.

You may also find of interest the following ...


6plex (6x1) confocal + deconvolution+spectral unmixing (compensation):

Histo-cytometry: a method for highly multiplex quantitative tissue
imaging analysis applied to dendritic cell subset microanatomy in lymph
nodes. </pubmed/22863836> Gerner MY, Kastenmuller W, Ifrim I, Kabat J,
Germain RN. Immunity. 2012 Aug 24;37(2):364-76. PMID: 22863836
Note: CompuCyte has been offering "flow like scatterplots from slides"
for a long time - with optional full resolution images since ~2005 in
the iCys/iCyt series.


100plex (2x50) plex:
Next-generation biomarkers based on 100-parameter functional
super-resolution microscopy TIS. </pubmed/22209707>* Schubert W*,
Gieseler A, Krusche A, Serocka P, Hillert R. New Biotechnol. 2012.
29(5):599-610. PMID: 22209707
Note: "functional super-resolution" is not super-resolution in the STED
and precision localization nanoscope senses of the phrase.


1plex (see Smith & Micheva's earlier papers for reiterative multiplexing):

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using
Bayesian Restoration of Fluorescence Images from Ultrathin Specimens.
</pubmed/22956902> Wang G, Smith SJ. PLoS Comput Biol. 2012.
8(8):e1002671. PMID: 22956902
Note: the SIM and STED nanoscopes - and/or operators - seemed to be
having days ... pixel size of STED could have been much better - I used
25 or 30 nm pixel size for the Leica CW-STED demo last year. also a bit
unfair not deconvolving the STED result (and sad they did not use HyD
photon counting mode)  ... Array Tomography is a great match for Leica's
STED, since the section thinness makes Leica's lack of Z-improvement moot).


15 (1x15) plex:

http://www.chipcytometry.com/technology.phtml
http://chipcytometry.com/Blog/?p=256
an earlier version (much surpassed as of ISAC 2011) was published in
PubMed 19006067.



Vendors - if you are offering 10plex (or more) or thinking about
offering 10plex (or more) multiplex immunofluorescence/ISH/FPs/etc,
please contact me offline. I am co-organizing a multiplex imaging
workshop in December and can use your help. Vendors and Academics ...
workshop will be no cost to attend - one day in Houston. I anticipate
mostly locals (in Houston) will be at the event. Dr. Maric is one of the
speakers and we are planning to have Dr. Christian Hennig of
ChipCytometry.com to give a seminar by webex.

Sincerely,

George

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Dear Confocal listserv,

On 9/16/2012 I posted to the listserv that colleagues and I were
organizing an one day multiplex imaging workshop in December.
I am writing today (10/24/24) that we are postponing the workshop to the
middle of 2013.

-

I hope you find of use something I posted on the Internet yesterday ...

Tattletales: multiplexing biosensors and/or gene activity reporters in
live cells

http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg 
<http://home.earthlink.net/%7Epubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg>

Some Tattletales image analysis related content is at the bottom of a
second poster (most of which is on cell motility - I lookforward in the
future to combining the two methods):

http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_TAM_and_Tattletales_TIK_Public_Domain.jpg 
<http://home.earthlink.net/%7Epubspectra/McNamara_20121023Tue_TAM_and_Tattletales_TIK_Public_Domain.jpg>

My apology for the ugly URLs - I'm better with microscopes and
nanoscopes than web site design.

If I don't reply anytime soon it may be because I will be presenting
these posters at two HHMI Janelia Farm conferences Oct 28-Nov 7.


enjoy,

George

George McNamara
University of Miami

Context: if GFP is expressed at (say) 1000 copies per cell, and the cell
is 1,000 pixels in size with equal amount of autofluorescence per pixel,
the GFP signal will be very weak (1000 GFP vs 2000 total for the cell).
If all 1000 GFP molecules are all localized to one pixel ("locus"), that
pixel is 1000 GFP vs 1001 total, and each pixel in the rest of the cell
is 0 GFP vs 1 autofluorescence. Same total cell signal in either case (a
bummer for flow cytometers, good for ImageStream), but 1000 GFP in one
spot will be easy to image.
The cell nucleus has lots of room for other loci of other colors and
combinations of colors (addresses and reporters).
In addition to addresses, each locus could have a fluorescent biosensor
or the output of a promoter driving expression of a fluorescent
protein(s) - and/or luciferase(s).

While looking up what biosensors might be fun to use, I came across
http://jcs.biologists.org/content/125/3/743.full (Meng, Sachs 2012 J
Cell Sci) - including expression of a spectrin-cpstFRET
insertion-spectrin protein that is apparently ~2500 amino acids (but
maybe longer). So, Venus6 (under 1500 amino acids),
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0038209 
(Nguyen ... Vogel 2012 PLoS One) is not even close to what is expressed
by (some) human cells. Those are tiny - while looking up spectrin's
length in NCBI Protein, I came across an entry for a related protein,
Nesprin, http://www.ncbi.nlm.nih.gov/protein/Q8NF91.3 with 9,797 amino
acids - implying potential for expressing (approximately) FP40mer single
polypeptide (if barrels were 'stacked' end to end, 40mer * 4 nm = 160
nm).Nesprin is small compared to Titin,
http://www.ncbi.nlm.nih.gov/protein/Q8WZ42.4 with 34,350 amino acids -
implying potential for expressing (approximately) FP140mer single
polypeptide (if barrels were 'stacked' end to end, 40mer * 4 nm = 560 nm
... of course could just keep going to 250mer ... 1 um stacked).
nlsFP40mer or nlsFP140mer should be a lot of fun to watch (1) being
synthesized [might want to use FastTimer for that] and (2) entering
nuclear pore.

I also see Tattletales as a great match to the MSIM system developed by
Andrew York ... Hari Shroff, NIBIB (or if a commercial SIM vendor would
implement Andrew ... Hari's improvements)
http://code.google.com/p/msim/
http://www.nature.com/nmeth/journal/v9/n7/full/nmeth.2025.html
Especially once the airy spots (movie 2 of the google code page) are
supported by GPU enabled real time deconvolution (and more images
acquired), followed by GPU enabled real time SIM calculations (and
multichannel please). In addition to 4D MSIM nanoscopy (with 'hand
picked' lenses), the system could also be used for optogenetics, spot
photobleaching/activation/conversion/switching, and for high speed
Tattletales-loci illumination combined with multiple region of interest
readout from scientific CMOS camera(s). The U does have an NIH S10
nanoscope grant proposal in - any of the nanoscopes would complement
Tattletales nicely.


On 9/16/2012 4:13 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocal Listserv,
>
> who is interested in doing 10plex immunofluorescence imaging?
> (vendors - if you are offering or thinking of offering 10+plex
> imaging, see bottom of message).
>
> If you are, I encourage you to check out Dr. Dragan Maric's (10x1)
> webinar on September 24 -
>
> http://www.denovosoftware.com/site/UpcomingWebinars.shtml
>
>
>      FCS Express 4 Image Cytometry Special Applications: Introduction
>      to FCS Express and Application Examples from Dragan Maric, PhD.
>
> We know that it can be difficult to try new software on your own, so
> we have scheduled a live online demo with an application example from
> Dragan Maric, Ph.D. NIH, to help you out. Agenda: De Novo Software:
> Getting Started in FCS Express Image Cytometry. 30 minutes. Dragan
> Maric, Ph.D., Staff Scientist, National Institutes of Health:
> Quantitative in situ mapping of emerging neural cell phenotypes during
> early rat brain development using FCS Express Image Cytometry.
>
> You may also find of interest the following ...
>
>
> 6plex (6x1) confocal + deconvolution+spectral unmixing (compensation):
>
> Histo-cytometry: a method for highly multiplex quantitative tissue
> imaging analysis applied to dendritic cell subset microanatomy in
> lymph nodes. </pubmed/22863836> Gerner MY, Kastenmuller W, Ifrim I,
> Kabat J, Germain RN. Immunity. 2012 Aug 24;37(2):364-76. PMID: 22863836
> Note: CompuCyte has been offering "flow like scatterplots from slides"
> for a long time - with optional full resolution images since ~2005 in
> the iCys/iCyt series.
>
>
> 100plex (2x50) plex:
> Next-generation biomarkers based on 100-parameter functional
> super-resolution microscopy TIS. </pubmed/22209707>* Schubert W*,
> Gieseler A, Krusche A, Serocka P, Hillert R. New Biotechnol. 2012.
> 29(5):599-610. PMID: 22209707
> Note: "functional super-resolution" is not super-resolution in the
> STED and precision localization nanoscope senses of the phrase.
>
>
> 1plex (see Smith & Micheva's earlier papers for reiterative
> multiplexing):
>
> Sub-diffraction Limit Localization of Proteins in Volumetric Space
> Using Bayesian Restoration of Fluorescence Images from Ultrathin
> Specimens. </pubmed/22956902> Wang G, Smith SJ. PLoS Comput Biol.
> 2012. 8(8):e1002671. PMID: 22956902
> Note: the SIM and STED nanoscopes - and/or operators - seemed to be
> having days ... pixel size of STED could have been much better - I
> used 25 or 30 nm pixel size for the Leica CW-STED demo last year. also
> a bit unfair not deconvolving the STED result (and sad they did not
> use HyD photon counting mode)  ... Array Tomography is a great match
> for Leica's STED, since the section thinness makes Leica's lack of
> Z-improvement moot).
>
>
> 15 (1x15) plex:
>
> http://www.chipcytometry.com/technology.phtml
> http://chipcytometry.com/Blog/?p=256
> an earlier version (much surpassed as of ISAC 2011) was published in
> PubMed 19006067.
>
>
>
> Vendors - if you are offering 10plex (or more) or thinking about
> offering 10plex (or more) multiplex immunofluorescence/ISH/FPs/etc,
> please contact me offline. I am co-organizing a multiplex imaging
> workshop in December and can use your help. Vendors and Academics ...
> workshop will be no cost to attend - one day in Houston. I anticipate
> mostly locals (in Houston) will be at the event. Dr. Maric is one of
> the speakers and we are planning to have Dr. Christian Hennig of
> ChipCytometry.com to give a seminar by webex.
>
> Sincerely,
>
> George
>