HBSS Autofluoresence?
Locked
|
*****
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. ***** We are trying to image live cultured cells in Hanks Balanced Salt solution without phenol red. It's widefield imaging with a standard Chroma GFP filter set and I'm seeing something I've never observed before. There's very high background all over the field which quickly fades within a few seconds. If I stop the acquisition briefly and resume, the background goes right back up by about 25%. I've never seen this before. What could be the source of this in HBSS, or is it something else? Chris |
|
|
*****
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 Chris Does medium alone behave the same? Could the cells be secreting something that bleaches/goes to the dark state? That would explain the recovery. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Hälsovägen 7C, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Chris O'Connell Sent: den 15 januari 2019 20:00 To: [hidden email] Subject: HBSS Autofluoresence? ***** 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. ***** We are trying to image live cultured cells in Hanks Balanced Salt solution without phenol red. It's widefield imaging with a standard Chroma GFP filter set and I'm seeing something I've never observed before. There's very high background all over the field which quickly fades within a few seconds. If I stop the acquisition briefly and resume, the background goes right back up by about 25%. I've never seen this before. What could be the source of this in HBSS, or is it something else? Chris När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>. |
 
|
simon walker (BI)-2 |
Re: HBSS Autofluoresence?
|
|
In reply to this post by Chris O'Connell
*****
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. We’ve seen this a lot and come to the conclusion it’s riboflavin. I think it sticks to the glass/plastic on which the cells are growing and briefly fluoresces before bleaching. It’s replenished from the solution, so once the imaging has stopped the signal recovers. Using low riboflavin medium should resolve the issue. Simon Sent from my mobile > On 15 Jan 2019, at 19:03, Chris O'Connell <[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. > ***** > > We are trying to image live cultured cells in Hanks Balanced Salt solution without phenol red. It's widefield imaging with a standard Chroma GFP filter set and I'm seeing something I've never observed before. There's very high background all over the field which quickly fades within a few seconds. If I stop the acquisition briefly and resume, the background goes right back up by about 25%. I've never seen this before. What could be the source of this in HBSS, or is it something else? > > Chris The information transmitted in this email is directed only to the addressee. If you received this in error, please contact the sender and delete this email from your system. The contents of this e-mail are the views of the sender and do not necessarily represent the views of the Babraham Institute. Full conditions at: www.babraham.ac.uk<http://www.babraham.ac.uk/terms> |
|
|
In reply to this post by Chris O'Connell
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi All I use a DMEM based media with phenol red, for neural cell cultures. Luckily it does not seem to matter in our case (td-tomato and EOS2) and has the advantage that we can monitor pH. In fact, it’s how I realised the gas regulator was not working on the new spinning disc....the media was blue-pink after imaging instead of salmon pink. Best wishes Julia ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of MODEL, MICHAEL [[hidden email]] Sent: 16 January 2019 00:08 To: [hidden email] Subject: Re: HBSS Autofluoresence? **vendor reply** ***** 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. ***** If they are using HBSS, there shouldn't be any riboflavin. I would try HBSS from another bottle. ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Kilgore, Jason A. <[hidden email]> Sent: Tuesday, January 15, 2019 3:50 PM To: [hidden email] Subject: Re: HBSS Autofluoresence? **vendor reply** ***** 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 - Listserv Lists at the University of Minnesota<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. ***** ** Vendor reply ** We've seen the autofluorescence from riboflavin as well. It's part of what prompted us at Thermo Fisher to release our FluoroBrite DMEM and our Live Cell Imaging Solution (a HEPES derivative), which lack the autofluorescent components as well as phenol red (which can partially quench visible wavelength dyes). Jason Jason A. Kilgore Technical Application Scientist Molecular Probes / EVOS Tech Support Life Sciences Solutions -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Simon Walker Sent: Tuesday, January 15, 2019 12:13 PM To: [hidden email] Subject: Re: HBSS Autofluoresence? 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=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=Cq1a4MFZYpPDjPlNKXJqTvwiXJWUEXcO7KXa-UWXDyw&s=KPN4M-DdHcWHd0Qc_vsMdWUQjYrt74nH5fmnOw0ixY8&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=Cq1a4MFZYpPDjPlNKXJqTvwiXJWUEXcO7KXa-UWXDyw&s=NzQvOuTgiZmbdrlrYCxPpRpUIg1DC02GmW8PWlTxf-Y&e= and include the link in your posting. ***** Hi. We’ve seen this a lot and come to the conclusion it’s riboflavin. I think it sticks to the glass/plastic on which the cells are growing and briefly fluoresces before bleaching. It’s replenished from the solution, so once the imaging has stopped the signal recovers. Using low riboflavin medium should resolve the issue. Simon Sent from my mobile > On 15 Jan 2019, at 19:03, Chris O'Connell <[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=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhI > l8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=Cq > 1a4MFZYpPDjPlNKXJqTvwiXJWUEXcO7KXa-UWXDyw&s=KPN4M-DdHcWHd0Qc_vsMdWUQjY > rt74nH5fmnOw0ixY8&e= Post images on > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=Cq1a4MFZYpPDjPlNKXJqTvwiXJWUEXcO7KXa-UWXDyw&s=NzQvOuTgiZmbdrlrYCxPpRpUIg1DC02GmW8PWlTxf-Y&e= and include the link in your posting. > ***** > > We are trying to image live cultured cells in Hanks Balanced Salt solution without phenol red. It's widefield imaging with a standard Chroma GFP filter set and I'm seeing something I've never observed before. There's very high background all over the field which quickly fades within a few seconds. If I stop the acquisition briefly and resume, the background goes right back up by about 25%. I've never seen this before. What could be the source of this in HBSS, or is it something else? > > Chris The information transmitted in this email is directed only to the addressee. If you received this in error, please contact the sender and delete this email from your system. The contents of this e-mail are the views of the sender and do not necessarily represent the views of the Babraham Institute. Full conditions at: www.babraham.ac.uk<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.babraham.ac.uk_terms&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=Cq1a4MFZYpPDjPlNKXJqTvwiXJWUEXcO7KXa-UWXDyw&s=JjA0cRCfZXW6fdLp4zjRFbVXqcZHWGa19K1a3VUkMm4&e=> |
|
|
In reply to this post by simon walker (BI)-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. ***** Vendor comment: https://www.tebu-bio.com/blog/2014/03/28/lower-background-fluorescence-in-live-cell-imaging/ The link above, from 2014, supports Simon's riboflavin conclusion and how phenol red serves to quench it. MIstakenly people have felt that phenol itself was autofluorescent, but instead it is a quencher and thus can disturb overall fluorescence as well. And, though not written by us, the link does reference a product our company, Marker Gene, has been offering for over five years to positive reviews: Opti-Klear. No riboflavin or phenol red and some of what cells need to thrive for imaging sessions as long as 4 hours, without the need for CO2. Mike Ignatius markergene.com Eugene, Oregon On Tue, Jan 15, 2019 at 12:20 PM Simon Walker <[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. We’ve seen this a lot and come to the conclusion it’s riboflavin. I > think it sticks to the glass/plastic on which the cells are growing and > briefly fluoresces before bleaching. It’s replenished from the solution, so > once the imaging has stopped the signal recovers. Using low riboflavin > medium should resolve the issue. > Simon > > Sent from my mobile > > > On 15 Jan 2019, at 19:03, Chris O'Connell <[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. > > ***** > > > > We are trying to image live cultured cells in Hanks Balanced Salt > solution without phenol red. It's widefield imaging with a standard Chroma > GFP filter set and I'm seeing something I've never observed before. > There's very high background all over the field which quickly fades within > a few seconds. If I stop the acquisition briefly and resume, the > background goes right back up by about 25%. I've never seen this before. > What could be the source of this in HBSS, or is it something else? > > > > Chris > The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT > Registered Charity No. 1053902. > The information transmitted in this email is directed only to the > addressee. If you received this in error, please contact the sender and > delete this email from your system. The contents of this e-mail are the > views of the sender and do not necessarily represent the views of the > Babraham Institute. Full conditions at: www.babraham.ac.uk< > http://www.babraham.ac.uk/terms> > -- Ignatius Family 225 East 39th Ave Eugene OR 97405 541 525 4076 [hidden email] |
|
|
In reply to this post by Chris O'Connell
*****
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. ***** Thanks for all the input, everyone regarding riboflavin. I didn't prep the samples, but there may be trace amounts of media left after they added HBSS. Maybe enough to cause what we are seeing so we can troubleshoot it. Chris |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |