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Re: fixable pH sensor vendor response and additional question...

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Kilgore, Jason A.

Re: fixable pH sensor vendor response and additional question...

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**Vendor Response**

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

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific

1-800-955-6288 then option 4, then option 3, then option 2.
Or dial direct at +1 541 335 0353
[hidden email]

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information. Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Douglas Richardson
Sent: Wednesday, January 24, 2018 8:44 AM
To: [hidden email]
Subject: Re: fixable pH sensor

CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.

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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:

Douglas Richardson

Re: fixable pH sensor vendor response and additional question...

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Hi Jason,

All of the fluorescent proteins I've worked with also use a similar
protonation reaction and are reversible (EGFP, EYFP, sepHlorin and
others....see Fig 1 here: doi:10.1038/s41467-017-00606-4
<https://www.nature.com/articles/s41467-017-00606-4>).

-Doug

On Wed, Jan 24, 2018 at 1:08 PM, Kilgore, Jason A. <
[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.
> *****
>
>
> **Vendor Response**
>
> 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
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Thermo Fisher Scientific
>
> 1-800-955-6288 then option 4, then option 3, then option 2.
> Or dial direct at +1 541 335 0353
> [hidden email]
>
> This communication is intended solely for the individual/entity to whom it
> is addressed. It may contain confidential or legally privileged
> information. Any unauthorized disclosure or copying is prohibited and may
> be unlawful. If you have received this communication in error, please
> notify the sender immediately and delete it from your system.
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Douglas Richardson
> Sent: Wednesday, January 24, 2018 8:44 AM
> To: [hidden email]
> Subject: Re: fixable pH sensor
>
> CAUTION: This email originated from outside of the organization. Do not
> click links or open attachments unless you recognize the sender and know
> the content is safe.
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> n-rb4fQCM&e= 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:
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhI
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> > 919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwnZ02M8oIuS1
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> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
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> n-rb4fQCM&e= 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:
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg
> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
> > > Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwnZ0
> > > 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=
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> > > qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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:
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg
> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
> > > Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwnZ0
> > > 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=
> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQt
> > > CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvP
> > > qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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:
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg
> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
> > > Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwnZ0
> > > 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=
> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQt
> > > CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvP
> > > qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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<https://urldefense.proofpoint.com/v2/url?u=http-3A__ki.se_en
> > > _bionut_welcome-2Dto-2Dthe-2Dlci-2Dfacility&d=DwIFaQ&c=q6k2DsTcEGCcC
> > > b_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-y
> > > ioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wqAvRxsrsRN
> > > JC2bSS68-xnCbCFJG4xO9BBOYA6bkImU&e=>
> >
>

Douglas Richardson

Re: fixable pH sensor vendor response and additional question...

In reply to this post by Kilgore, Jason A.

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Hi Jason,

Interesting question.

Hi Jason,

All of the fluorescent proteins (FPs) I've worked with also use a similar
protonation reaction and are reversible (EGFP, EYFP, sepHlorin and
others....see Fig 1 here: doi:10.1038/s41467-017-00606-4
<https://www.nature.com/articles/s41467-017-00606-4>). That experiment was
done with purified FPs in solution. I have never tried to place an FP in
an acidic buffer, fix it, neutralize the acid and see if the fluorescence
is recovered. My gut feeling is that PFA crosslinking of the FP would not
be sufficient to block deprotonation.

-Doug

-Doug

On Wed, Jan 24, 2018 at 1:08 PM, Kilgore, Jason A. <
[hidden email]> wrote:

Kilgore, Jason A.

Re: fixable pH sensor vendor response and additional question...

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Thank you, Doug. Your paper is excellent, and I wasn't aware of all of those FP options. It'll definitely be one for our records to share with researchers.

I suspect you are correct in your hypothesis that the protonation would still be reversible. Your proposed experiment would be worthwhile to try, I think, though it might be more pertinent to do so in an in-cell environment (considering crosslinking to intracellular proteins, as well as that a cellular assay would be more likely what a researcher would use them for).

Here in Tech Support at Molecular Probes, we get this very question relatively often. If you or anyone else who reads this could try the proposed experiments, and lets us know, we would add that info to our notes to share with other researchers who call us.

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific

1-800-955-6288 then option 4, then option 3, then option 2.
Or dial direct at +1 541 335 0353
[hidden email]

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information. Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Douglas Richardson
Sent: Wednesday, January 24, 2018 10:25 AM
To: [hidden email]
Subject: Re: fixable pH sensor **vendor response** and additional question...

CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.

*****
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*****

Hi Jason,

Interesting question.

Hi Jason,

All of the fluorescent proteins (FPs) I've worked with also use a similar protonation reaction and are reversible (EGFP, EYFP, sepHlorin and others....see Fig 1 here: doi:10.1038/s41467-017-00606-4 <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_s41467-2D017-2D00606-2D4&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=l0QGXb_wkcXUVo1Ldp_VkPiJks56Oxo5JdmIdXFQ_JQ&e=>). That experiment was done with purified FPs in solution. I have never tried to place an FP in an acidic buffer, fix it, neutralize the acid and see if the fluorescence is recovered. My gut feeling is that PFA crosslinking of the FP would not be sufficient to block deprotonation.

-Doug

-Doug

On Wed, Jan 24, 2018 at 1:08 PM, Kilgore, Jason A. < [hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhI
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> i58L0SM2SPuPXGA2o&e= Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=Eo6tqOpCiES_Qy_DYRZ_7otDVbzJ4NUdJ2WrDYjpBoQ&e= and include the link in your posting.
> *****
>
>
> **Vendor Response**
>
> 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
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Thermo Fisher Scientific
>
> 1-800-955-6288 then option 4, then option 3, then option 2.
> Or dial direct at +1 541 335 0353
> [hidden email]
>
> This communication is intended solely for the individual/entity to
> whom it is addressed. It may contain confidential or legally
> privileged information. Any unauthorized disclosure or copying is
> prohibited and may be unlawful. If you have received this
> communication in error, please notify the sender immediately and delete it from your system.
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Douglas Richardson
> Sent: Wednesday, January 24, 2018 8:44 AM
> To: [hidden email]
> Subject: Re: fixable pH sensor
>
> CAUTION: This email originated from outside of the organization. Do
> not click links or open attachments unless you recognize the sender
> and know the content is safe.
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCc
> Cb_
> WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-
> DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBbl
> hY& s=d_8H0QyvTLpwpRwnZ02M8oIuS1pmaq3jaJideuPDRhc&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
> imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&
> r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-
> MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7R
> n-rb4fQCM&e= 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:
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg
> > i-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6h
> > hI
> > l8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=
> > 5V
> > 919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwnZ02M8oIu
> > S1
> > pmaq3jaJideuPDRhc&e= Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
> imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&
> r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-
> MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7R
> n-rb4fQCM&e= 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:
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_
> > > cg
> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtV
> > > Sz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjT
> > > Ly
> > > Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwn
> > > Z0 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&
> > > d=
> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
> > > Qt
> > > CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNn
> > > vP qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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:
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_
> > > cg
> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtV
> > > Sz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjT
> > > Ly
> > > Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwn
> > > Z0 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&
> > > d=
> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
> > > Qt
> > > CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNn
> > > vP qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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:
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_
> > > cg
> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtV
> > > Sz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjT
> > > Ly
> > > Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwn
> > > Z0 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&
> > > d=
> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
> > > Qt
> > > CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNn
> > > vP qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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<https://urldefense.proofpoint.com/v2/url?u=http-3A__ki.se_
> > > en
> > > _bionut_welcome-2Dto-2Dthe-2Dlci-2Dfacility&d=DwIFaQ&c=q6k2DsTcEGC
> > > cC
> > > b_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV
> > > -y
> > > ioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wqAvRxsrs
> > > RN JC2bSS68-xnCbCFJG4xO9BBOYA6bkImU&e=>
> >
>

Lemasters, John J.

Re: fixable pH sensor vendor response and additional question...

*****
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The structure of LysoTracker Red has lots of amines that can react with aldehydes for fixation and cross-linking. Unlike LysoSensor dyes, LysoTracker Red fluorescence is not pH-dependent. Rather LysoTracker Red accumulates into acidic compartments like other weak bases to increase the fluorescence of the compartment (e.g., lysosomes).

My friend Brad Chazotte has a methods paper on this subject:

Labeling Lysosomes in Live Cells with LysoTracker. Brad Chazotte. In Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.

https://www.ncbi.nlm.nih.gov/pubmed/21285271
http://cshprotocols.cshlp.org/content/2011/2/pdb.prot5571.long
http://cshprotocols.cshlp.org/content/2011/2/pdb.prot5571.abstract

Hi Brad! It's been years. Maybe you can share your paper with the group.

John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 139
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: 843-876-2353
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kilgore, Jason A.
Sent: Wednesday, January 24, 2018 2:32 PM
To: [hidden email]
Subject: Re: fixable pH sensor **vendor response** and additional question...

*****
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.
*****

Thank you, Doug. Your paper is excellent, and I wasn't aware of all of those FP options. It'll definitely be one for our records to share with researchers.

I suspect you are correct in your hypothesis that the protonation would still be reversible. Your proposed experiment would be worthwhile to try, I think, though it might be more pertinent to do so in an in-cell environment (considering crosslinking to intracellular proteins, as well as that a cellular assay would be more likely what a researcher would use them for).

Here in Tech Support at Molecular Probes, we get this very question relatively often. If you or anyone else who reads this could try the proposed experiments, and lets us know, we would add that info to our notes to share with other researchers who call us.

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific

1-800-955-6288 then option 4, then option 3, then option 2.
Or dial direct at +1 541 335 0353
[hidden email]

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information. Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Douglas Richardson
Sent: Wednesday, January 24, 2018 10:25 AM
To: [hidden email]
Subject: Re: fixable pH sensor **vendor response** and additional question...

CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.

*****
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=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=Eo6tqOpCiES_Qy_DYRZ_7otDVbzJ4NUdJ2WrDYjpBoQ&e= and include the link in your posting.
*****

Hi Jason,

Interesting question.

Hi Jason,

All of the fluorescent proteins (FPs) I've worked with also use a similar protonation reaction and are reversible (EGFP, EYFP, sepHlorin and others....see Fig 1 here: doi:10.1038/s41467-017-00606-4 <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_s41467-2D017-2D00606-2D4&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=l0QGXb_wkcXUVo1Ldp_VkPiJks56Oxo5JdmIdXFQ_JQ&e=>). That experiment was done with purified FPs in solution. I have never tried to place an FP in an acidic buffer, fix it, neutralize the acid and see if the fluorescence is recovered. My gut feeling is that PFA crosslinking of the FP would not be sufficient to block deprotonation.

-Doug

-Doug

On Wed, Jan 24, 2018 at 1:08 PM, Kilgore, Jason A. < [hidden email]> wrote:

-------------------------------------------------------------------------
This message was secured via TLS by MUSC.

Nuno Moreno

Re: fixable pH sensor vendor response and additional question...

*****
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.
*****

Many thanks.

I was always thinking in a protonation. This might be a good trick for Sylvie, not measuring directly the pH but the aggregation of LysoTracker in acidic compartments.

best
Nuno

> On 24 Jan 2018, at 20:14, Lemasters, John J. <[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.
> *****
>
> The structure of LysoTracker Red has lots of amines that can react with aldehydes for fixation and cross-linking. Unlike LysoSensor dyes, LysoTracker Red fluorescence is not pH-dependent. Rather LysoTracker Red accumulates into acidic compartments like other weak bases to increase the fluorescence of the compartment (e.g., lysosomes).
>
> My friend Brad Chazotte has a methods paper on this subject:
>
> Labeling Lysosomes in Live Cells with LysoTracker. Brad Chazotte. In Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.
>
> https://www.ncbi.nlm.nih.gov/pubmed/21285271
> http://cshprotocols.cshlp.org/content/2011/2/pdb.prot5571.long
> http://cshprotocols.cshlp.org/content/2011/2/pdb.prot5571.abstract
>
> Hi Brad! It's been years. Maybe you can share your paper with the group.
>
> John
>
> --
> John J. Lemasters, MD, PhD
> Professor and GlaxoSmithKline Distinguished Endowed Chair
> Director, Center for Cell Death, Injury & Regeneration
> Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology
> Medical University of South Carolina
> DD504 Drug Discovery Building
> 70 President Street, MSC 139
> Charleston, SC 29425
>
> Office: 843-876-2360
> Lab: 843-876-2354
> Fax: 843-876-2353
> Email: [hidden email]
> http://academicdepartments.musc.edu/ccdir
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kilgore, Jason A.
> Sent: Wednesday, January 24, 2018 2:32 PM
> To: [hidden email]
> Subject: Re: fixable pH sensor **vendor response** and additional question...
>
>
>
> *****
> 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.
> *****
>
> Thank you, Doug. Your paper is excellent, and I wasn't aware of all of those FP options. It'll definitely be one for our records to share with researchers.
>
> I suspect you are correct in your hypothesis that the protonation would still be reversible. Your proposed experiment would be worthwhile to try, I think, though it might be more pertinent to do so in an in-cell environment (considering crosslinking to intracellular proteins, as well as that a cellular assay would be more likely what a researcher would use them for).
>
> Here in Tech Support at Molecular Probes, we get this very question relatively often. If you or anyone else who reads this could try the proposed experiments, and lets us know, we would add that info to our notes to share with other researchers who call us.
>
> Jason
>
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Thermo Fisher Scientific
>
> 1-800-955-6288 then option 4, then option 3, then option 2.
> Or dial direct at +1 541 335 0353
> [hidden email]
>
> This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information. Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Douglas Richardson
> Sent: Wednesday, January 24, 2018 10:25 AM
> To: [hidden email]
> Subject: Re: fixable pH sensor **vendor response** and additional question...
>
> CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=eJqZomSGmERRufFPc0159hlQQMi58L0SM2SPuPXGA2o&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=Eo6tqOpCiES_Qy_DYRZ_7otDVbzJ4NUdJ2WrDYjpBoQ&e= and include the link in your posting.
> *****
>
> Hi Jason,
>
> Interesting question.
>
> Hi Jason,
>
> All of the fluorescent proteins (FPs) I've worked with also use a similar protonation reaction and are reversible (EGFP, EYFP, sepHlorin and others....see Fig 1 here: doi:10.1038/s41467-017-00606-4 <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_s41467-2D017-2D00606-2D4&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=l0QGXb_wkcXUVo1Ldp_VkPiJks56Oxo5JdmIdXFQ_JQ&e=>). That experiment was done with purified FPs in solution. I have never tried to place an FP in an acidic buffer, fix it, neutralize the acid and see if the fluorescence is recovered. My gut feeling is that PFA crosslinking of the FP would not be sufficient to block deprotonation.
>
> -Doug
>
> -Doug
>
> On Wed, Jan 24, 2018 at 1:08 PM, Kilgore, Jason A. < [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
>> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhI
>> l8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJ
>> sd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=eJqZomSGmERRufFPc0159hlQQM
>> i58L0SM2SPuPXGA2o&e= Post images on
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=zJsd8Kfbim0LR8z9sFDPPnu1rpQCWanuuRWHGWthbdQ&s=Eo6tqOpCiES_Qy_DYRZ_7otDVbzJ4NUdJ2WrDYjpBoQ&e= and include the link in your posting.
>> *****
>>
>>
>> **Vendor Response**
>>
>> 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
>>
>> Jason A. Kilgore
>> Technical Application Scientist
>> Molecular Probes / EVOS Tech Support
>> Thermo Fisher Scientific
>>
>> 1-800-955-6288 then option 4, then option 3, then option 2.
>> Or dial direct at +1 541 335 0353
>> [hidden email]
>>
>> This communication is intended solely for the individual/entity to
>> whom it is addressed. It may contain confidential or legally
>> privileged information. Any unauthorized disclosure or copying is
>> prohibited and may be unlawful. If you have received this
>> communication in error, please notify the sender immediately and delete it from your system.
>>
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]]
>> On Behalf Of Douglas Richardson
>> Sent: Wednesday, January 24, 2018 8:44 AM
>> To: [hidden email]
>> Subject: Re: fixable pH sensor
>>
>> CAUTION: This email originated from outside of the organization. Do
>> not click links or open attachments unless you recognize the sender
>> and know the content is safe.
>>
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.
>> umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCc
>> Cb_
>> WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-
>> DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBbl
>> hY& s=d_8H0QyvTLpwpRwnZ02M8oIuS1pmaq3jaJideuPDRhc&e=
>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
>> imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&
>> r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-
>> MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7R
>> n-rb4fQCM&e= 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:
>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg
>>> i-
>>> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6h
>>> hI
>>> l8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=
>>> 5V
>>> 919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwnZ02M8oIu
>>> S1
>>> pmaq3jaJideuPDRhc&e= Post images on
>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
>> imgur.com&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&
>> r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-
>> MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7R
>> n-rb4fQCM&e= 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:
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_
>>>> cg
>>>> i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtV
>>>> Sz
>>>> 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjT
>>>> Ly
>>>> Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwn
>>>> Z0 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&
>>>> d=
>>>> DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
>>>> Qt
>>>> CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNn
>>>> vP qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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:
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_
>>>> cg
>>>> i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtV
>>>> Sz
>>>> 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjT
>>>> Ly
>>>> Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwn
>>>> Z0 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&
>>>> d=
>>>> DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
>>>> Qt
>>>> CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNn
>>>> vP qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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:
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_
>>>> cg
>>>> i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtV
>>>> Sz
>>>> 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjT
>>>> Ly
>>>> Q&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=d_8H0QyvTLpwpRwn
>>>> Z0 2M8oIuS1pmaq3jaJideuPDRhc&e= Post images on
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&
>>>> d=
>>>> DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
>>>> Qt
>>>> CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNn
>>>> vP qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= 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<https://urldefense.proofpoint.com/v2/url?u=http-3A__ki.se_
>>>> en
>>>> _bionut_welcome-2Dto-2Dthe-2Dlci-2Dfacility&d=DwIFaQ&c=q6k2DsTcEGC
>>>> cC
>>>> b_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV
>>>> -y
>>>> ioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvPqFjBblhY&s=wqAvRxsrs
>>>> RN JC2bSS68-xnCbCFJG4xO9BBOYA6bkImU&e=>
>>>
>>
>
>
>
>
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