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Weird issue with FRET after acceptor PB

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Avi Jacob

Weird issue with FRET after acceptor PB

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Hi all,

Got a weird issue I cannot explain.

We are doing acceptor photobleaching FRET with a CFP-YFP pair, on fixed
slides, on Leica widefield with scanner.
We are getting great initial bleaching of the YFP acceptor, however, there
is an almost immediate recovery of fluorescence in the bleached ROI.
This is happening also in the YFP only control.

Can this possibly be because of the mounting medium? What else can be a
mobile fraction on a fixed slide?
The signal is good, has the expected expression pattern.

Cheers,
Avi

--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit
Bar-Ilan University, Ramat-Gan 5290002, Israel
http://tinyurl.com/BIU-Microscopy

Steffen Dietzel

Re: Weird issue with FRET after acceptor PB

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Hi Avi,

that is weird. I would try to figure out if this is due to diffusion of
unbleached YFP (or some contaminant in the mounting medium) or due to
actual recovery of dark proteins becoming fluorescent again. Bleaching
of a large area might give a clue, or a FLIP experiment.

Good hunting

Steffen

Am 19.07.2018 um 09:33 schrieb Avi Jacob:

> *****
> 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 all,
>
> Got a weird issue I cannot explain.
>
> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on fixed
> slides, on Leica widefield with scanner.
> We are getting great initial bleaching of the YFP acceptor, however, there
> is an almost immediate recovery of fluorescence in the bleached ROI.
> This is happening also in the YFP only control.
>
> Can this possibly be because of the mounting medium? What else can be a
> mobile fraction on a fixed slide?
> The signal is good, has the expected expression pattern.
>
> Cheers,
> Avi
>
> --
> Avi Jacob, Ph.D.
> Head of The Kanbar Light Microscopy Unit
> Bar-Ilan University, Ramat-Gan 5290002, Israel
> http://tinyurl.com/BIU-Microscopy
>

--
------------------------------------------------------------
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

Kevin Dean

Re: Weird issue with FRET after acceptor PB

In reply to this post by Avi Jacob

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This is a common problem with fluorescent proteins. Reversible photobleaching, or dark-state conversion. You can find quite a few papers on the topic once you know the keywords to search for. One shameless self-reference: https://www.sciencedirect.com/science/article/pii/S0006349511007867

If you are working with fixed specimens, I would recommend just photobleaching for longer.

Best,
Kevin

On Jul 19, 2018, at 2:33 AM, Avi Jacob <[hidden email]<mailto:[hidden email]>> wrote:

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Hi all,

Got a weird issue I cannot explain.

We are doing acceptor photobleaching FRET with a CFP-YFP pair, on fixed
slides, on Leica widefield with scanner.
We are getting great initial bleaching of the YFP acceptor, however, there
is an almost immediate recovery of fluorescence in the bleached ROI.
This is happening also in the YFP only control.

Can this possibly be because of the mounting medium? What else can be a
mobile fraction on a fixed slide?
The signal is good, has the expected expression pattern.

Cheers,
Avi

--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit
Bar-Ilan University, Ramat-Gan 5290002, Israel
http://tinyurl.com/BIU-Microscopy

________________________________

UT Southwestern

Medical Center

The future of medicine, today.

George McNamara

Re: Weird issue with FRET after acceptor PB

In reply to this post by Avi Jacob

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Hi Avi,

FRET of FP pairs after fixation is a complete waste of time (don't
believe any literature that claims it is useful -- even if authored by
confocal listserv members). By extension, FRET between fluorescent
antibodies (Fab's or nanobodies would be smaller, so potentially bigger
dynamic range) of epitopes detected after fixation may be subject to the
same issue(s).

The simple test is compare your live vs fixed by acceptor photobleaching.

The best way to do FRET quantitation is FLIM on live cells (and again,
waste of time to do FLIM-FRET measurements of FP's on fixed cells).
Traditionally this has been donor FLIM only -- I suggest measuring donor
and acceptor FLIM simultaneously is better, and several companies now
offer 30+ million counts per second photon counting (see p.s.).

George

p.s. disclosure: I may be hosting Leica FALCON (Sept) and ISS FastFLIM
(October?) demonstrations/workshops here at JHU.

I'm also now (as of this email) thinking I should attend the "Advanced
TCSPC Techniques" workshop in Bethesda Oct 9-10, 2018, that Wolfgang
Becker recently posted about.

https://www.eventbrite.com/e/12th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-sciences-tickets-46820517428

TCSPC has historically been "painfully slow, small field of view" FLIM (
under 1 million photon counts per second ... which would be average ~1
photon per pixel per second if imaging 1000x1000 pixels, and more
usefully ~100 counts per pixel per second for 100x100 pixels)). I am
intrigued by FALCON, FastFLIM, and similar from Becker&Hickl and
probably others (PicoQuant) because a lot faster (>=30 million counts
per counting channel - different companies may have different ways to up
the count rate).

FYI - I am also intrigued by the potential of using "fast counting"
electronics for simple photon counting at ~30x faster than I can do now
on our current SP8 HyD's
http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscope
(I have not updated FALCON visit dates - yet). That is, not -- yet --
investing in pulsed lasers and simply 'count faster'.

On 7/19/2018 3:33 AM, Avi Jacob wrote:

> *****
> confocal microscopy listserv
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> Got a weird issue I cannot explain.
>
> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on fixed
> slides, on Leica widefield with scanner.
> We are getting great initial bleaching of the YFP acceptor, however, there
> is an almost immediate recovery of fluorescence in the bleached ROI.
> This is happening also in the YFP only control.
>
> Can this possibly be because of the mounting medium? What else can be a
> mobile fraction on a fixed slide?
> The signal is good, has the expected expression pattern.
>
> Cheers,
> Avi
>
> --
> Avi Jacob, Ph.D.
> Head of The Kanbar Light Microscopy Unit
> Bar-Ilan University, Ramat-Gan 5290002, Israel
> http://tinyurl.com/BIU-Microscopy

--

George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures

Martin Wessendorf-2

Re: Weird issue with FRET after acceptor PB

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*****

Hey, George!

It'd be helpful if you could offer some references or give your
reasoning for thinking that fixed-tissue FRET is a waste of time.

Thanks!

Martin Wessendorf

On 7/19/2018 8:52 AM, George McNamara 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 Avi,
>
> FRET of FP pairs after fixation is a complete waste of time (don't
> believe any literature that claims it is useful -- even if authored by
> confocal listserv members). By extension, FRET between fluorescent
> antibodies (Fab's or nanobodies would be smaller, so potentially
> bigger dynamic range) of epitopes detected after fixation may be
> subject to the same issue(s).
>
> The simple test is compare your live vs fixed by acceptor photobleaching.
>
> The best way to do FRET quantitation is FLIM on live cells (and again,
> waste of time to do FLIM-FRET measurements of FP's on fixed cells).
> Traditionally this has been donor FLIM only -- I suggest measuring
> donor and acceptor FLIM simultaneously is better, and several
> companies now offer 30+ million counts per second photon counting (see
> p.s.).
>
>
> George
>
> p.s. disclosure: I may be hosting Leica FALCON (Sept) and ISS FastFLIM
> (October?) demonstrations/workshops here at JHU.
>
> I'm also now (as of this email) thinking I should attend the "Advanced
> TCSPC Techniques" workshop in Bethesda Oct 9-10, 2018, that Wolfgang
> Becker recently posted about.
>
> https://www.eventbrite.com/e/12th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-sciences-tickets-46820517428
>
>
> TCSPC has historically been "painfully slow, small field of view" FLIM
> ( under 1 million photon counts per second ... which would be average
> ~1 photon per pixel per second if imaging 1000x1000 pixels, and more
> usefully ~100 counts per pixel per second for 100x100 pixels)). I am
> intrigued by FALCON, FastFLIM, and similar from Becker&Hickl and
> probably others (PicoQuant) because a lot faster (>=30 million counts
> per counting channel - different companies may have different ways to
> up the count rate).
>
> FYI - I am also intrigued by the potential of using "fast counting"
> electronics for simple photon counting at ~30x faster than I can do
> now on our current SP8 HyD's
> http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscope
> (I have not updated FALCON visit dates - yet). That is, not -- yet --
> investing in pulsed lasers and simply 'count faster'.
>
>
> On 7/19/2018 3:33 AM, Avi Jacob wrote:
>> *****
>> confocal microscopy listserv
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> Hi all,
>>
>> Got a weird issue I cannot explain.
>>
>> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on fixed
>> slides, on Leica widefield with scanner.
>> We are getting great initial bleaching of the YFP acceptor, however,
>> there
>> is an almost immediate recovery of fluorescence in the bleached ROI.
>> This is happening also in the YFP only control.
>>
>> Can this possibly be because of the mounting medium? What else can be a
>> mobile fraction on a fixed slide?
>> The signal is good, has the expected expression pattern.
>>
>> Cheers,
>> Avi
>>
>> --
>> Avi Jacob, Ph.D.
>> Head of The Kanbar Light Microscopy Unit
>> Bar-Ilan University, Ramat-Gan 5290002, Israel
>> http://tinyurl.com/BIU-Microscopy
>

--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

Periasamy, Ammasi (ap3t)-2

Re: Weird issue with FRET after acceptor PB

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*****

George,
I do not agree with you, it's waste of time to do FRET with fixed cells. As you know there are number of papers published on acceptor photobleaching not only in live cells but also in fixed cells.
For example, RNA-protein interaction there is no way you can work with live cells.
Rehman, S., Gladman, J.T., Periasamy, A., Sun, Y. and Mahadevan, M.S. (2014) Development of an AP-FRET based analysis for characterinzing RNA-protein interactions in myotonic dystrophy (DM1). PLoS ONE 9(4):e95957.

On FLIM-FRET, we have tested for fixed cells (not published), the lifetime changes only after a week or two of fixation. Yes, FLIM has to be used for live specimens. For some experiment you do not have a choice except to use fixed cells for FRET.
Hope this helps.
Ammasi

Dr. Ammasi Periasamy
Professor & Center Director,
WM Keck Center for Cellular Imaging,
Department of Biology, Univeristy of Virginia,
90 Geldard Dr., Charlottesville, VA 22904, USA.

http://www.kcci.virginia.edu/people/profile/ap3t
Phone: (434) 243-7602 or 982-4869
Fax: (434) 982-5210
E-mail: [hidden email]

FRET/FLIM Workshop-March 11-15, 2019: http://www.kcci.virginia.edu/workshop

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Martin Wessendorf
Sent: Thursday, July 19, 2018 10:57 AM
To: [hidden email]
Subject: Re: Weird issue with FRET after acceptor PB

*****
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.
*****

Hey, George!

It'd be helpful if you could offer some references or give your reasoning for thinking that fixed-tissue FRET is a waste of time.

Thanks!

Martin Wessendorf

On 7/19/2018 8:52 AM, George McNamara 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 Avi,
>
> FRET of FP pairs after fixation is a complete waste of time (don't
> believe any literature that claims it is useful -- even if authored by
> confocal listserv members). By extension, FRET between fluorescent
> antibodies (Fab's or nanobodies would be smaller, so potentially
> bigger dynamic range) of epitopes detected after fixation may be
> subject to the same issue(s).
>
> The simple test is compare your live vs fixed by acceptor photobleaching.
>
> The best way to do FRET quantitation is FLIM on live cells (and again,
> waste of time to do FLIM-FRET measurements of FP's on fixed cells).
> Traditionally this has been donor FLIM only -- I suggest measuring
> donor and acceptor FLIM simultaneously is better, and several
> companies now offer 30+ million counts per second photon counting (see
> p.s.).
>
>
> George
>
> p.s. disclosure: I may be hosting Leica FALCON (Sept) and ISS FastFLIM
> (October?) demonstrations/workshops here at JHU.
>
> I'm also now (as of this email) thinking I should attend the "Advanced
> TCSPC Techniques" workshop in Bethesda Oct 9-10, 2018, that Wolfgang
> Becker recently posted about.
>
> https://www.eventbrite.com/e/12th-annual-workshop-on-advanced-tcspc-te
> chniques-in-the-biomedical-sciences-tickets-46820517428
>
>
> TCSPC has historically been "painfully slow, small field of view" FLIM
> ( under 1 million photon counts per second ... which would be average
> ~1 photon per pixel per second if imaging 1000x1000 pixels, and more
> usefully ~100 counts per pixel per second for 100x100 pixels)). I am
> intrigued by FALCON, FastFLIM, and similar from Becker&Hickl and
> probably others (PicoQuant) because a lot faster (>=30 million counts
> per counting channel - different companies may have different ways to
> up the count rate).
>
> FYI - I am also intrigued by the potential of using "fast counting"
> electronics for simple photon counting at ~30x faster than I can do
> now on our current SP8 HyD's
> http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscop
> e (I have not updated FALCON visit dates - yet). That is, not -- yet
> -- investing in pulsed lasers and simply 'count faster'.
>
>
> On 7/19/2018 3:33 AM, Avi Jacob wrote:
>> *****
>> confocal microscopy listserv
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> Hi all,
>>
>> Got a weird issue I cannot explain.
>>
>> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on
>> fixed slides, on Leica widefield with scanner.
>> We are getting great initial bleaching of the YFP acceptor, however,
>> there is an almost immediate recovery of fluorescence in the bleached
>> ROI.
>> This is happening also in the YFP only control.
>>
>> Can this possibly be because of the mounting medium? What else can be
>> a mobile fraction on a fixed slide?
>> The signal is good, has the expected expression pattern.
>>
>> Cheers,
>> Avi
>>
>> --
>> Avi Jacob, Ph.D.
>> Head of The Kanbar Light Microscopy Unit Bar-Ilan University,
>> Ramat-Gan 5290002, Israel http://tinyurl.com/BIU-Microscopy
>

George McNamara

Re: Weird issue with FRET after acceptor PB

In reply to this post by Martin Wessendorf-2

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Martin,

thanks alot for the prompt.

I am pasting below just selected -- by me -- text from the abstracts. I
note that this (i) is not comprehensive, (ii) the texts are not in
agreement (nor are the methods the same, re: fixation, mounting media,
use or not of nail polish sealer).

I'll also 'double down' up here on my "FRET of FP pairs after fixation
is a complete waste of time" with: each cell should be used as its own
control, in a time series, under identical conditions (live!)
conditions, i.e. before or after stimulation. That is, every single
cell, even if it is from an expanded clone, or even if a small clone,
such as the 'HeLa tetrad' approach used in fig F2 of
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012539/figure/F2/ (which
was not a FRET paper, and I still like this approach to efficient
quantitation of cell fluorescence.

I am also going to point out up top here that for FP biosensors (the
original post does not mention whether their experiments were FP
biosensors, or simply proximity assay, or other) that single FP, i.e.
GCaMP7 dark --> bright on Ca++ binding, is the future AND I am
enthusiastic about the new dLight1 GPCR FP biosensor paper see
https://www.ncbi.nlm.nih.gov/pubmed/29853555 (even though they used
GCaMP6 instead of 7) because it implies every GPCR can be turned into a
dLight-like FP biosensor (dLight1 optimation in main text, 7
un-optimized in supplemental) (note: I am not connected to the dLight work).

OK, back to "fixed cell FRET is a waste of time" light survey:

Reminder: I am selectively quoting from the abstracts. If anyone wants
more, read the full abstract and papers.

https://www.ncbi.nlm.nih.gov/pubmed/25179491

Effect of*fixation*procedures on the fluorescence lifetimes of Aequorea
victoria derived fluorescent proteins.
<https://www.ncbi.nlm.nih.gov/pubmed/25179491>

Joosen L, Hink MA, Gadella TW Jr, Goedhart J.

J Microsc. 2014 Dec;256(3):166-76. doi: 10.1111/jmi.12168. Epub 2014 Sep 1.

PMID:
25179491

However, the effect offixation methods and mounting media on
fluorescence lifetime is poorly documented so far. Here, we demonstrate
thatfixationby formaldehyde or methanol itself does not affect the
lifetime of fluorescent proteins produced in cells but that several
widely used mounting media decrease the fluorescence lifetime by up to
20%. It is shown that fixed cells producing Aequorea victoria derived
fluorescent proteins mounted in Tris buffer have fluorescence lifetimes
indistinguishable from values measured in living cells. Tris buffer also
allows accurate Förster Resonance Energy Transfer quantification in
fixed cells, as shown with an mTurquoise2-SYFP2 fusion protein.
Moreover, identical lifetime contrasts are measured in living and fixed
cells mounted in Tris buffer after introducing a single plasmid
expressing two lifetime variants of cyan fluorescent proteins, each
targeted to different locations in the cell.

https://www.ncbi.nlm.nih.gov/pubmed/?term=fret+fixation+artifacts

(3 of 4 hits summarized below)

https://www.ncbi.nlm.nih.gov/pubmed/21490932

Some secrets of fluorescent proteins: distinct bleaching in various
mounting fluids and photoactivation of cyan fluorescent proteins at
YFP-excitation. <https://www.ncbi.nlm.nih.gov/pubmed/21490932>

Malkani N, Schmid JA.

PLoS One. 2011 Apr 7;6(4):e18586. doi: 10.1371/journal.pone.0018586.

PMID:
21490932

When we applied a commonly usedFRETmicroscopy technique--the increase in
donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP),
we obtained good signals in live cells, but very weak signals for the
same samples afterfixationand mounting in commercial microscopy mounting
fluids. This observation could be traced back to much faster bleaching
of CFP in these mounting media. Strikingly, the opposite effect of the
mounting fluid was observed for YFP and also for other proteins such as
Cerulean, TFP or Venus. The changes in photostability of CFP and YFP
were not caused by thefixationbut directly dependent on the mounting
fluid. Furthermore we made the interesting observation that the
CFP-fluorescence intensity increases by about 10-15% after illumination
at the YFP-excitation wavelength--a phenomenon, which was also observed
for Cerulean. This photoactivation of cyan fluorescent proteins at the
YFP-excitation can cause false-positive signals in theFRET-microscopy
technique that is based on bleaching of a yellowFRETacceptor.

https://www.ncbi.nlm.nih.gov/pubmed/18359791

Resonance energy transfer in cells: a new look at*fixation*effect and
receptor aggregation on cell membrane.
<https://www.ncbi.nlm.nih.gov/pubmed/18359791>

Anikovsky M, Dale L, Ferguson S, Petersen N.

Biophys J. 2008 Aug;95(3):1349-59. doi: 10.1529/biophysj.107.124313.
Epub 2008 Mar 21.

PMID:
18359791

We have considered systems in whichFREToccurs as intramolecular and/or
intermolecular process. The proposed dynamicFRETmodel shows that in the
case of intermolecular process the degree of aggregation only slightly
affects the energy transfer efficiency. ...This dual dependence often
leads to ambiguity. In this article, we show howFRETefficiency can be
significantly reduced even in highly coupled system through
conformational restrictions in the donor-acceptor pair. Importantly,
such restrictions can be imposed on the system by cellfixation, a
procedure routinely used when conductingFRETmeasurements.

https://www.ncbi.nlm.nih.gov/pubmed/18453757

*Fixation*, mounting and sealing with nail polish of cell specimens lead
to incorrect*FRET*measurements using acceptor photobleaching.
<https://www.ncbi.nlm.nih.gov/pubmed/18453757>

Rodighiero S, Bazzini C, Ritter M, Fürst J, Botta G, Meyer G, Paulmichl M.

Cell Physiol Biochem. 2008;21(5-6):489-98. doi: 10.1159/000129642. Epub
2008 Apr 24.

PMID:
18453757

... the calculation of theFRETefficiency by de-quenching the donor
cyan-fluorescent-protein (CFP) emission due to acceptor-photobleaching
leads to erroneous estimate of theFRETefficiency in fixed, mounted and
sealed specimens. The acceptor photobleaching leads to a modification of
the donor cyan-fluorescent-protein, which shows then a strong emission,
thus mimicking functional interaction between CFP (donor) and
yellow-fluorescent-protein (YFP; acceptor). Moreover, the procedure of
acceptor photobleaching masks physiological (non random) interaction
between molecules within the fixed, mounted and sealed cell.

//

another weakness of fixed cell FRET is that it is not readily
'translatable' to either mouse (or other model systems) in vivo imaging,
and especially not to human clinical fluorescence in vivo.

enjoy,

George

p.s. above is all fluorescent proteins. Funky things can happen to small
molecules. As one example, Cy3 can readily undergo (at times a
researcher may not be thinking about ... lifetime could be used to
measure) "Protein-inducedfluorescenceenhancement(PIFE)" -- see
https://www.ncbi.nlm.nih.gov/pubmed/26263254 for an entry point to PIFE
(the abstract does not make it clear that "cis-Cy3" is dark, "trans-Cy3"
is the fluorescent form ... stated in body of that text).

p.p.s. not that I'm recommending it (since the whole point of my posts
to is to avoid doing "fixation FRET"), the "best" approach might be to
do on the microscope (carefully): simply aspirate the medium, add
appropriate dilution fixative, fix [optimum or at least consistent
time?], wash, stick in a simple buffer or optiimum (?) pH (Joosen et al
liked Tris, but abstract did not disclose pH) and measure. Maybe:
thoroughly aerate the imaging medium (in some consistent way: good luck)
to maximize O2. A better use of scope time: image cells live.

On 7/19/2018 10:56 AM, Martin Wessendorf 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.
> *****
>
> Hey, George!
>
> It'd be helpful if you could offer some references or give your
> reasoning for thinking that fixed-tissue FRET is a waste of time.
>
> Thanks!
>
> Martin Wessendorf
>
>
>
>
> On 7/19/2018 8:52 AM, George McNamara 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 Avi,
>>
>> FRET of FP pairs after fixation is a complete waste of time (don't
>> believe any literature that claims it is useful -- even if authored
>> by confocal listserv members). By extension, FRET between fluorescent
>> antibodies (Fab's or nanobodies would be smaller, so potentially
>> bigger dynamic range) of epitopes detected after fixation may be
>> subject to the same issue(s).
>>
>> The simple test is compare your live vs fixed by acceptor
>> photobleaching.
>>
>> The best way to do FRET quantitation is FLIM on live cells (and
>> again, waste of time to do FLIM-FRET measurements of FP's on fixed
>> cells). Traditionally this has been donor FLIM only -- I suggest
>> measuring donor and acceptor FLIM simultaneously is better, and
>> several companies now offer 30+ million counts per second photon
>> counting (see p.s.).
>>
>>
>> George
>>
>> p.s. disclosure: I may be hosting Leica FALCON (Sept) and ISS
>> FastFLIM (October?) demonstrations/workshops here at JHU.
>>
>> I'm also now (as of this email) thinking I should attend the
>> "Advanced TCSPC Techniques" workshop in Bethesda Oct 9-10, 2018, that
>> Wolfgang Becker recently posted about.
>>
>> https://www.eventbrite.com/e/12th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-sciences-tickets-46820517428
>>
>>
>> TCSPC has historically been "painfully slow, small field of view"
>> FLIM ( under 1 million photon counts per second ... which would be
>> average ~1 photon per pixel per second if imaging 1000x1000 pixels,
>> and more usefully ~100 counts per pixel per second for 100x100
>> pixels)). I am intrigued by FALCON, FastFLIM, and similar from
>> Becker&Hickl and probably others (PicoQuant) because a lot faster
>> (>=30 million counts per counting channel - different companies may
>> have different ways to up the count rate).
>>
>> FYI - I am also intrigued by the potential of using "fast counting"
>> electronics for simple photon counting at ~30x faster than I can do
>> now on our current SP8 HyD's
>> http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscope
>> (I have not updated FALCON visit dates - yet). That is, not -- yet --
>> investing in pulsed lasers and simply 'count faster'.
>>
>>
>> On 7/19/2018 3:33 AM, Avi Jacob wrote:
>>> *****
>>> confocal microscopy listserv
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hi all,
>>>
>>> Got a weird issue I cannot explain.
>>>
>>> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on fixed
>>> slides, on Leica widefield with scanner.
>>> We are getting great initial bleaching of the YFP acceptor, however,
>>> there
>>> is an almost immediate recovery of fluorescence in the bleached ROI.
>>> This is happening also in the YFP only control.
>>>
>>> Can this possibly be because of the mounting medium? What else can be a
>>> mobile fraction on a fixed slide?
>>> The signal is good, has the expected expression pattern.
>>>
>>> Cheers,
>>> Avi
>>>
>>> --
>>> Avi Jacob, Ph.D.
>>> Head of The Kanbar Light Microscopy Unit
>>> Bar-Ilan University, Ramat-Gan 5290002, Israel
>>> http://tinyurl.com/BIU-Microscopy
>>
>

George McNamara

Re: Weird issue with FRET after acceptor PB

In reply to this post by Periasamy, Ammasi (ap3t)-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 Peri,

I am happy to discuss the merits of fixed cell FRET imaging at a
bar/restaurant at M&M 2018, Aug 5-9, Baltimore,
https://www.microscopy.org/mandm/2018

and, if I attend, the Bethesda Advanced TCSPC meeting in Bethesda in
October.

As for characterizing RNA-protein interactions, maybe FRET is not the
optimal method. At least for genetically engineered systems (which
practically all FP fusion protein experiments are), a different design
may be better, for MS2/PP7 system(s), example:

https://www.ncbi.nlm.nih.gov/pubmed/24402470

Background free imaging of single mRNAs in live cells using split
fluorescent proteins. <https://www.ncbi.nlm.nih.gov/pubmed/24402470>

Wu B, Chen J,*Singer*RH.

Sci Rep. 2014 Jan 9;4:3615. doi: 10.1038/srep03615.

PMID:
24402470

See also:

https://www.ncbi.nlm.nih.gov/pubmed/29345990

Imaging mRNA In Vivo, from Birth to Death.
<https://www.ncbi.nlm.nih.gov/pubmed/29345990>

Tutucci E, Livingston NM,*Singer RH*,*Wu B*.

Annu Rev Biophys. 2018 May 20;47:85-106. doi:
10.1146/annurev-biophys-070317-033037. Epub 2018 Jan 18.

PMID:
29345990

https://www.ncbi.nlm.nih.gov/pubmed/27313041

Translation dynamics of single mRNAs in live cells and neurons.
<https://www.ncbi.nlm.nih.gov/pubmed/27313041>

*Wu B*, Eliscovich C, Yoon YJ,*Singer RH*.

Science. 2016 Jun 17;352(6292):1430-5. doi: 10.1126/science.aaf1084.
Epub 2016 May 5.

PMID:
27313041

Disclosure: Prof. Bin Wu is now my supervisor at JHU (I am writing this
on my own time).

enjoy,

George

On 7/19/2018 11:43 AM, Periasamy, Ammasi (ap3t) 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.
> *****
>
> George,
> I do not agree with you, it's waste of time to do FRET with fixed cells. As you know there are number of papers published on acceptor photobleaching not only in live cells but also in fixed cells.
> For example, RNA-protein interaction there is no way you can work with live cells.
> Rehman, S., Gladman, J.T., Periasamy, A., Sun, Y. and Mahadevan, M.S. (2014) Development of an AP-FRET based analysis for characterinzing RNA-protein interactions in myotonic dystrophy (DM1). PLoS ONE 9(4):e95957.
>
> On FLIM-FRET, we have tested for fixed cells (not published), the lifetime changes only after a week or two of fixation. Yes, FLIM has to be used for live specimens. For some experiment you do not have a choice except to use fixed cells for FRET.
> Hope this helps.
> Ammasi
>
> Dr. Ammasi Periasamy
> Professor & Center Director,
> WM Keck Center for Cellular Imaging,
> Department of Biology, Univeristy of Virginia,
> 90 Geldard Dr., Charlottesville, VA 22904, USA.
>
> http://www.kcci.virginia.edu/people/profile/ap3t
> Phone: (434) 243-7602 or 982-4869
> Fax: (434) 982-5210
> E-mail: [hidden email]
>
> FRET/FLIM Workshop-March 11-15, 2019: http://www.kcci.virginia.edu/workshop
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Martin Wessendorf
> Sent: Thursday, July 19, 2018 10:57 AM
> To: [hidden email]
> Subject: Re: Weird issue with FRET after acceptor PB
>
> *****
> 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.
> *****
>
> Hey, George!
>
> It'd be helpful if you could offer some references or give your reasoning for thinking that fixed-tissue FRET is a waste of time.
>
> Thanks!
>
> Martin Wessendorf
>
>
>
>
> On 7/19/2018 8:52 AM, George McNamara 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 Avi,
>>
>> FRET of FP pairs after fixation is a complete waste of time (don't
>> believe any literature that claims it is useful -- even if authored by
>> confocal listserv members). By extension, FRET between fluorescent
>> antibodies (Fab's or nanobodies would be smaller, so potentially
>> bigger dynamic range) of epitopes detected after fixation may be
>> subject to the same issue(s).
>>
>> The simple test is compare your live vs fixed by acceptor photobleaching.
>>
>> The best way to do FRET quantitation is FLIM on live cells (and again,
>> waste of time to do FLIM-FRET measurements of FP's on fixed cells).
>> Traditionally this has been donor FLIM only -- I suggest measuring
>> donor and acceptor FLIM simultaneously is better, and several
>> companies now offer 30+ million counts per second photon counting (see
>> p.s.).
>>
>>
>> George
>>
>> p.s. disclosure: I may be hosting Leica FALCON (Sept) and ISS FastFLIM
>> (October?) demonstrations/workshops here at JHU.
>>
>> I'm also now (as of this email) thinking I should attend the "Advanced
>> TCSPC Techniques" workshop in Bethesda Oct 9-10, 2018, that Wolfgang
>> Becker recently posted about.
>>
>> https://www.eventbrite.com/e/12th-annual-workshop-on-advanced-tcspc-te
>> chniques-in-the-biomedical-sciences-tickets-46820517428
>>
>>
>> TCSPC has historically been "painfully slow, small field of view" FLIM
>> ( under 1 million photon counts per second ... which would be average
>> ~1 photon per pixel per second if imaging 1000x1000 pixels, and more
>> usefully ~100 counts per pixel per second for 100x100 pixels)). I am
>> intrigued by FALCON, FastFLIM, and similar from Becker&Hickl and
>> probably others (PicoQuant) because a lot faster (>=30 million counts
>> per counting channel - different companies may have different ways to
>> up the count rate).
>>
>> FYI - I am also intrigued by the potential of using "fast counting"
>> electronics for simple photon counting at ~30x faster than I can do
>> now on our current SP8 HyD's
>> http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscop
>> e (I have not updated FALCON visit dates - yet). That is, not -- yet
>> -- investing in pulsed lasers and simply 'count faster'.
>>
>>
>> On 7/19/2018 3:33 AM, Avi Jacob wrote:
>>> *****
>>> confocal microscopy listserv
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hi all,
>>>
>>> Got a weird issue I cannot explain.
>>>
>>> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on
>>> fixed slides, on Leica widefield with scanner.
>>> We are getting great initial bleaching of the YFP acceptor, however,
>>> there is an almost immediate recovery of fluorescence in the bleached
>>> ROI.
>>> This is happening also in the YFP only control.
>>>
>>> Can this possibly be because of the mounting medium? What else can be
>>> a mobile fraction on a fixed slide?
>>> The signal is good, has the expected expression pattern.
>>>
>>> Cheers,
>>> Avi
>>>
>>> --
>>> Avi Jacob, Ph.D.
>>> Head of The Kanbar Light Microscopy Unit Bar-Ilan University,
>>> Ramat-Gan 5290002, Israel http://tinyurl.com/BIU-Microscopy

Nicolai.Urban@mpfi.org

Re: Weird issue with FRET after acceptor PB

*****
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 Avi,

getting back to your initial question (fluorescence recovery after photobleaching), there might be an explanation beyond having a mobile fraction of dye in your fixated sample. Have you considered that instead of actually (irreversibly) photobleaching the YFP, you are actually pumping it into a long-lived dark state, from which it can recover, given time? YFP has been shown to exhibit these dark-states (added some links below), which expresses itself as blinking or as recovery of fluorescence over time/after near-UV irradiation.

It is fairly common for numerous FPs, and could explain the recovery despite everything being fixated and immobile. You could test it by either trying to induce recovery with a 405 nm laser (or similar), or possibly trying to bleach using much harsher illumination.

Best of luck with your experiments,!

Nicolai Urban

>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Dr. Nicolai T. Urban
Max Planck Florida Institute
Jupiter, 33458 FL, USA

>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Links concerning YFP dark-states:
Ha & Tinnefeld; Photophysics of Fluorescence Probes for Single Molecule Biophysics and Super-Resolution Imaging; Annu Rev Phys Chem. 2012; 63: 595–617.; 10.1146/annurev-physchem-032210-103340
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736144/

Dickson RM, Cubitt AB, Tsien RY, Moerner WE. On/off blinking and switching behavior of single molecules of green fluorescent protein. Nature. 1997;388:355–358.

Biteen JS, Thompson MA, Tselentis NK, Bowman GR, Shapiro L, Moerner WE. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat Methods. 2008;5:947–949.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655310/
>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of George McNamara
Sent: Donnerstag, 19. Juli 2018 12:32
To: [hidden email]
Subject: Re: Weird issue with FRET after acceptor PB

*****
To join, leave or search the confocal microscopy listserv, go to:
https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293404512&sdata=NBx0ticPz9cCs68Tplgc6misH05lf%2Bqupn7oHAWZ%2Bok%3D&reserved=0
Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293404512&sdata=DXc%2B7aVGZu%2F3pSrpyW8ACo71J87My%2F9f0vEy7EThlWI%3D&reserved=0 and include the link in your posting.
*****

Hi Peri,

I am happy to discuss the merits of fixed cell FRET imaging at a bar/restaurant at M&M 2018, Aug 5-9, Baltimore,
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.microscopy.org%2Fmandm%2F2018&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293404512&sdata=cjJEItNF6ukYY%2BtvtX0hjEEi1OdW6DURH5U6XOW43rg%3D&reserved=0

and, if I attend, the Bethesda Advanced TCSPC meeting in Bethesda in October.

As for characterizing RNA-protein interactions, maybe FRET is not the optimal method. At least for genetically engineered systems (which practically all FP fusion protein experiments are), a different design may be better, for MS2/PP7 system(s), example:

https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F24402470&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293404512&sdata=%2BLMIBh1CGkrW3eTff31ls3H4c%2B768H%2BuwmmgVzOYcrU%3D&reserved=0

Background free imaging of single mRNAs in live cells using split fluorescent proteins. <https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F24402470&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=IxJNdL9WCblXiyJI5H%2FhyZ%2FGjf9GHeBOxohMEhJyimE%3D&reserved=0>

Wu B, Chen J,*Singer*RH.

Sci Rep. 2014 Jan 9;4:3615. doi: 10.1038/srep03615.

PMID:
24402470

See also:

https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29345990&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=xXBHnrtX6eeRtyhETHL6%2BjjxZCdmYp7HEory%2FRVs4n8%3D&reserved=0

Imaging mRNA In Vivo, from Birth to Death.
<https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F29345990&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=xXBHnrtX6eeRtyhETHL6%2BjjxZCdmYp7HEory%2FRVs4n8%3D&reserved=0>

Tutucci E, Livingston NM,*Singer RH*,*Wu B*.

Annu Rev Biophys. 2018 May 20;47:85-106. doi:
10.1146/annurev-biophys-070317-033037. Epub 2018 Jan 18.

PMID:
29345990

https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F27313041&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=WltnGFClLoTg0Mwm0wbWCyFQUOyCx27fw0awOdoXSuQ%3D&reserved=0

Translation dynamics of single mRNAs in live cells and neurons.
<https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F27313041&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=WltnGFClLoTg0Mwm0wbWCyFQUOyCx27fw0awOdoXSuQ%3D&reserved=0>

*Wu B*, Eliscovich C, Yoon YJ,*Singer RH*.

Science. 2016 Jun 17;352(6292):1430-5. doi: 10.1126/science.aaf1084.
Epub 2016 May 5.

PMID:
27313041

Disclosure: Prof. Bin Wu is now my supervisor at JHU (I am writing this on my own time).

enjoy,

George

On 7/19/2018 11:43 AM, Periasamy, Ammasi (ap3t) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.
> umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNi
> colai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517d
> b44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=EQ1QsQWu
> NihXfIP3IoIVS4oVgFb8z9rqmJJiQLEyjCA%3D&reserved=0
> Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=eNVq8k41GUXSOPpTozqymSXH7GaeX6nzd0%2FBMWYtptY%3D&reserved=0 and include the link in your posting.
> *****
>
> George,
> I do not agree with you, it's waste of time to do FRET with fixed cells. As you know there are number of papers published on acceptor photobleaching not only in live cells but also in fixed cells.
> For example, RNA-protein interaction there is no way you can work with live cells.
> Rehman, S., Gladman, J.T., Periasamy, A., Sun, Y. and Mahadevan, M.S. (2014) Development of an AP-FRET based analysis for characterinzing RNA-protein interactions in myotonic dystrophy (DM1). PLoS ONE 9(4):e95957.
>
> On FLIM-FRET, we have tested for fixed cells (not published), the lifetime changes only after a week or two of fixation. Yes, FLIM has to be used for live specimens. For some experiment you do not have a choice except to use fixed cells for FRET.
> Hope this helps.
> Ammasi
>
> Dr. Ammasi Periasamy
> Professor & Center Director,
> WM Keck Center for Cellular Imaging,
> Department of Biology, Univeristy of Virginia,
> 90 Geldard Dr., Charlottesville, VA 22904, USA.
>
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.kc
> ci.virginia.edu%2Fpeople%2Fprofile%2Fap3t&data=02%7C01%7CNicolai.U
> rban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a
> 5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=Neg%2Fk4hxDpHfg
> ZqjXF%2BHxOK%2Fu1Nbqy5rWnf%2FFqTvM5E%3D&reserved=0
> Phone: (434) 243-7602 or 982-4869
> Fax: (434) 982-5210
> E-mail: [hidden email]
>
> FRET/FLIM Workshop-March 11-15, 2019:
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.kc
> ci.virginia.edu%2Fworkshop&data=02%7C01%7CNicolai.Urban%40MPFI.ORG
> %7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0
> %7C0%7C1%7C636676147293414520&sdata=YsGpRRJy3QLTpGltlcgvKla7I1m4T6
> 3sw53MD3kDZ3w%3D&reserved=0
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Martin Wessendorf
> Sent: Thursday, July 19, 2018 10:57 AM
> To: [hidden email]
> Subject: Re: Weird issue with FRET after acceptor PB
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.
> umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNi
> colai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517d
> b44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=EQ1QsQWu
> NihXfIP3IoIVS4oVgFb8z9rqmJJiQLEyjCA%3D&reserved=0
> Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=eNVq8k41GUXSOPpTozqymSXH7GaeX6nzd0%2FBMWYtptY%3D&reserved=0 and include the link in your posting.
> *****
>
> Hey, George!
>
> It'd be helpful if you could offer some references or give your reasoning for thinking that fixed-tissue FRET is a waste of time.
>
> Thanks!
>
> Martin Wessendorf
>
>
>
>
> On 7/19/2018 8:52 AM, George McNamara wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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>> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7C
>> Nicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b455
>> 17db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293414520&sdata=EQ1Q
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>> *****
>>
>> Hi Avi,
>>
>> FRET of FP pairs after fixation is a complete waste of time (don't
>> believe any literature that claims it is useful -- even if authored
>> by confocal listserv members). By extension, FRET between fluorescent
>> antibodies (Fab's or nanobodies would be smaller, so potentially
>> bigger dynamic range) of epitopes detected after fixation may be
>> subject to the same issue(s).
>>
>> The simple test is compare your live vs fixed by acceptor photobleaching.
>>
>> The best way to do FRET quantitation is FLIM on live cells (and
>> again, waste of time to do FLIM-FRET measurements of FP's on fixed cells).
>> Traditionally this has been donor FLIM only -- I suggest measuring
>> donor and acceptor FLIM simultaneously is better, and several
>> companies now offer 30+ million counts per second photon counting
>> (see p.s.).
>>
>>
>> George
>>
>> p.s. disclosure: I may be hosting Leica FALCON (Sept) and ISS
>> FastFLIM
>> (October?) demonstrations/workshops here at JHU.
>>
>> I'm also now (as of this email) thinking I should attend the
>> "Advanced TCSPC Techniques" workshop in Bethesda Oct 9-10, 2018, that
>> Wolfgang Becker recently posted about.
>>
>> https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
>> eventbrite.com%2Fe%2F12th-annual-workshop-on-advanced-tcspc-te&da
>> ta=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952
>> ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293424528&a
>> mp;sdata=ttRqqObOWCjK5ggEHEibEXUK63WXhZS%2B7ojmWqjpmpA%3D&reserve
>> d=0
>> chniques-in-the-biomedical-sciences-tickets-46820517428
>>
>>
>> TCSPC has historically been "painfully slow, small field of view"
>> FLIM ( under 1 million photon counts per second ... which would be
>> average
>> ~1 photon per pixel per second if imaging 1000x1000 pixels, and more
>> usefully ~100 counts per pixel per second for 100x100 pixels)). I am
>> intrigued by FALCON, FastFLIM, and similar from Becker&Hickl and
>> probably others (PicoQuant) because a lot faster (>=30 million counts
>> per counting channel - different companies may have different ways to
>> up the count rate).
>>
>> FYI - I am also intrigued by the potential of using "fast counting"
>> electronics for simple photon counting at ~30x faster than I can do
>> now on our current SP8 HyD's
>> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fconfo
>> cal.jhu.edu%2Fcurrent-equipment%2Fleica-sp8-confocal-microscop&da
>> ta=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952
>> ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293424528&a
>> mp;sdata=s6dXLzMAQYm%2FHuYEM2LupsU5g1tREIdsGmQBz%2FONKQw%3D&reser
>> ved=0 e (I have not updated FALCON visit dates - yet). That is, not
>> -- yet
>> -- investing in pulsed lasers and simply 'count faster'.
>>
>>
>> On 7/19/2018 3:33 AM, Avi Jacob wrote:
>>> *****
>>> confocal microscopy listserv
>>> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist
>>> s.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%
>>> 7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b
>>> 45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293424528&sdata=
>>> zDhjMDioiI5hlV2A0rqfQ0ni8F63lf0%2F2MMhQjSxD10%3D&reserved=0
>>> Post images on
>>> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C1%7C636676147293424528&sdata=qmdVJYi0B5JBc1iFbka7UJxbdzn697HSLSCSFJgxl7A%3D&reserved=0 and include the link in your posting.
>>> *****
>>>
>>> Hi all,
>>>
>>> Got a weird issue I cannot explain.
>>>
>>> We are doing acceptor photobleaching FRET with a CFP-YFP pair, on
>>> fixed slides, on Leica widefield with scanner.
>>> We are getting great initial bleaching of the YFP acceptor, however,
>>> there is an almost immediate recovery of fluorescence in the
>>> bleached ROI.
>>> This is happening also in the YFP only control.
>>>
>>> Can this possibly be because of the mounting medium? What else can
>>> be a mobile fraction on a fixed slide?
>>> The signal is good, has the expected expression pattern.
>>>
>>> Cheers,
>>> Avi
>>>
>>> --
>>> Avi Jacob, Ph.D.
>>> Head of The Kanbar Light Microscopy Unit Bar-Ilan University,
>>> Ramat-Gan 5290002, Israel
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>>> url.com%2FBIU-Microscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG
>>> %7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603e
>>> d0%7C0%7C0%7C636676147293424528&sdata=EjB1kDzvrUq7thw7T9KGaVnlve
>>> o240xM2HUAi73pp48%3D&reserved=0

--

George McNamara, PhD
Baltimore, MD 21231
[hidden email]
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July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fonlinelibrary.wiley.com%2Fdoi%2F10.1002%2Fcphg.42%2Fabstract&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C90c7108baa08405e4cc308d5ed952ab4%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636676147293424528&sdata=oFy8s8J0cOI0%2FeTWMzAgRwas%2FOW4bfqd2sv1Im64mmw%3D&reserved=0
supporting materials direct link is
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figures at
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