http://confocal-microscopy-list.275.s1.nabble.com/Re-fixable-pH-sensor-vendor-response-and-additional-question-tp7587838.html
I agree with Doug. Though the LysoSensor dextran is "fixable" in the sense that the dextran portion has amine groups that are crosslinked with formaldehyde, and will thus be retained after fixation, the pH-response is *not* fixable and will reflect the pH of the subsequent buffers. You could put the fixed cells into a pH 4 buffer to visualize the localization of the dextran, but not be able to see what the pH was prior to the fixation.
To my knowledge, there aren't any pH indicator organic dyes that will have a pH *response* that will be irreversible or fixable.
HOWEVER, I am not familiar with pH-sensitive fluorescent proteins. A question for the rest of the listserv: Would pH-responsive fluorescent proteins be fixable and retain the pH response?
Jason A. Kilgore
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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!
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> *****
>
> 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:
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> > 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:
> >
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> > 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:
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> > 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<
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