Detecting charge using fluorescence

[Dun, Alison]

*****
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 have a query for a user, they would like to look at the change in charge on the membrane of bacteria (gram negative). Does anyone know of a way to do this using fluorescence at all?

Thanks!
Ali

Please note: Working hours: Tuesday - Friday 8am - 4pm

Dr. Alison Dun
Edinburgh Super Resolution Imaging Consortium (ESRIC)
3.29 William Perkin Building
Heriot-Watt University
Edinburgh
EH14 4AS
Office: [phone]
Mobile: [phone]

http://www.esric.org
Follow us on Twitter @ESRIC_Imaging




________________________________

Heriot-Watt University is The Times & The Sunday Times International University of the Year 2018

Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With campuses and students across the entire globe we span the world, delivering innovation and educational excellence in business, engineering, design and the physical, social and life sciences.

This email is generated from the Heriot-Watt University Group, which includes:

  1.  Heriot-Watt University, a Scottish charity registered under number SC000278
  2.  Edinburgh Business School a Charity Registered in Scotland, SC026900. Edinburgh Business School is a company limited by guarantee, registered in Scotland with registered number SC173556 and registered office at Heriot-Watt University Finance Office, Riccarton, Currie, Midlothian, EH14 4AS
  3.  Heriot- Watt Services Limited (Oriam), Scotland's national performance centre for sport. Heriot-Watt Services Limited is a private limited company registered is Scotland with registered number SC271030 and registered office at Research & Enterprise Services Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS.

The contents (including any attachments) are confidential. If you are not the intended recipient of this e-mail, any disclosure, copying, distribution or use of its contents is strictly prohibited, and you should please notify the sender immediately and then delete it (including any attachments) from your system.

[mmodel]

*****
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 Alison,


Anionic DiBAC is a standard reagent for mammalian cells, and it may also work for bacteria. Many other positively charged chemicals, including mitochondrial potential probes, accumulate in cells more or less according to the Nernst equation (some nonspecific binding is possible of course). Not all of them are suitable for potential measurements in cells containing mitochondria because those probes end up in more negative mitochondria (~ negative 200 mV). But since bacteria do not have mitochondria, mitochondrial probes may work.


If the user needs quantitative data, things get more tricky because to do proper calibration, the membrane must be completely depolaraized.


Mike


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Dun, Alison <[hidden email]>
Sent: Wednesday, September 12, 2018 6:47 AM
To: [hidden email]
Subject: Detecting charge using fluorescence

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
lists.umn.edu
[hidden email]: listserv archives. confocalmicroscopy



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

Hi,

I have a query for a user, they would like to look at the change in charge on the membrane of bacteria (gram negative). Does anyone know of a way to do this using fluorescence at all?

Thanks!
Ali

Please note: Working hours: Tuesday - Friday 8am - 4pm

Dr. Alison Dun
Edinburgh Super Resolution Imaging Consortium (ESRIC)
3.29 William Perkin Building
Heriot-Watt University
Edinburgh
EH14 4AS
Office: [phone]
Mobile: [phone]

http://www.esric.org
Follow us on Twitter @ESRIC_Imaging




________________________________

Heriot-Watt University is The Times & The Sunday Times International University of the Year 2018

Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With campuses and students across the entire globe we span the world, delivering innovation and educational excellence in business, engineering, design and the physical, social and life sciences.

This email is generated from the Heriot-Watt University Group, which includes:

  1.  Heriot-Watt University, a Scottish charity registered under number SC000278
  2.  Edinburgh Business School a Charity Registered in Scotland, SC026900. Edinburgh Business School is a company limited by guarantee, registered in Scotland with registered number SC173556 and registered office at Heriot-Watt University Finance Office, Riccarton, Currie, Midlothian, EH14 4AS
  3.  Heriot- Watt Services Limited (Oriam), Scotland's national performance centre for sport. Heriot-Watt Services Limited is a private limited company registered is Scotland with registered number SC271030 and registered office at Research & Enterprise Services Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS.

The contents (including any attachments) are confidential. If you are not the intended recipient of this e-mail, any disclosure, copying, distribution or use of its contents is strictly prohibited, and you should please notify the sender immediately and then delete it (including any attachments) from your system.

*****
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 Alison,

Without knowing anything more about your user's desired application, have you considered using voltage sensitive fluorescent proteins or dyes to do this (such as the ASAP, Arclight, Ace sensor FP families)? I know that they are frequently used in neurons and similar environments, and I have had some success using them as an alternative to calcium sensors (in primary mouse neurons). I only really have had experience with ASAP2, which worked quite well, but there are surely newer variants to try out.

The signal-to-noise ratio can be tricky to handle, depending on how large the change in charge is that you would be dealing with as well as the time scales involved. Also, some of the fluorescent dyes have been shown to be fairly cytotoxic (I quickly stopped working with those).

I am not sure if this would be an option, but regardless, good luck for you and your user!

Best,

Nicolai Urban

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


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL
Sent: Mittwoch, 12. September 2018 07:27
To: [hidden email]
Subject: Re: Detecting charge using fluorescence

*****
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&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260146031&amp;sdata=lA8zXpoEgkzoA62QflMIEwMSrpjSxJyA93sIarA9YBM%3D&amp;reserved=0
Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260146031&amp;sdata=VnOOXIFGf%2FPeKsNeAdP7flQwA2YNZ3JpKwYe%2FyDTJEc%3D&amp;reserved=0 and include the link in your posting.
*****

Hello Alison,


Anionic DiBAC is a standard reagent for mammalian cells, and it may also work for bacteria. Many other positively charged chemicals, including mitochondrial potential probes, accumulate in cells more or less according to the Nernst equation (some nonspecific binding is possible of course). Not all of them are suitable for potential measurements in cells containing mitochondria because those probes end up in more negative mitochondria (~ negative 200 mV). But since bacteria do not have mitochondria, mitochondrial probes may work.


If the user needs quantitative data, things get more tricky because to do proper calibration, the membrane must be completely depolaraized.


Mike


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Dun, Alison <[hidden email]>
Sent: Wednesday, September 12, 2018 6:47 AM
To: [hidden email]
Subject: Detecting charge using fluorescence

*****
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&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=ziPbjM83ldEXim4hWNIXjoaYfGZu6QT%2FFjU4RIsc2GU%3D&amp;reserved=0

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=ziPbjM83ldEXim4hWNIXjoaYfGZu6QT%2FFjU4RIsc2GU%3D&amp;reserved=0>
lists.umn.edu
[hidden email]: listserv archives. confocalmicroscopy



Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=HscO47w%2Fn3F9pJp6n%2FEJ21SNk0C4m5GnLudOc0%2F3DaM%3D&amp;reserved=0 and include the link in your posting.
*****

Hi,

I have a query for a user, they would like to look at the change in charge on the membrane of bacteria (gram negative). Does anyone know of a way to do this using fluorescence at all?

Thanks!
Ali

Please note: Working hours: Tuesday - Friday 8am - 4pm

Dr. Alison Dun
Edinburgh Super Resolution Imaging Consortium (ESRIC)
3.29 William Perkin Building
Heriot-Watt University
Edinburgh
EH14 4AS
Office: [phone]
Mobile: [phone]

https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.esric.org&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=%2Fn%2FJTxaFGy39ieUFv2pQQadC8x6Qm%2F9WP%2F5um5AWCJo%3D&amp;reserved=0
Follow us on Twitter @ESRIC_Imaging




________________________________

Heriot-Watt University is The Times & The Sunday Times International University of the Year 2018

Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With campuses and students across the entire globe we span the world, delivering innovation and educational excellence in business, engineering, design and the physical, social and life sciences.

This email is generated from the Heriot-Watt University Group, which includes:

  1.  Heriot-Watt University, a Scottish charity registered under number SC000278
  2.  Edinburgh Business School a Charity Registered in Scotland, SC026900. Edinburgh Business School is a company limited by guarantee, registered in Scotland with registered number SC173556 and registered office at Heriot-Watt University Finance Office, Riccarton, Currie, Midlothian, EH14 4AS
  3.  Heriot- Watt Services Limited (Oriam), Scotland's national performance centre for sport. Heriot-Watt Services Limited is a private limited company registered is Scotland with registered number SC271030 and registered office at Research & Enterprise Services Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS.

The contents (including any attachments) are confidential. If you are not the intended recipient of this e-mail, any disclosure, copying, distribution or use of its contents is strictly prohibited, and you should please notify the sender immediately and then delete it (including any attachments) from your system.

[Dun, Alison]

*****
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 very much for your replies, a few things for us to try now,
Cheers
Ali

Please note: Working hours: Tuesday - Friday 8am - 4pm

Dr. Alison Dun
Edinburgh Super Resolution Imaging Consortium (ESRIC)
3.29 William Perkin Building
Heriot-Watt University
Edinburgh
EH14 4AS
Office: [phone]
Mobile: [phone]

http://www.esric.org
Follow us on Twitter @ESRIC_Imaging



On 12 Sep 2018, at 15:27, [hidden email]<mailto:[hidden email]> <[hidden email]<mailto:[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<http://www.imgur.com/> and include the link in your posting.
*****

Hi Alison,

Without knowing anything more about your user's desired application, have you considered using voltage sensitive fluorescent proteins or dyes to do this (such as the ASAP, Arclight, Ace sensor FP families)? I know that they are frequently used in neurons and similar environments, and I have had some success using them as an alternative to calcium sensors (in primary mouse neurons). I only really have had experience with ASAP2, which worked quite well, but there are surely newer variants to try out.

The signal-to-noise ratio can be tricky to handle, depending on how large the change in charge is that you would be dealing with as well as the time scales involved. Also, some of the fluorescent dyes have been shown to be fairly cytotoxic (I quickly stopped working with those).

I am not sure if this would be an option, but regardless, good luck for you and your user!

Best,

Nicolai Urban

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


-----Original Message-----
From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> On Behalf Of MODEL, MICHAEL
Sent: Mittwoch, 12. September 2018 07:27
To: [hidden email]<mailto:[hidden email]>
Subject: Re: Detecting charge using fluorescence

*****
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&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260146031&amp;sdata=lA8zXpoEgkzoA62QflMIEwMSrpjSxJyA93sIarA9YBM%3D&amp;reserved=0
Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260146031&amp;sdata=VnOOXIFGf%2FPeKsNeAdP7flQwA2YNZ3JpKwYe%2FyDTJEc%3D&amp;reserved=0 and include the link in your posting.
*****

Hello Alison,


Anionic DiBAC is a standard reagent for mammalian cells, and it may also work for bacteria. Many other positively charged chemicals, including mitochondrial potential probes, accumulate in cells more or less according to the Nernst equation (some nonspecific binding is possible of course). Not all of them are suitable for potential measurements in cells containing mitochondria because those probes end up in more negative mitochondria (~ negative 200 mV). But since bacteria do not have mitochondria, mitochondrial probes may work.


If the user needs quantitative data, things get more tricky because to do proper calibration, the membrane must be completely depolaraized.


Mike


________________________________
From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> on behalf of Dun, Alison <[hidden email]<mailto:[hidden email]>>
Sent: Wednesday, September 12, 2018 6:47 AM
To: [hidden email]<mailto:[hidden email]>
Subject: Detecting charge using fluorescence

*****
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&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=ziPbjM83ldEXim4hWNIXjoaYfGZu6QT%2FFjU4RIsc2GU%3D&amp;reserved=0

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.umn.edu/><https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=ziPbjM83ldEXim4hWNIXjoaYfGZu6QT%2FFjU4RIsc2GU%3D&amp;reserved=0>
lists.umn.edu<http://lists.umn.edu/>
[hidden email]<mailto:[hidden email]>: listserv archives. confocalmicroscopy



Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=HscO47w%2Fn3F9pJp6n%2FEJ21SNk0C4m5GnLudOc0%2F3DaM%3D&amp;reserved=0 and include the link in your posting.
*****

Hi,

I have a query for a user, they would like to look at the change in charge on the membrane of bacteria (gram negative). Does anyone know of a way to do this using fluorescence at all?

Thanks!
Ali

Please note: Working hours: Tuesday - Friday 8am - 4pm

Dr. Alison Dun
Edinburgh Super Resolution Imaging Consortium (ESRIC)
3.29 William Perkin Building
Heriot-Watt University
Edinburgh
EH14 4AS
Office: [phone]
Mobile: [phone]

https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.esric.org&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cae3f208abae44aeb134208d618a2a9b9%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636723484260156035&amp;sdata=%2Fn%2FJTxaFGy39ieUFv2pQQadC8x6Qm%2F9WP%2F5um5AWCJo%3D&amp;reserved=0
Follow us on Twitter @ESRIC_Imaging




________________________________

Heriot-Watt University is The Times & The Sunday Times International University of the Year 2018

Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With campuses and students across the entire globe we span the world, delivering innovation and educational excellence in business, engineering, design and the physical, social and life sciences.

This email is generated from the Heriot-Watt University Group, which includes:

 1.  Heriot-Watt University, a Scottish charity registered under number SC000278
 2.  Edinburgh Business School a Charity Registered in Scotland, SC026900. Edinburgh Business School is a company limited by guarantee, registered in Scotland with registered number SC173556 and registered office at Heriot-Watt University Finance Office, Riccarton, Currie, Midlothian, EH14 4AS
 3.  Heriot- Watt Services Limited (Oriam), Scotland's national performance centre for sport. Heriot-Watt Services Limited is a private limited company registered is Scotland with registered number SC271030 and registered office at Research & Enterprise Services Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS.

The contents (including any attachments) are confidential. If you are not the intended recipient of this e-mail, any disclosure, copying, distribution or use of its contents is strictly prohibited, and you should please notify the sender immediately and then delete it (including any attachments) from your system.

[Seamus Holden-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.
*****

Dear Ali

My colleague Henrik Strahl does a lot of work on membrane potential imaging in bacteria so I forwarded this to him.
Here's his reply:

" Assuming "charge on the membrane" membrane potential (and not surface charge)

DiSC3(5) solved in DMSO
-2 uM final concentration at cells
-maintain 1% DMSO while staining
-5 min incubation directly in medium under shaking is enough
-positive control for depolarisation is 10ug/ml polymyxin B (incubated together with the dye for 5min)

While nothing is published with Ecoli, I am happy to help out if he gets in touch with email.
Here is the protocol for Bsub:
https://www.frontiersin.org/articles/10.3389/fcell.2016.00029/full"
"

Best wishes
Seamus

Dr Seamus Holden
Wellcome Trust Sir Henry Dale Fellow
Newcastle University
Centre for Bacterial Cell Biology
NE2 4AX, United Kingdom

Phone: [phone]
https://blogs.ncl.ac.uk/seamusholden/


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Dun, Alison
Sent: 12 September 2018 11:47
To: [hidden email]
Subject: Detecting charge using fluorescence

*****
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 have a query for a user, they would like to look at the change in charge on the membrane of bacteria (gram negative). Does anyone know of a way to do this using fluorescence at all?

Thanks!
Ali

Please note: Working hours: Tuesday - Friday 8am - 4pm

Dr. Alison Dun
Edinburgh Super Resolution Imaging Consortium (ESRIC)
3.29 William Perkin Building
Heriot-Watt University
Edinburgh
EH14 4AS
Office: [phone]
Mobile: [phone]

http://www.esric.org
Follow us on Twitter @ESRIC_Imaging




________________________________

Heriot-Watt University is The Times & The Sunday Times International University of the Year 2018

Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With campuses and students across the entire globe we span the world, delivering innovation and educational excellence in business, engineering, design and the physical, social and life sciences.

This email is generated from the Heriot-Watt University Group, which includes:

  1.  Heriot-Watt University, a Scottish charity registered under number SC000278
  2.  Edinburgh Business School a Charity Registered in Scotland, SC026900. Edinburgh Business School is a company limited by guarantee, registered in Scotland with registered number SC173556 and registered office at Heriot-Watt University Finance Office, Riccarton, Currie, Midlothian, EH14 4AS
  3.  Heriot- Watt Services Limited (Oriam), Scotland's national performance centre for sport. Heriot-Watt Services Limited is a private limited company registered is Scotland with registered number SC271030 and registered office at Research & Enterprise Services Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS.

The contents (including any attachments) are confidential. If you are not the intended recipient of this e-mail, any disclosure, copying, distribution or use of its contents is strictly prohibited, and you should please notify the sender immediately and then delete it (including any attachments) from your system.

[Rusty Nicovich]

*****
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.
*****

Alex Ma at UNSW published a FRET sensor that detects membrane charge in
mammalian cells:

https://www.nature.com/articles/nbt.3828

(I contributed a bit of analysis to that paper, but Dr Ma is the expert).

Not sure how it translates to bacteria, but it is a fluorescence-based
readout of the phenomenon in question.

Thanks,
Rusty

On Thu, Sep 13, 2018 at 2:25 AM, Seamus Holden <
[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.
> *****
>
> Dear Ali
>
> My colleague Henrik Strahl does a lot of work on membrane potential
> imaging in bacteria so I forwarded this to him.
> Here's his reply:
>
> " Assuming "charge on the membrane" membrane potential (and not surface
> charge)
>
> DiSC3(5) solved in DMSO
> -2 uM final concentration at cells
> -maintain 1% DMSO while staining
> -5 min incubation directly in medium under shaking is enough
> -positive control for depolarisation is 10ug/ml polymyxin B (incubated
> together with the dye for 5min)
>
> While nothing is published with Ecoli, I am happy to help out if he gets
> in touch with email.
> Here is the protocol for Bsub:
> https://www.frontiersin.org/articles/10.3389/fcell.2016.00029/full"
> "
>
> Best wishes
> Seamus
>
> Dr Seamus Holden
> Wellcome Trust Sir Henry Dale Fellow
> Newcastle University
> Centre for Bacterial Cell Biology
> NE2 4AX, United Kingdom
>
> Phone: [phone]
> https://blogs.ncl.ac.uk/seamusholden/
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Dun, Alison
> Sent: 12 September 2018 11:47
> To: [hidden email]
> Subject: Detecting charge using fluorescence
>
> *****
> 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 have a query for a user, they would like to look at the change in charge
> on the membrane of bacteria (gram negative). Does anyone know of a way to
> do this using fluorescence at all?
>
> Thanks!
> Ali
>
> Please note: Working hours: Tuesday - Friday 8am - 4pm
>
> Dr. Alison Dun
> Edinburgh Super Resolution Imaging Consortium (ESRIC)
> 3.29 William Perkin Building
> Heriot-Watt University
> Edinburgh
> EH14 4AS
> Office: [phone]
> Mobile: [phone]
>
> http://www.esric.org
> Follow us on Twitter @ESRIC_Imaging
>
>
>
>
> ________________________________
>
> Heriot-Watt University is The Times & The Sunday Times International
> University of the Year 2018
>
> Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With
> campuses and students across the entire globe we span the world, delivering
> innovation and educational excellence in business, engineering, design and
> the physical, social and life sciences.
>
> This email is generated from the Heriot-Watt University Group, which
> includes:
>
>   1.  Heriot-Watt University, a Scottish charity registered under number
> SC000278
>   2.  Edinburgh Business School a Charity Registered in Scotland,
> SC026900. Edinburgh Business School is a company limited by guarantee,
> registered in Scotland with registered number SC173556 and registered
> office at Heriot-Watt University Finance Office, Riccarton, Currie,
> Midlothian, EH14 4AS
>   3.  Heriot- Watt Services Limited (Oriam), Scotland's national
> performance centre for sport. Heriot-Watt Services Limited is a private
> limited company registered is Scotland with registered number SC271030 and
> registered office at Research & Enterprise Services Heriot-Watt University,
> Riccarton, Edinburgh, EH14 4AS.
>
> The contents (including any attachments) are confidential. If you are not
> the intended recipient of this e-mail, any disclosure, copying,
> distribution or use of its contents is strictly prohibited, and you should
> please notify the sender immediately and then delete it (including any
> attachments) from your system.
>

[Davide Calebiro]

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

Dear all,

Is there anyone who could advise about air conditioning for a microscopy facility?

I have recently put together a rather  advanced 4-camera TIRF system for single molecule and superresolution microscopy and I have realised that the temperature in the brand new room dedicated to the microscope is fluctuating of 2 degrees every 10 min, which is creating major problems with x-.y stability (despite the microscope being partially surrounded by an enclosure with temperature control).

I am struggling to obtain details about the installed air conditioning system (unfortunately estates did not consult me). But I have found out that apparently they have installed a fan coil unit that can both cool and heat, which I do not think is appropriate or very clever.

 What type of air conditioning would you recommend for a delicate setup? What is a reasonable expectation in terms of temperature stability in the room within say 30 min? eg +/- 0.5C? or is it reasonable to ask for better? Which other parameters (eg humidity) and specifications should I consider?

Thank you.

Bw
Davide

Davide Calebiro
University of Birmingham

Sent from my iPhone
>>

[Jeremy Adler-4]

*****
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.
*****

Try putting your microscope in an air tight enclosure - air tight is significantly better than a leaky enclosure and in our case almost completely eliminated the cyclical changes produced by the air conditioning.


Reducing image distortions due to temperature‐related microscope stage drift<https://onlinelibrary.wiley.com/doi/10.1046/j.1365-2818.2003.01160.x>

     *   J. Adler<https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Adler%2C+J>

     *   S. N. Pagakis <https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Pagakis%2C+S+N>

Journal of Microscopy<https://onlinelibrary.wiley.com/journal/13652818>Volume 210, Issue 2<https://onlinelibrary.wiley.com/toc/13652818/210/2>



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Davide Calebiro <[hidden email]>
Sent: 16 September 2018 22:36:05
To: [hidden email]
Subject: Advice about air conditioning

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

Dear all,

Is there anyone who could advise about air conditioning for a microscopy facility?

I have recently put together a rather  advanced 4-camera TIRF system for single molecule and superresolution microscopy and I have realised that the temperature in the brand new room dedicated to the microscope is fluctuating of 2 degrees every 10 min, which is creating major problems with x-.y stability (despite the microscope being partially surrounded by an enclosure with temperature control).

I am struggling to obtain details about the installed air conditioning system (unfortunately estates did not consult me). But I have found out that apparently they have installed a fan coil unit that can both cool and heat, which I do not think is appropriate or very clever.

 What type of air conditioning would you recommend for a delicate setup? What is a reasonable expectation in terms of temperature stability in the room within say 30 min? eg +/- 0.5C? or is it reasonable to ask for better? Which other parameters (eg humidity) and specifications should I consider?

Thank you.

Bw
Davide

Davide Calebiro
University of Birmingham

Sent from my iPhone
>>








Nar du har kontakt med oss pa Uppsala universitet med e-post sa innebar det att vi behandlar dina personuppgifter. For att lasa mer om hur vi gor det kan du lasa har: http://www.uu.se/om-uu/dataskydd-personuppgifter/

E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/om-uu/dataskydd-personuppgifter/

[Davide Calebiro]

*****
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 Jeremy!

Interesting paper! We see exactly those type of cyclic changes in x-y correlating with room temperature that you are reporting!

The stage, objective and entire core of the microscope are enclosed in an Okolab cage incubator. The temperature measured inside the cage incubator is very stable, so the cage incubator seems to be rather tight. But there are parts like the quadruple beam splitter and the four cameras than are outside the incubator. I do not know if those parts are responsible for the drift (theoretically not very likely, though not sure), but putting everything inside an incubator might be tricky....

Bw
Davide



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of [hidden email]
Sent: 17 September 2018 09:27
To: [hidden email]
Subject: Re: Advice about air conditioning

*****
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.
*****

Try putting your microscope in an air tight enclosure - air tight is significantly better than a leaky enclosure and in our case almost completely eliminated the cyclical changes produced by the air conditioning.


Reducing image distortions due to temperature‐related microscope stage drift<https://onlinelibrary.wiley.com/doi/10.1046/j.1365-2818.2003.01160.x>

     *   J. Adler<https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Adler%2C+J

     *   S. N. Pagakis <https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Pagakis%2C+S+N>

Journal of Microscopy<https://onlinelibrary.wiley.com/journal/13652818>Volume 210, Issue 2<https://onlinelibrary.wiley.com/toc/13652818/210/2>



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Davide Calebiro <[hidden email]>
Sent: 16 September 2018 22:36:05
To: [hidden email]
Subject: Advice about air conditioning

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

Dear all,

Is there anyone who could advise about air conditioning for a microscopy facility?

I have recently put together a rather  advanced 4-camera TIRF system for single molecule and superresolution microscopy and I have realised that the temperature in the brand new room dedicated to the microscope is fluctuating of 2 degrees every 10 min, which is creating major problems with x-.y stability (despite the microscope being partially surrounded by an enclosure with temperature control).

I am struggling to obtain details about the installed air conditioning system (unfortunately estates did not consult me). But I have found out that apparently they have installed a fan coil unit that can both cool and heat, which I do not think is appropriate or very clever.

 What type of air conditioning would you recommend for a delicate setup? What is a reasonable expectation in terms of temperature stability in the room within say 30 min? eg +/- 0.5C? or is it reasonable to ask for better? Which other parameters (eg humidity) and specifications should I consider?

Thank you.

Bw
Davide

Davide Calebiro
University of Birmingham

Sent from my iPhone
>>








Nar du har kontakt med oss pa Uppsala universitet med e-post sa innebar det att vi behandlar dina personuppgifter. For att lasa mer om hur vi gor det kan du lasa har: http://www.uu.se/om-uu/dataskydd-personuppgifter/

E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/om-uu/dataskydd-personuppgifter/