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Hi George,
so where's the monomeric or tandem dimer AausFP1? They apparently do exist,
but the sequence hasn't been published yet.
And, btw, AausFP1 is on Addgene...



On Sat, Nov 14, 2020 at 8:46 AM George McNamara <[log in to unmask]>
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.
> *****
>
> Hi Alby,
>
> The STELLARIS (and SP8, SP5) X1 ports should also work well for
> fluorescence polarization aka fluorescence anisotropy imaging microscopy
> (FAIM). Other microscopes could also "do" FPol=FAIM. Which in turn
> enables various quantitative applications, including:
>
> * fluorescence anisotropy FP biosensors, see (pubmed search): rizzo
> zhang biosensors
>
> * multiphoton fluorescence anisotropy for imaging bound-to-target (high
> FA) vs free (low FA) fluorescdent drugs, Vinegnoi, Weiddeler et al find
> BODIPY-drug conjugates work well, see review
> https://pubmed.ncbi.nlm.nih.gov/29410158/
>
> * cytoplasmic viscosity, see verkman Fluorescence anisotropy viscosity
>
> ----
>
> Nathan Shaner et al's AausFP1 paper finally out, 5x brighter than EGFP
> (or 10x brighter if tandem dimer), narrow excitation and emission
> spectral peaks (making Leica's white light laser * AOBS very interesting
> light source [some other vendors also offer WLL, for example ISS]
>
>
> https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000936
>
> if aa sequence only in figures (tedious to extract for codon
> optimization) see text files at
> https://www.biorxiv.org/content/10.1101/677344v2.supplementary-material
>
> tdAausFP1 at 10x brighter than EGFP will be only a little brighter than
> Steve Vogel's V6 ("VVVVVV" at http://www.addgene.org/27813 ), so
> hopefully Steve or someone on listserv (Alby) will make AausFP1_6 etc.
> Steve has published a paper that might resonate with Alby (and others)
> on Venus dimers coherence, https://pubmed.ncbi.nlm.nih.gov/31060812/ ...
> so another use of tdAausFP1 (possibly wit hdifferent length linkers)
> would be a better test FP-FP for that. A nice control would be
> AausFP1(green)-AausFP1(yellow) [g-y, y-g, various linkers), as well as
> being outstanding FRET series (a'la Steve's C5V etc in addgene).
>
>
> enjoy,
>
> George
>
>
> On 11/14/2020 2:34 AM, Alberto Diaspro 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.
> > *****
> >
> > Great George... from Italy I plan using Grappa, in the meanwhile we work
> from remote with our brand new Stellaris 8 TAU-STED...amazing! ...also
> adding dimensions by means of Mueller matrix elements like circular
> dichroism outside absorrtion bands...
> > Let's tink more, learning a lot in the global pandemic era from the 16
> dimensions - some redundant - scattered by the sample
> >
> > Stay safe, take care of your mask...after touching clean your hands
> before a caress to your loved ones... I miss Sissi our beloved 14 years old
> Cavalier King Charles...i know it is another story ...so lets go to the
> microscope...
> >
> > All the best
> > ALby
> > -------------------------
> > Alberto Diaspro
> > Dipartimento di Fisica, UNIGE
> > Dipartimento di Nanofisica, IIT
> >
> > https://peerj.com/Diaspro/
> > ——————————————
> >
> >
> >
> >
> > Il giorno 14 nov 2020, alle ore 02:06, George McNamara <
> [log in to unmask]> ha scritto:
> >
> > *****
> > 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.
> > *****
> >
> >
> > Leica Science Week 2020 had nice presentation on this by James Marr,
> Non-traditional imaging: a microscopist with a microscope during a global
> pandemic
> >
> > (the Doritos data was tasty!)
> >
> > https://web.cvent.com/event/16597ab5-db93-48e9-9820-0974d58f2497/summary
> (or ask your local leica rep for a URL ... presentations online for a
> couple of months).
> >
> > Also good: Scott Fraser's talk "Keynote: Adding Dimensions to Multiplex
> Molecular Imaging "on spectral phasors meet flim phasors (aka "set phasors
> to stun", though would have liked to have seen one hemisphere [hemi-globe?]
> graphic combining both).  I was pleased to hear from Scott that he finally
> disavows 32 channel detector (for reasons I will not detail here). I
> disagree that 4 detectors is enough and suggest that Leica STELLARIS 5 (or
> 8) with 5 "S" detectors inside plus 4 on X1 port (ok, 8 would be even
> better, but don't want to put the Leica engineers or S10 reviewers into
> cardiac arrest) [S = SiPM]. With respect to STELLARIS, U.S. (and Canada?)
> labs and cores staff can check out
> https://www.leica-microsystems.com/stellaris-award (disclosure: I already
> applied and am currently hosting a "5" demo). I also enjoyed Prof. Emily
> Mace's NK cells (NCAM matters) keynote.
> >
> > ***
> >
> > I also have a GMO free tip: sterilize (70% EtOH works best, at home
> could go with vodka) finger nail, scrape some cheek cells, deposit on
> microscope slide and spread out ... either air dry or wet mount uwith a
> coverglass (the bacteria are easier to see by wt mount).
> >
> > happy 2020 and stay safe,
> >
> > George
> >
> >
> > On 11/11/2020 8:08 AM, Oshel, Philip Eugene 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.
> >> *****
> >>
> >> Pond/ditch water. Between the protists and the micro/meso scale
> animals, you'll be lost in your slides. Especially if you have DIC or
> darkfield.
> >>
> >> Phil
> >> -------------
> >> Philip Oshel
> >> Imaging Facility Director
> >> Biology Department
> >> 1304 Biosciences
> >> 1455 Calumet Ct.
> >> Central Michigan University
> >> Mt. Pleasant, MI 48859
> >> 989 774-3576 office
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List <[log in to unmask]> on
> behalf of Mika Ruonala <[log in to unmask]>
> >> Reply-To: Confocal Microscopy List <[log in to unmask]>
> >> Date: Wednesday, 11November, 2020 at 04:43
> >> To: "[log in to unmask]" <
> [log in to unmask]>
> >> Subject: [External] GMO-free Specimen for Demonstrations
> >>
> >>      *****
> >>      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 Listserver colleagues,
> >>      whenever I have some time from other stuff I'm always happy to
> image all kind of customer slides with my confocal setup built in my Man
> Cave (Axiovert 200M, Clarity laser-free confocal etc.). However, it gets a
> bit boring to always image fixed slides, no matter how exciting stuff is
> provided.
> >>
> >>      Since I also have the environmental system from Bioptechs I'm
> wondering if there is an abundant source for suitable fluorescent
> live-specimen that would not require GMO security and/or cell culture
> facility. I do have a source for GMO free cells from a friendly lab and
> pumping them full of vital dyes is one option, but still is not something
> that could be done relatively spontaneously but does require some
> preparations.
> >>
> >>      Any suggestions would be most welcome!
> >>
> >>      Best,
> >>
> >>      Mika
> >>
>


-- 
-- 
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth