Posted by
Nicolai.Urban@mpfi.org on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Tetraspeck-beads-for-PSFs-vendor-reply-tp7588497p7588511.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
Hi Jean,
most polystyrene fluorescent beads are susceptible to different mounting media, which can ruin the fluorescence of the beads within anything from minutes to days. Depending on the mounting media used, you can observe the beads swelling, the dye bleeding out into the media or the fluorescence simply getting quenched. This is not really an issue, however, if you either mount the beads in distilled water or air. This might cause small aberrations (refractive index mismatch, reflections at the coverslip-medium interface), but they tend to be minimal. If mounting in water, just prepare as usual and use water instead of your typical mounting medium. If using air, I typically used a little bit of mounting medium (spread of in a ring-like manner around the circumference of the coverslip), to form an air bubble on the inside, but still keep them isolated from the surrounding environment.
Mounted this way, I have maintained some fluorescent bead samples for several years now, with only a minor decrease in fluorescence intensity. Just keep them at a stable room temperature and out of any light, and they should be fairly stable. Just make sure not to drop them... which was the bane of most of my long-lived samples.
In my experience, having a reliable sample, of which you know precisely what it looks like on your microscope(s) is more important than having absolutely perfect PSFs, but remaking your samples every couple of days or weeks.
Good luck!
[hidden email]
>>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<<
Dr. Nicolai T. Urban
Max Planck Florida Institute
Jupiter, 33458 FL, USA
-----Original Message-----
From: Confocal Microscopy List <
[hidden email]> On Behalf Of Kilgore, Jason A.
Sent: Montag, 6. August 2018 02:00
To:
[hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**
*****
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&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803533904&sdata=Y0ljWknnOSMcLyYNMumYrRqlgHuUT%2FxIZX3DVLVKSXQ%3D&reserved=0Post images on
https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803533904&sdata=r8t%2FK%2FWBRtBFvORGJumvdgBCSjQMfqCxdq2gPCUbYfM%3D&reserved=0 and include the link in your posting.
*****
That would make sense. You see, most of the Molecular Probes fluorescent microspheres utilize BODIPY dyes, which are quite lipophilic. Thus, it wouldn't surprise me that they might be drawn out by oils.
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 MODEL, MICHAEL
Sent: Saturday, August 04, 2018 6:01 AM
To:
[hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**
Thanks to this discussion I realized what may be going on in our experiments as well. We are trying to image nonfluorescent Spherotech particles in slightly mismatched Cargille oils, and noticed that some oils produce something around the beads. I used to think it was trapped air but now understand that polystyrene gets partially dissolved in some of these oils.
Mike
________________________________
From: Confocal Microscopy List <
[hidden email]> on behalf of MODEL, MICHAEL <
[hidden email]>
Sent: Saturday, August 4, 2018 8:36 AM
To:
[hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**
*****
To join, leave or search the confocal microscopy listserv, go to:
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3D2sJQG16l24gxeA6DBeyaFYN7qoGvWnXfpGDTD5O-iDw%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803533904&sdata=bNkiqbGm7HiTn2k5URUafwGBDSkhED3wdp8Mhajr%2FYE%3D&reserved=0=LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3D2sJQG16l24gxeA6DBeyaFYN7qoGvWnXfpGDTD5O-iDw%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803533904&sdata=bNkiqbGm7HiTn2k5URUafwGBDSkhED3wdp8Mhajr%2FYE%3D&reserved=0=>
lists.umn.edu
[hidden email]: listserv archives. confocalmicroscopy
Post images on
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3DdUrbX4VrVqWyfa4aWtxfj_D2EmsBl6Qx54Nr0kNb72M%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&sdata=Cm7zZcHxE0qEIeYJ5Rc4pBaOdE13giUMsUdZtlOausE%3D&reserved=0= and include the link in your posting.
*****
I think you may simply keep dry beads on a coverslip and there is no need in a mounting medium. No spherical aberration would accumulate over a 0.1 um depth
Mike Model
________________________________
From: Confocal Microscopy List <
[hidden email]> on behalf of Andreas Bruckbauer <
[hidden email]>
Sent: Saturday, August 4, 2018 4:01 AM
To:
[hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**
*****
To join, leave or search the confocal microscopy listserv, go to:
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3D2sJQG16l24gxeA6DBeyaFYN7qoGvWnXfpGDTD5O-iDw%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&sdata=TKeWG3t1Bywh0JoD6Ts24NyhQmBE8xJiqzqrWPwaVWw%3D&reserved=0=LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3D2sJQG16l24gxeA6DBeyaFYN7qoGvWnXfpGDTD5O-iDw%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&sdata=TKeWG3t1Bywh0JoD6Ts24NyhQmBE8xJiqzqrWPwaVWw%3D&reserved=0=>
lists.umn.edu
[hidden email]: listserv archives. confocalmicroscopy
Post images on
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3DdUrbX4VrVqWyfa4aWtxfj_D2EmsBl6Qx54Nr0kNb72M%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&sdata=Cm7zZcHxE0qEIeYJ5Rc4pBaOdE13giUMsUdZtlOausE%3D&reserved=0= and include the link in your posting.
*****
Thanks Jean for raising this issue, I have a similar problem, the dye diffuses out of the beads and leaves a large spot around them. It can happen very quickly when the slides get to warm and the current heatwave is not helping. Putting them in the fridge is also not an option because of drift once put back on the microscope.
This is with mounting medium from Moleculr Probes for beads.
No solution yet, I don't think it used to be like this, have you changed the beads?
Aren't 100 nm beads too big for PSF measurements on the Elyra? 40 nm should be better.
Best wishes
Andreas
Sent from my phone
> On 3 Aug 2018, at 22:05, Kilgore, Jason A. <
[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furlde> fense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-&
> data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62
> 063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&a
> mp;sdata=2DlVDcjPD1Gczop%2B%2BnuaURDW%2B8r8vVmkPqIZKLMBpso%3D&rese
> rved=0
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=q6k2DsTcEGCcCb_WtVSz6hhI
> l8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=7L
> M14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8&s=2sJQG16l24gxeA6DBeyaFYN7qo
> GvWnXfpGDTD5O-iDw&e= Post images on
>
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIF-g%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3D7LM14j0RRNKXNBbNA6rOihPlP1-soxMJ0ZS6FlUeWz8%26s%3DdUrbX4VrVqWyfa4aWtxfj_D2EmsBl6Qx54Nr0kNb72M%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&sdata=Cm7zZcHxE0qEIeYJ5Rc4pBaOdE13giUMsUdZtlOausE%3D&reserved=0= and include the link in your posting.
> *****
>
>
> ** Vendor reply **
>
> Polystyrene microspheres, such as the TetraSpecks, will swell in the presence of certain solvents. So you'll want to use an aqueous-based mounting media, preferably one that cures (such as ProLong products or Fluoromount-G) instead of using an organic solvent-based one, like Cytoseal-60. When dye-labeled microspheres like these swell, the dye is free to diffuse out of the polystyrene matrix.
>
> Cheers,
>
> 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 Jean Ross
> Sent: Friday, August 03, 2018 12:49 PM
> To:
[hidden email]
> Subject: Tetraspeck beads for PSFs
>
> 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://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furlde> fense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-&
> data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62
> 063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&a
> mp;sdata=2DlVDcjPD1Gczop%2B%2BnuaURDW%2B8r8vVmkPqIZKLMBpso%3D&rese
> rved=0
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz6hhI
> l8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=F8
> IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWzz6NEeoPt
> sEIhu0PMk_Stm2Gko&e= Post images on
>
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIBaQ%26c%3Dq6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU%26r%3DMVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ%26m%3DF8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU%26s%3DO2T49sxgjykKvUtqbrfKaSktZURKhrNNnPd3CQna8Gc%26e&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C4372055327a54fba751408d5fb62063d%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636691320803543916&sdata=YBRBOD%2Bwq%2BsKWcnPhHZXQYE8Bvm9p3xfUE1EmDjcOKo%3D&reserved=0= and include the link in your posting.
> *****
>
> Hi Everyone,
>
> I have been preparing 0.1um Tetraspeck bead slides to use when measuring point spread functions on our Zeiss Elyra PS1 super resolution microscope
> and Zeiss LSM880 confocal with Airyscan. The problem is that over time
> the beads appear to swell to about 3 times in size and lose much of
> their fluorescence. I have tried many different mounting medias
> (hardening and
> nonhardening) all with the same result. Has anyone else experienced this problem? Any suggestions to solve the problem would be appreciated.
>
> Thanks,
> Jean
>
> --
> Jean Ross
> Delaware Biotechnology Institute, BioImaging Center University of
> Delaware
> 15 Innovation Way
> Suite 117
> Newark, DE 19711
> Phone: (302)831-0620
> Fax: (302)831-4841