GMO-free Specimen for Demonstrations

[ICIT]

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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

[Oshel, Philip Eugene]

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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 <[hidden email]> on behalf of Mika Ruonala <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Wednesday, 11November, 2020 at 04:43
To: "[hidden email]" <[hidden email]>
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
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    *****

    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

[Craig Brideau]

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If you have any flowers around you can simply smack them against a slide
and you will get excellent pollen samples as well. Riffing off Philip's
pond water idea, perhaps a small terrarium with aquatic plants and animals?
You can sample the algae and water periodically and see what you get. A
typical fish tank would probably have some creatures in it beyond the fish.
Craig

On Wed, Nov 11, 2020 at 6:08 AM Oshel, Philip Eugene <[hidden email]>
wrote:

> *****
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> 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 <[hidden email]> on
> behalf of Mika Ruonala <[hidden email]>
> Reply-To: Confocal Microscopy List <[hidden email]>
> Date: Wednesday, 11November, 2020 at 04:43
> To: "[hidden email]" <[hidden email]>
> 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
>
>

[Steffen Dietzel]

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Mika,

I second Phil's suggestion. For bright field or other non-fluorescent
approaches, take some slimy stuff out of a pond, it will be full of
protists, dashing around.

If you need fluorescence, the easiest is algae or moss leaves, strong
red fluorescence from chlorophyll. Neither have air inside their leafs
like higher plants that will be difficult on the optical path. pond
water may also contain chloroplast containing protists or small algea
such as Euglena or Volvox. But you have to be lucky or knowledgeable to
find these in sufficient quantities.

Some lichen also show fluorescence in other parts of the spectrum. If
you take a walk at dawn with a UV-flashlight, you will find some. Not
sure that will be more entertaining than fixed samples though, they
don't move much :-) Going with the UV-Flashlight through your dark
kitchen (spices, fruit surfaces, ...) and fridge also might turn up a
few things. My kids where excited, at the time.

And scorpions show blue fluorescence. But there may be reasons other
than GMO regulations that you don't want them alive in your microscope room.

Easier is pollen. Some of them are quite big, Hibiscus around 150 nm.
Pumpkin is also big.  My feeling is that bigger flowers carry bigger
pollen, but I may be wrong. They are hydrophobic and thus solve nicely
directly in immersion oil. Finally a sample where you can have a
mounting medium with an Ri of 1.518.


If you are thinking more about fluorescence in cells, nile red might be
a noteworthy option. It stains membranes.

Have fun!

Steffen


Am 11.11.2020 um 14:08 schrieb Oshel, Philip Eugene:

> *****
> 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 <[hidden email]> on
> behalf of Mika Ruonala <[hidden email]>
> Reply-To: Confocal Microscopy List <[hidden email]>
> Date: Wednesday, 11November, 2020 at 04:43
> To: "[hidden email]" <[hidden email]>
> 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
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Glenn Merrill-Skoloff]

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A note on pond water: adding some glycerol will slow the critters down a little.

-- Glenn

Glenn Merrill-Skoloff
Laboratory Manager, Intravital Core Director
Division of Hemostasis and Thrombosis, BILH
3 Blackfan Circle, CLS 942
Boston, MA  02115

617-735-4040

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Oshel, Philip Eugene
Sent: Wednesday, November 11, 2020 8:08 AM
To: [hidden email]
Subject: Re: [External] GMO-free Specimen for Demonstrations

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=WknmpdNpvrlj2B5K1aWVqL1SOiF30547pqSuOmtwXTQ&r=42NmABDnL7YbxR4oFPmDN-5XK3Kw7RY6XnrLkSD93J4&m=PBsArX0b5oFVv-HY6nBFe_45Gd6lggZf-PkXnioPaVw&s=SOO_lavBMgj70gI-vMmCyxyLvMRr8jm7xqbAkHTAQJA&e=  and include the link in your posting.
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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 <[hidden email]> on behalf of Mika Ruonala <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Wednesday, 11November, 2020 at 04:43
To: "[hidden email]" <[hidden email]>
Subject: [External] GMO-free Specimen for Demonstrations

    *****
    To join, leave or search the confocal microscopy listserv, go to:
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    Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=WknmpdNpvrlj2B5K1aWVqL1SOiF30547pqSuOmtwXTQ&r=42NmABDnL7YbxR4oFPmDN-5XK3Kw7RY6XnrLkSD93J4&m=PBsArX0b5oFVv-HY6nBFe_45Gd6lggZf-PkXnioPaVw&s=SOO_lavBMgj70gI-vMmCyxyLvMRr8jm7xqbAkHTAQJA&e=  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


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[daj1u06]

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In my experience, pretty much anything and everything will fluoresce and give an interesting confocal image if you find the right colour to excite with and detect - a quick look down the eyepieces in std fluorescence mode with the different cubes will tell you which colours excite and which colours the fluorescence is, then set your system up to match these - we have Leica's with true spectral detection so this is a breeze. We had an arts project a few years ago where a professional artist went around community groups and heritage sites in a local city and, with owner's consent, took tiny samples from treasured possessions or historical artefacts, and the story behind them. We did 100s of samples and only had 1 failure we couldn't get anything worthwhile from - have a look at www.cityforensics.com for the product of this project - nothing was stained, all simply placed on slides and imaged and apart from recolouring the blank backgrounds on a few of the images, there are all simply max projecxtions straight from the system. So you could just get your punters to "donate" or loan something they have on them, any fabric, paper, wood etc. is always a winner. If you have reflectance mode, then any bit of jewellery or old coin (remember those, pre Covid!) simply place on a slide and image at low, dry mag, can produce stunning pictures from the microscratches.

For plant material, soaking and mounting in perflourodecalin will take out the air and anything with photosynthetic pigments will fluoresce with pretty much any colour excitation. Anything with different mixes of photosynthetic pigments and multi colour excitation gives great tonal images (cellulose autofluoresceces. Pollen also great, - a pot of mixed "bee granules" from a health food store will keep you busy for hours, looking for the different types. Any mixed spices or fruit teas, dry, limply piled up on a slide also really good.

If you can get some zooplankton, fix and stain with rose bengal, congo red or carmine, also great and really 3D and last for ever.

My general recommendation is to keep it simple, use something the punter can relate to / donates, use low mag and dry mounts wherever possible.

The ability to play is the greatest perk of our imaging jobs.

[Olivier Bardot-2]

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Hello
Just take honey, add some ethanol (70% is fine), mix and centrifuge. Repeat twice.
Add some glycerol and put on slice.

[Colin Haig]

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Sounds tasty. Make mine a slice of lime.
Seriously though, that's so simple. Thanks for sharing.
-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of olivier bardot
Sent: November 13, 2020 3:43 AM
To: [hidden email]
Subject: Re: GMO-free Specimen for Demonstrations

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Hello
Just take honey, add some ethanol (70% is fine), mix and centrifuge. Repeat twice.
Add some glycerol and put on slice.

[George McNamara]

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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 <[hidden email]> on behalf of Mika Ruonala <[hidden email]>
> Reply-To: Confocal Microscopy List <[hidden email]>
> Date: Wednesday, 11November, 2020 at 04:43
> To: "[hidden email]" <[hidden email]>
> 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
>

[Alberto Diaspro-2]

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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 <[hidden email]> ha scritto:

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*****


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 <[hidden email]> on behalf of Mika Ruonala <[hidden email]>
> Reply-To: Confocal Microscopy List <[hidden email]>
> Date: Wednesday, 11November, 2020 at 04:43
> To: "[hidden email]" <[hidden email]>
> 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
>

[George McNamara]

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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 <[hidden email]> 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 <[hidden email]> on behalf of Mika Ruonala <[hidden email]>
>> Reply-To: Confocal Microscopy List <[hidden email]>
>> Date: Wednesday, 11November, 2020 at 04:43
>> To: "[hidden email]" <[hidden email]>
>> 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
>>

[Benjamin Smith]

*****
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.
*****

On the topic of fluorescence polarization you can also use the scan-field
rotator to rotate the excitation polarization.  We combined the polarizer
in the detector path of the Leica SP8 with the scan field polarizer to do
an automated 2D scan of fluorescence polarization in nanocomposites.

On Sat, Nov 14, 2020 at 5:46 AM George McNamara <[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.
> *****
>
> 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 <
> [hidden email]> 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 <[hidden email]> on
> behalf of Mika Ruonala <[hidden email]>
> >> Reply-To: Confocal Microscopy List <[hidden email]>
> >> Date: Wednesday, 11November, 2020 at 04:43
> >> To: "[hidden email]" <
> [hidden email]>
> >> 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
> >>
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Zdenek Svindrych-2]

*****
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 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 <[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.
> *****
>
> 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 <
> [hidden email]> 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 <[hidden email]> on
> behalf of Mika Ruonala <[hidden email]>
> >> Reply-To: Confocal Microscopy List <[hidden email]>
> >> Date: Wednesday, 11November, 2020 at 04:43
> >> To: "[hidden email]" <
> [hidden email]>
> >> 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