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Re: Tetraspeck beads for PSFs vendor reply

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Kilgore, Jason A.

Re: Tetraspeck beads for PSFs vendor reply

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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 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.

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=O2T49sxgjykKvUtqbrfKaSktZURKhrNNnPd3CQna8Gc&e= and include the link in your posting.
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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

0000001ed7f52e4a-dmarc-request

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:
>
> *****
> 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 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://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWzz6NEeoPtsEIhu0PMk_Stm2Gko&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=O2T49sxgjykKvUtqbrfKaSktZURKhrNNnPd3CQna8Gc&e= 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

James D. Manton

Re: Tetraspeck beads for PSFs vendor reply

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> Aren’t 100 nm beads too big for PSF measurements on the Elyra? 40 nm should be better.
There are a couple of methods that allow you to use larger beads, and
hence benefit from the increased signal, which I like but which seem to
be infrequently used. Both these approaches are detailed in an excellent
article from Hanser et al. in the Journal of Microscopy
(https://doi.org/10.1111/j.0022-2720.2004.01393.x).

The first method relies on knowing the Fourier transform of the bead
shape and dividing the measured optical transfer function by this
distribution (as what you have actually measured is the true
instrumental point spread function convolved with the intensity
distribution of the bead). For a uniformly filled spherical bead, the
Fourier domain distribution function, b(k), is 3h(πkd), where d is the
bead diameter and h(x) = sin(x) / x^3 - cos(x) / x^2.

The second method, and the main focus of the Hanser et al. article, is
generating a pupil function via phase retrieval and using this to
produce a simulated point spread function. As the final PSF is
simulated, no noise corrupts the result, which can aid in deconvolution
if the only available experimental PSFs have a low SNR. In addition,
Zernike polynomials can be fitted to this pupil function to provide a
quantitative measure of aberrations present within the microscope. Once
again, the final OTF is produced by dividing the inferred OTF by the
bead distribution.

While the first method is relatively simple to implement, the second is
quite complex, especially for high numerical aperture objectives in
which a vectorial theory of diffraction must be used. The second also
relies on an empirical scaling function to better match experimental
results to those generated by the theory, as high spatial frequencies
are over-emphasised in the phase retrieval process. For this reason,
unless I want a measure of the aberrations present in a system, I
generally stick with just dividing the experimentally-measured OTF by
the Fourier transform of the bead.

Best wishes,
James

mmodel

Re: Tetraspeck beads for PSFs vendor reply

In reply to this post by 0000001ed7f52e4a-dmarc-request

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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

mmodel

Re: Tetraspeck beads for PSFs vendor reply

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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**

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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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Post images on http://www.imgur.com 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

0000001ed7f52e4a-dmarc-request

Re: Tetraspeck beads for PSFs vendor reply

In reply to this post by mmodel

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>I think you may simply keep dry >beads on a coverslip

The bead may be 100 nm, but you want to measure the full PSF of about 4 micron, so proper mounting is important.

Andreas

Sent from my phone

> On 4 Aug 2018, at 13:36, MODEL, MICHAEL <[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.
> *****
>
> 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:
> 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.
> *****
>
> 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:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com 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://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWzz6NEeoPtsEIhu0PMk_Stm2Gko&e=
>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=O2T49sxgjykKvUtqbrfKaSktZURKhrNNnPd3CQna8Gc&e= 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

mmodel

Re: Tetraspeck beads for PSFs vendor reply

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Andreas,

As far as I understand, spherical aberration due to refractive mismatch becomes noticeable only at larger depths (for example, Booth and Wilson, J Biomed Optics, 6, 266-272, 2001)

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Andreas Bruckbauer <[hidden email]>
Sent: Saturday, August 4, 2018 12:25 PM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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*****

>I think you may simply keep dry >beads on a coverslip

The bead may be 100 nm, but you want to measure the full PSF of about 4 micron, so proper mounting is important.

Andreas

Sent from my phone

0000001ed7f52e4a-dmarc-request

Re: Tetraspeck beads for PSFs vendor reply

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Well observing a bead through the microscope and checking for symmetry when focussing up and down will tell. Alternatively you can go to the SVI webpage and calculate theoretical PSFs with the Nyquist calculator https://svi.nl/NyquistCalculator (tick the box for calculating PSF), the differences between water and mounting medium are visible and important for deconvolution and SIM.

best wishes

Andreas

-----Original Message-----
From: MODEL, MICHAEL <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, 5 Aug 2018 14:23
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

*****
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*****

Andreas,

As far as I understand, spherical aberration due to refractive mismatch becomes noticeable only at larger depths (for example, Booth and Wilson, J Biomed Optics, 6, 266-272, 2001)

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Andreas Bruckbauer <[hidden email]>
Sent: Saturday, August 4, 2018 12:25 PM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

*****
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*****

>I think you may simply keep dry >beads on a coverslip

The bead may be 100 nm, but you want to measure the full PSF of about 4 micron, so proper mounting is important.

Andreas

Sent from my phone

mmodel

Re: Tetraspeck beads for PSFs vendor reply

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Interesting. But something is not right there. Their calculator gives a difference between n=1 and n=1.5 for a zero depth. A zero depth means that fluorescent emission does not pass through the "wrong" medium on its way to the objective (unless it is a 4pi microscope), so its refractive index shouldn't matter. Am I missing something?

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Andreas Bruckbauer <[hidden email]>
Sent: Sunday, August 5, 2018 10:44 AM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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*****

Well observing a bead through the microscope and checking for symmetry when focussing up and down will tell. Alternatively you can go to the SVI webpage and calculate theoretical PSFs with the Nyquist calculator https://svi.nl/NyquistCalculator (tick the box for calculating PSF), the differences between water and mounting medium are visible and important for deconvolution and SIM.

best wishes

Andreas

-----Original Message-----
From: MODEL, MICHAEL <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, 5 Aug 2018 14:23
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

*****
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*****

Andreas,

As far as I understand, spherical aberration due to refractive mismatch becomes noticeable only at larger depths (for example, Booth and Wilson, J Biomed Optics, 6, 266-272, 2001)

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Andreas Bruckbauer <[hidden email]>
Sent: Saturday, August 4, 2018 12:25 PM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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>I think you may simply keep dry >beads on a coverslip

The bead may be 100 nm, but you want to measure the full PSF of about 4 micron, so proper mounting is important.

Andreas

Sent from my phone

0000001ed7f52e4a-dmarc-request

Re: Tetraspeck beads for PSFs vendor reply

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You are focussing quite a bit into the air at n=1, but you are right, the fluorescence still comes from zero depth. Maybe it is the normal fish tank effect, the length of the psf is larger by a factor of 1.5 when focussing into air and together with the enhanced gamma and false color it looks like this?

best wishes

Andreas

-----Original Message-----
From: MODEL, MICHAEL <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, 5 Aug 2018 16:42
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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Interesting. But something is not right there. Their calculator gives a difference between n=1 and n=1.5 for a zero depth. A zero depth means that fluorescent emission does not pass through the "wrong" medium on its way to the objective (unless it is a 4pi microscope), so its refractive index shouldn't matter. Am I missing something?

Mike

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Well observing a bead through the microscope and checking for symmetry when focussing up and down will tell. Alternatively you can go to the SVI webpage and calculate theoretical PSFs with the Nyquist calculator https://svi.nl/NyquistCalculator (tick the box for calculating PSF), the differences between water and mounting medium are visible and important for deconvolution and SIM.

best wishes

Andreas

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Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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Andreas,

As far as I understand, spherical aberration due to refractive mismatch becomes noticeable only at larger depths (for example, Booth and Wilson, J Biomed Optics, 6, 266-272, 2001)

Mike

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>I think you may simply keep dry >beads on a coverslip

The bead may be 100 nm, but you want to measure the full PSF of about 4 micron, so proper mounting is important.

Andreas

Sent from my phone

Kilgore, Jason A.

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

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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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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

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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

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> 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
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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
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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

Nicolai.Urban@mpfi.org

Re: Tetraspeck beads for PSFs vendor reply

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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

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Sent: Montag, 6. August 2018 02:00
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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
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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

________________________________
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Sent: Saturday, August 4, 2018 8:36 AM
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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

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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 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.
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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 click links or open attachments unless you recognize the sender and know the content is safe.
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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

Zdenek Svindrych-2

Re: Tetraspeck beads for PSFs

In reply to this post by mmodel

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Hi Mike,
indeed, it seems that Huygens PSF calculator does not work in this case (oil
immersion, air sample, zero distance from coverslip). Even if you reduce NA
below one (effective NA cannot be higher than one if sample is in air and is
outside the TIR zone), the PSFs (1.5 vs 1.0 sample RI) look drastically
different.
In fact, full 3D electromagnetic treatment would be needed to calculate the
exact PSF, I'm afraid. For a 100 nm bead the result should be quite similar
to the ideal case, but for 200 nm beads some of the fluorescence may need to
travel through quite a bit of air (and polystyrene) so the near-field
coupling may be reduced (you'll get less supercritical-angle fluorescence,
that is, the light with NA>1).

More importantly, there is a reflection from the air-glass interface (the
glass slide), which is typically only few microns away from the bead. This
may also distort the PSF!

That being said, I don't have a reliable protocol for embedding tetraspeck
beads in a permanent slide...

Best, zdenek

--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
email: [hidden email]

---------- Původní e-mail ----------
Od: MODEL, MICHAEL <[hidden email]>
Komu: [hidden email]
Datum: 5. 8. 2018 11:45:21
Předmět: Re: Tetraspeck beads for PSFs **vendor reply**
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Interesting. But something is not right there. Their calculator gives a
difference between n=1 and n=1.5 for a zero depth. A zero depth means that
fluorescent emission does not pass through the "wrong" medium on its way to
the objective (unless it is a 4pi microscope), so its refractive index
shouldn't matter. Am I missing something?

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf
of Andreas Bruckbauer <[hidden email]>
Sent: Sunday, August 5, 2018 10:44 AM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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Well observing a bead through the microscope and checking for symmetry when
focussing up and down will tell. Alternatively you can go to the SVI webpage
and calculate theoretical PSFs with the Nyquist calculator https://svi.nl/
NyquistCalculator (tick the box for calculating PSF), the differences
between water and mounting medium are visible and important for
deconvolution and SIM.

best wishes

Andreas

-----Original Message-----
From: MODEL, MICHAEL <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, 5 Aug 2018 14:23
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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Andreas,

As far as I understand, spherical aberration due to refractive mismatch
becomes noticeable only at larger depths (for example, Booth and Wilson, J
Biomed Optics, 6, 266-272, 2001)

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf
of Andreas Bruckbauer <[hidden email]>
Sent: Saturday, August 4, 2018 12:25 PM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs **vendor reply**

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>I think you may simply keep dry >beads on a coverslip

The bead may be 100 nm, but you want to measure the full PSF of about 4
micron, so proper mounting is important.

Andreas

Sent from my phone

> On 4 Aug 2018, at 13:36, MODEL, MICHAEL <[hidden email]> wrote:
>
> *****
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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**
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> [hidden email]: listserv archives. confocalmicroscopy
>
>
>
> Post images on http://www.imgur.com 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. <Jason.Kilgore@THERMOFISHER.
COM> 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 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:
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PMk_Stm2Gko&e=
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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
"

Craig Brideau

Re: Tetraspeck beads for PSFs vendor reply

In reply to this post by Nicolai.Urban@mpfi.org

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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**
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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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]
>
> This communication is intended solely for the individual/entity to whom it
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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
>
>
>
> ________________________________
> 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**
>
> *****
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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**
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> and include the link in your posting.
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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:
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
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> 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
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> 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
>

Jerry Chao

Re: Tetraspeck beads for PSFs vendor reply

In reply to this post by Kilgore, Jason A.

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Hello all. For those who are interested we carried out a relatively extensive study on the influence of bead size in the context of estimating the location of a bead by fitting a PSF. In short, we found that beads with a diameter of up to 100 nm yielded very similar results, suggesting that beads 100 nm or less in diameter would be suitable for PSF measurements. (https://doi.org/10.1371/journal.pone.0134112)

Best regards,
Jerry

Jerry Chao, Ph.D.
Department of Biomedical Engineering
Texas A&M University
Email: [hidden email]

Steffen Dietzel

Re: Tetraspeck beads for PSFs

In reply to this post by mmodel

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Michael,

with 100 nm beads your fluorescence may pass up to 100 nm through the
'wrong' medium, and your excitation light for several microns, when you
record the PSF in 3D. So if the bead is embedded in air (n=1), when you
focus below the bead maybe the excitation PSF is such that in can cause
this difference?

Steffen

Am 05.08.2018 um 17:41 schrieb MODEL, MICHAEL:

> *****
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> Interesting. But something is not right there. Their calculator gives a difference between n=1 and n=1.5 for a zero depth. A zero depth means that fluorescent emission does not pass through the "wrong" medium on its way to the objective (unless it is a 4pi microscope), so its refractive index shouldn't matter. Am I missing something?
>
>
> Mike
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Andreas Bruckbauer <[hidden email]>
> Sent: Sunday, August 5, 2018 10:44 AM
> To: [hidden email]
> Subject: Re: Tetraspeck beads for PSFs **vendor reply**
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
>
> Well observing a bead through the microscope and checking for symmetry when focussing up and down will tell. Alternatively you can go to the SVI webpage and calculate theoretical PSFs with the Nyquist calculator https://svi.nl/NyquistCalculator (tick the box for calculating PSF), the differences between water and mounting medium are visible and important for deconvolution and SIM.
>
> best wishes
>
> Andreas
>
>
>
> -----Original Message-----
> From: MODEL, MICHAEL <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Sun, 5 Aug 2018 14:23
> Subject: Re: Tetraspeck beads for PSFs **vendor reply**
>
> *****
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> Andreas,
>
>
> As far as I understand, spherical aberration due to refractive mismatch becomes noticeable only at larger depths (for example, Booth and Wilson, J Biomed Optics, 6, 266-272, 2001)
>
>
> Mike
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Andreas Bruckbauer <[hidden email]>
> Sent: Saturday, August 4, 2018 12:25 PM
> To: [hidden email]
> Subject: Re: Tetraspeck beads for PSFs **vendor reply**
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
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> *****
>
>> I think you may simply keep dry >beads on a coverslip
> The bead may be 100 nm, but you want to measure the full PSF of about 4 micron, so proper mounting is important.
>
> Andreas
>
> Sent from my phone
>
>> On 4 Aug 2018, at 13:36, MODEL, MICHAEL <[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.
>> *****
>>
>> 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:
>> 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.
>> *****
>>
>> 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:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com 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]
>>>
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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
>>>
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>>>
>>> *****
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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

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de