Confocal Microscopy List

fixable pH sensor

Classic

List

Threaded

8 messages Options

Sylvie Le Guyader

fixable pH sensor

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello everyone

Would anyone know of a fixable pH sensor for lysosomes that is excited in the visible range and not in the UV? We only know of the Lysosensor Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want to use.

thanks

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 8 524 811 72
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>

mmodel

Re: fixable pH sensor

*****
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.
*****

Sylvie,

Do you want to measure pH in chemically fixed cells? (Sorry, this is not an answer to your question but a counter-question)

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Wednesday, January 24, 2018 7:25 AM
To: [hidden email]
Subject: fixable pH sensor

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello everyone

Would anyone know of a fixable pH sensor for lysosomes that is excited in the visible range and not in the UV? We only know of the Lysosensor Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want to use.

thanks

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 8 524 811 72
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>

Sylvie Le Guyader

Re: fixable pH sensor

*****
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 Mike

Yes we want to measure the Lysosome pH in cells infected with a BSL3 virus but we image them in a non BSL3 imaging facility so the cells must be fixed before imaging.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 8 524 811 72
LCI website

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: den 24 januari 2018 15:46
To: [hidden email]
Subject: Re: fixable pH sensor

*****
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.
*****

Sylvie,

Do you want to measure pH in chemically fixed cells? (Sorry, this is not an answer to your question but a counter-question)

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Wednesday, January 24, 2018 7:25 AM
To: [hidden email]
Subject: fixable pH sensor

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello everyone

Would anyone know of a fixable pH sensor for lysosomes that is excited in the visible range and not in the UV? We only know of the Lysosensor Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want to use.

thanks

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 8 524 811 72
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>

Nuno Moreno

Re: fixable pH sensor

*****
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

I see no point on measuring pH with fixed cell. Upon fixation you should have an immediate dissipation of any acidic compartment, specially with protons that move very fast. Even you can measure something I would doubt about the quality of those results. It is hard enough without fixation :)

...Buy a microscope for the BLS3

regards
Nuno Moreno

> On 24 Jan 2018, at 15:49, Sylvie Le Guyader <[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 Mike
>
> Yes we want to measure the Lysosome pH in cells infected with a BSL3 virus but we image them in a non BSL3 imaging facility so the cells must be fixed before imaging.
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: +46 (0) 73 733 5008
> office: +46 (0) 8 524 811 72
> LCI website
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
> Sent: den 24 januari 2018 15:46
> To: [hidden email]
> Subject: Re: fixable pH sensor
>
> *****
> 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.
> *****
>
> Sylvie,
>
> Do you want to measure pH in chemically fixed cells? (Sorry, this is not an answer to your question but a counter-question)
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
> Sent: Wednesday, January 24, 2018 7:25 AM
> To: [hidden email]
> Subject: fixable pH sensor
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello everyone
>
> Would anyone know of a fixable pH sensor for lysosomes that is excited in the visible range and not in the UV? We only know of the Lysosensor Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want to use.
>
> thanks
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: +46 (0) 73 733 5008
> office: +46 (0) 8 524 811 72
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>

Douglas Richardson

Re: fixable pH sensor

*****
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 Sylvie,

I just had this discussion with a user yesterday as well. All of the pH
sensor that I know work based on a reversible protonation reaction that
shifts the fluorophore's fluorescent properties. Because this reaction is
reversible, as soon as you perforate the membrane during fixation, the pH
probe is going to read out the pH of whatever buffer/mounting media the
cells are sitting in.

If anyone knows of an irreversible pH sensor, I'd be interested to hear
about it!

-Doug

On Wed, Jan 24, 2018 at 11:04 AM, Nuno Moreno <[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
>
> I see no point on measuring pH with fixed cell. Upon fixation you should
> have an immediate dissipation of any acidic compartment, specially with
> protons that move very fast. Even you can measure something I would doubt
> about the quality of those results. It is hard enough without fixation :)
>
> ...Buy a microscope for the BLS3
>
> regards
> Nuno Moreno
>
> > On 24 Jan 2018, at 15:49, Sylvie Le Guyader <[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 Mike
> >
> > Yes we want to measure the Lysosome pH in cells infected with a BSL3
> virus but we image them in a non BSL3 imaging facility so the cells must be
> fixed before imaging.
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> > Sylvie Le Guyader, PhD
> > Live Cell Imaging Facility Manager
> > Karolinska Institutet- Bionut Dpt
> > Hälsovägen 7,
> > Novum, G lift, floor 6
> > 14157 Huddinge
> > Sweden
> > mobile: +46 (0) 73 733 5008
> > office: +46 (0) 8 524 811 72
> > LCI website
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of MODEL, MICHAEL
> > Sent: den 24 januari 2018 15:46
> > To: [hidden email]
> > Subject: Re: fixable pH sensor
> >
> > *****
> > 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.
> > *****
> >
> > Sylvie,
> >
> > Do you want to measure pH in chemically fixed cells? (Sorry, this is not
> an answer to your question but a counter-question)
> >
> > Mike
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Sylvie Le Guyader
> > Sent: Wednesday, January 24, 2018 7:25 AM
> > To: [hidden email]
> > Subject: fixable pH sensor
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hello everyone
> >
> > Would anyone know of a fixable pH sensor for lysosomes that is excited
> in the visible range and not in the UV? We only know of the Lysosensor
> Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want
> to use.
> >
> > thanks
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> > Sylvie Le Guyader, PhD
> > Live Cell Imaging Facility Manager
> > Karolinska Institutet- Bionut Dpt
> > Hälsovägen 7,
> > Novum, G lift, floor 6
> > 14157 Huddinge
> > Sweden
> > mobile: +46 (0) 73 733 5008
> > office: +46 (0) 8 524 811 72
> > LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>
>

Dalton Leprich

Re: fixable pH sensor

*****
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 Sylvie,

Look into C-SNAFL and C-SNARF. They are ratiometric pH indicators, used
both extra- and intracellularly. I also believe you can use them single
excitation-dual emission, or dual excitation-single emission.

These are two good papers to reference (I can't attach the PDF):

Fluorescence spectroscopy of pH in vivo using a dual-emission fluorophore
(C-SNAFL-1) S. Mordon

Fluorescent Indicators for Intracellular pH Junyan Han and Kevin Burgess

Good luck,

Dalton Leprich

On Wed, Jan 24, 2018 at 12:54 PM, Dalton Leprich <[hidden email]> wrote:

> Hi Sylvie,
>
> Look into C-SNAFL and C-SNARF. They are ratiometric pH indicators, used
> both extra- and intracellularly. I also believe you can use them single
> excitation-dual emission, or dual excitation-single emission.
>
> Ive attached a couple of papers.
>
> Dalton
>
> On Wed, Jan 24, 2018 at 10:43 AM, Douglas Richardson <
> [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 Sylvie,
>>
>> I just had this discussion with a user yesterday as well. All of the pH
>> sensor that I know work based on a reversible protonation reaction that
>> shifts the fluorophore's fluorescent properties. Because this reaction is
>> reversible, as soon as you perforate the membrane during fixation, the pH
>> probe is going to read out the pH of whatever buffer/mounting media the
>> cells are sitting in.
>>
>> If anyone knows of an irreversible pH sensor, I'd be interested to hear
>> about it!
>>
>> -Doug
>>
>> On Wed, Jan 24, 2018 at 11:04 AM, Nuno Moreno <[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
>> >
>> > I see no point on measuring pH with fixed cell. Upon fixation you should
>> > have an immediate dissipation of any acidic compartment, specially with
>> > protons that move very fast. Even you can measure something I would
>> doubt
>> > about the quality of those results. It is hard enough without fixation
>> :)
>> >
>> > ...Buy a microscope for the BLS3
>> >
>> > regards
>> > Nuno Moreno
>> >
>> > > On 24 Jan 2018, at 15:49, Sylvie Le Guyader <[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 Mike
>> > >
>> > > Yes we want to measure the Lysosome pH in cells infected with a BSL3
>> > virus but we image them in a non BSL3 imaging facility so the cells
>> must be
>> > fixed before imaging.
>> > >
>> > > Med vänlig hälsning / Best regards
>> > >
>> > > Sylvie
>> > >
>> > > @@@@@@@@@@@@@@@@@@@@@@@@
>> > > Sylvie Le Guyader, PhD
>> > > Live Cell Imaging Facility Manager
>> > > Karolinska Institutet- Bionut Dpt
>> > > Hälsovägen 7,
>> > > Novum, G lift, floor 6
>> > > 14157 Huddinge
>> > > Sweden
>> > > mobile: +46 (0) 73 733 5008
>> > > office: +46 (0) 8 524 811 72
>> > > LCI website
>> > >
>> > >
>> > > -----Original Message-----
>> > > From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@lis
>> ts.umn.edu]
>> > On Behalf Of MODEL, MICHAEL
>> > > Sent: den 24 januari 2018 15:46
>> > > To: [hidden email]
>> > > Subject: Re: fixable pH sensor
>> > >
>> > > *****
>> > > 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.
>> > > *****
>> > >
>> > > Sylvie,
>> > >
>> > > Do you want to measure pH in chemically fixed cells? (Sorry, this is
>> not
>> > an answer to your question but a counter-question)
>> > >
>> > > Mike
>> > >
>> > > -----Original Message-----
>> > > From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@LIS
>> TS.UMN.EDU]
>> > On Behalf Of Sylvie Le Guyader
>> > > Sent: Wednesday, January 24, 2018 7:25 AM
>> > > To: [hidden email]
>> > > Subject: fixable pH sensor
>> > >
>> > > *****
>> > > To join, leave or search the confocal microscopy listserv, go to:
>> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > > Post images on http://www.imgur.com and include the link in your
>> > posting.
>> > > *****
>> > >
>> > > Hello everyone
>> > >
>> > > Would anyone know of a fixable pH sensor for lysosomes that is excited
>> > in the visible range and not in the UV? We only know of the Lysosensor
>> > Yellow/Blue dextran by Thermofisher we have no UV on the microscope we
>> want
>> > to use.
>> > >
>> > > thanks
>> > >
>> > > Med vänlig hälsning / Best regards
>> > >
>> > > Sylvie
>> > >
>> > > @@@@@@@@@@@@@@@@@@@@@@@@
>> > > Sylvie Le Guyader, PhD
>> > > Live Cell Imaging Facility Manager
>> > > Karolinska Institutet- Bionut Dpt
>> > > Hälsovägen 7,
>> > > Novum, G lift, floor 6
>> > > 14157 Huddinge
>> > > Sweden
>> > > mobile: +46 (0) 73 733 5008
>> > > office: +46 (0) 8 524 811 72
>> > > LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>
>> >
>>
>
>
>
> --
> Dalton J. Leprich
> PhD Student, Geobiology
> University of Minnesota, Earth Science
> [hidden email]
>

--
Dalton J. Leprich
PhD Student, Geobiology
University of Minnesota, Earth Science
[hidden email]

phil laissue-3

Re: fixable pH sensor

In reply to this post by Sylvie Le Guyader

*****
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.
*****

regarding FP-based pH sensors, we've used pHRed (Tantama, Hung & Yellen,
2011) for live plants, with very good results. This is ratiometric (dual
excitation/single emission). I don't think fixation would work.
PMID: 28663550

Has anyone on the list worked with pHuji?
PMID: 25385186
_________________________________________
Philippe Laissue, PhD
Royal Society Industry Fellow and MBL Whitman Center Scientist
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
website <https://laissue.github.io/>

On 24 January 2018 at 12:25, Sylvie Le Guyader <[hidden email]>
wrote:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello everyone
>
> Would anyone know of a fixable pH sensor for lysosomes that is excited in

the visible range and not in the UV? We only know of the Lysosensor
Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want
to use.

>
> thanks
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: +46 (0) 73 733 5008 <+46%2073%20733%2050%2008>
> office: +46 (0) 8 524 811 72 <+46%208%20524%20811%2072>
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-facility>

Oshel, Philip Eugene

Re: fixable pH sensor

*****
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.
*****

Even if pH sensor dyes or FPs were fixable, would it mean anything? Given the “acid wave” that precedes the diffusion front of the fixative as moves through the tissue or cells, surely any fixed pH indication would be artifactually low.

Phil
-------------
Philip Oshel
Imaging Facility Director
Biology Department
1304 Biosciences
1455 Calumet Ct.
Central Michigan University
Mt. Pleasant, MI 48859
989 774-3576 office
989 774-7567 lab

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> on behalf of phil laissue <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Thursday, 25January, 2018 at 05:32
To: "[hidden email]" <[hidden email]>
Subject: Re: fixable pH sensor

*****
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.
*****

regarding FP-based pH sensors, we've used pHRed (Tantama, Hung & Yellen,
2011) for live plants, with very good results. This is ratiometric (dual
excitation/single emission). I don't think fixation would work.
PMID: 28663550

Has anyone on the list worked with pHuji?
PMID: 25385186
_________________________________________
Philippe Laissue, PhD
Royal Society Industry Fellow and MBL Whitman Center Scientist
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
website <https://laissue.github.io/>

On 24 January 2018 at 12:25, Sylvie Le Guyader <[hidden email]>
wrote:

the visible range and not in the UV? We only know of the Lysosensor
Yellow/Blue dextran by Thermofisher we have no UV on the microscope we want
to use.