Confocal Microscopy List - Re: Tetraspeck beads for PSFs **vendor reply**

Re: Tetraspeck beads for PSFs vendor reply

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Tetraspeck-beads-for-PSFs-vendor-reply-tp7588497p7588514.html

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I have mounted beads in Sylgard 184 with no obvious issues with
fluorescence for long periods (years). What ever is in the mix (silicone +
hardener) does not appear to bother the beads.

Craig

On Mon, Aug 6, 2018 at 8:29 AM [hidden email] <
[hidden email]> wrote:

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> 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**
>
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> 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]
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> -----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
>
>
>
> ________________________________
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> Sent: Saturday, August 4, 2018 8:36 AM
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> Subject: Re: Tetraspeck beads for PSFs **vendor reply**
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> 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**
>
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>
> 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:
> >
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> >
> >
> > ** 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
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> >
> >
> >
> > -----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
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> >
> > 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
>