Re: Tetraspeck beads for PSFs (Experiment)

[mmodel]

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

I did some quick measurements on 0.17 um beads under confocal and an 60/1.4 oil objective. Yes, the z spread was smaller when the beads were in oil than when they were dry (even after a 0.66 scale adjustment). The xy dimensions were similar.
So I was wrong. Of course the full objective NA is not realized when light travels through air. How that plays out when the beads are so close to the surface I don't know but apparently it does matter.

Mike

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
Sent: Wednesday, August 8, 2018 6:17 AM
To: [hidden email]
Subject: Re: Tetraspeck beads for PSFs

*****
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.
*****

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:

> *****
> 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.
> *****
>
> 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:
> 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.
> *****
>
>
> 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**
>
> *****
> 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.
> *****
>
> 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:
> 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.
> *****
>
>> 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 [phone]
>>> [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_cg
>>> i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz
>>> 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
>>> Q&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWz
>>> z6NEeoPtsEIhu0PMk_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:  [phone]
>>> Fax:  [phone]

--
------------------------------------------------------------
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

[Sergey Tauger]

*****
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.
*****

Michael,

For calculations I prefer vectorial PSF model by Torok and Varga or its generalization by O.Haeberle ("Focusing of light through a stratified medium: a practical approach for computing microscope point spread functions", 2 parts: conventional and confocal microscopy). The model is available from COSMOS software by C.Preza. The results closely correspond to what I had with 80 nm beads and x60 NA 1.4 Oil objective; To avoid swelling and fading I used Apathy's mounting medium (aqueius, gum-arabic based, RI ~1.51, for receip please see article by J.A.Kiernan at http://publish.uwo.ca/~jkiernan/aqmount.htm). I'd recommend to give the model a try, the one in SVI Huygens software is simplified.

From my experience, coverslip thickness is also important. Most objectives are designed for #1.5 (170mkm) coverglasses, with acceptable error no more than 3mkm (167-173). If the glass is thicker or thinner, aplanatic point is shifted, and even if the object is just below the coverglass (up to 300-500 nm) its PSP becomes asymmetrical along Z-axis.

Best,
Sergey

08.08.2018, 20:03, "MODEL, MICHAEL" <[hidden email]>:

> *****
> 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 did some quick measurements on 0.17 um beads under confocal and an 60/1.4 oil objective. Yes, the z spread was smaller when the beads were in oil than when they were dry (even after a 0.66 scale adjustment). The xy dimensions were similar.
> So I was wrong. Of course the full objective NA is not realized when light travels through air. How that plays out when the beads are so close to the surface I don't know but apparently it does matter.
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
> Sent: Wednesday, August 8, 2018 6:17 AM
> To: [hidden email]
> Subject: Re: Tetraspeck beads for PSFs
>
> *****
> 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.
> *****
>
> 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:
>>  *****
>>  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.
>>  *****
>>
>>  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:
>>  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.
>>  *****
>>
>>  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**
>>
>>  *****
>>  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.
>>  *****
>>
>>  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:
>>  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.
>>  *****
>>
>>>  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 [phone]
>>>>  [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_cg
>>>>  i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz
>>>>  6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
>>>>  Q&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWz
>>>>  z6NEeoPtsEIhu0PMk_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: [phone]
>>>>  Fax: [phone]
>
> --
> ------------------------------------------------------------
> 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

[Remko Dijkstra]

*****
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 **

Dear all,

We like to take criticism seriously and will look more closely into our
online PSF calculator (https://svi.nl/NyquistCalculator) and see if we
can find anything that may explain the situation Mike initially described.
Although Mike already added "the full objective NA is not realized",
which indeed will effectively stretch the PSF, we would like to look
into this matter further.
I hope to be able to get back to you soon with a more detailed reply.

Kind regards,
On behalf of the SVI-Huygens support team,

Remko

***********************************************************
Remko Dijkstra, MSc
Imaging Specialist
Scientific Volume Imaging bv
Tel: + 31 35 642 1626
www.svi.nl
***********************************************************
For support matters contact: [hidden email]

On 08-08-18 19:02, MODEL, MICHAEL 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 did some quick measurements on 0.17 um beads under confocal and an 60/1.4 oil objective. Yes, the z spread was smaller when the beads were in oil than when they were dry (even after a 0.66 scale adjustment). The xy dimensions were similar.
> So I was wrong. Of course the full objective NA is not realized when light travels through air. How that plays out when the beads are so close to the surface I don't know but apparently it does matter.
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
> Sent: Wednesday, August 8, 2018 6:17 AM
> To: [hidden email]
> Subject: Re: Tetraspeck beads for PSFs
>
> *****
> 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.
> *****
>
> 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:
>> *****
>> 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.
>> *****
>>
>> 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:
>> 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.
>> *****
>>
>>
>> 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**
>>
>> *****
>> 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.
>> *****
>>
>> 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:
>> 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.
>> *****
>>
>>> 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 [phone]
>>>> [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_cg
>>>> i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz
>>>> 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
>>>> Q&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWz
>>>> z6NEeoPtsEIhu0PMk_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:  [phone]
>>>> Fax:  [phone]
> --
> ------------------------------------------------------------
> 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

[Remko Dijkstra]

*****
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 **

Dear all,

Aspromised, a more detailed reply on this subject.
Firstly, we would like to thank Mike, Andreas and Zdenek for observing
and reporting that our online Nyquist calculator page appears to display
an unexpected PSF in this case of extreme RI mismatch(oil-lens to air
embedding medium).

It is importantto note that theacquisition depth value does not have any
effect on the calculation of Nyquist rate values, and that PSF generator
in the Huygens software has always behaved as intended.

After your feedback we looked into the onlinePSF calculator, and we
realized that the definition of "zero position value"in the online user
interface was ambiguousandthat this is a source for confusion. For
optimal spherical aberration correction during Huygens deconvolution,
the PSF focus needs to be generated at the center of each brick
volume.As a result, the 'focus' of the generated PSF has some small
offset to the defined coverslip position. With an average practical
RI mismatch this offset hardly causes any visualdifference, but in an
extreme RI mismatch case even a small offset causes asevere spherical
aberration. For displaying the PSF 'at the coverslip position' in the
online calculator this behavioris clearly confusing, as we can conclude
from this thread.

Today we improved the online PSF calculator to eliminate any confusion
on the "zero depth". When you now set the acquisition depth at zero, the
online calculator actually generates the PSF at the coverslip surface.
At the same time we took the opportunity to improve the online
calculator by adding the Objective Quality setting parameter and Bessel
Beam and Lattice Light Sheet models, to makethe available parameters
more consistent with those recently developed in the Huygens
Softwareuser interfaces.
Feel free to try out the updated online PSF generator here:
https://svi.nl/NyquistCalculator

As an educational tool, wehave also created an interactive example that
demonstrates the effect of RI mismatch on the PSF shape at zero depth
("at the coverslip position"), and an additional example that shows the
effects at different acquisition depths. You can find these interactive
examples on the following page: https://svi.nl/TheoreticalPsf

Additionally, we would like to emphasize -- contrary to some earlier
suggestions -- that the Huygens Software uses advanced vector-based EM
diffraction theory to generate the theoretical 3D PSFs including
spherical aberration. The Huygens PSF generator algorithm is fully
multi-threaded, and is able to generate even very large PSF's very
efficiently and accurately. The Huygens Software can generate PSF's for
a wide rangeof microscope models, including: widefield, confocal, 4pi,
multi-photon, spinning-disk, STED and various light-sheet systems.
Through our website and newsletter we will keep you up to datewith the
latest developments, including the introduction of PSF models for new
microscope systems.

Lastly: in case you are interested in an experimental approach to
determinegeometrical distortion(fish-tank effect) caused by RI mismatch,
then I would recommend: Besseling et.al., 'Methods to calibrate and
scale axial distances in confocal microscopy as a function of refractive
index', https://doi.org/10.1111/jmi.12194

Kind regards,
on behalf of the SVI-Huygens support team,

Remko Dijkstra

***********************************************************
Remko Dijkstra, MSc
Imaging Specialist
Scientific Volume Imaging bv
Tel: + 31 35 642 1626
www.svi.nl
***********************************************************
For support matters contact: [hidden email]

On 14-08-18 08:24, Remko Dijkstra 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 **
>
> Dear all,
>
> We like to take criticism seriously and will look more closely into
> our online PSF calculator (https://svi.nl/NyquistCalculator) and see
> if we can find anything that may explain the situation Mike initially
> described.
> Although Mike already added "the full objective NA is not realized",
> which indeed will effectively stretch the PSF, we would like to look
> into this matter further.
> I hope to be able to get back to you soon with a more detailed reply.
>
> Kind regards,
> On behalf of the SVI-Huygens support team,
>
> Remko
>
> ***********************************************************
> Remko Dijkstra, MSc
> Imaging Specialist
> Scientific Volume Imaging bv
> Tel: + 31 35 642 1626
> www.svi.nl
> ***********************************************************
> For support matters contact: [hidden email]
>
> On 08-08-18 19:02, MODEL, MICHAEL 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 did some quick measurements on 0.17 um beads under confocal and an
>> 60/1.4 oil objective. Yes, the z spread was smaller when the beads
>> were in oil than when they were dry (even after a 0.66 scale
>> adjustment). The xy dimensions were similar.
>> So I was wrong. Of course the full objective NA is not realized when
>> light travels through air. How that plays out when the beads are so
>> close to the surface I don't know but apparently it does matter.
>>
>> Mike
>>
>> -----Original Message-----
>> From: Confocal Microscopy List <[hidden email]> On
>> Behalf Of Steffen Dietzel
>> Sent: Wednesday, August 8, 2018 6:17 AM
>> To: [hidden email]
>> Subject: Re: Tetraspeck beads for PSFs
>>
>> *****
>> 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.
>> *****
>>
>> 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:
>>> *****
>>> 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.
>>> *****
>>>
>>> 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:
>>> 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.
>>> *****
>>>
>>>
>>> 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**
>>>
>>> *****
>>> 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.
>>> *****
>>>
>>> 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:
>>> 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.
>>> *****
>>>
>>>> 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 [phone]
>>>>> [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_cg
>>>>> i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=q6k2DsTcEGCcCb_WtVSz
>>>>> 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
>>>>> Q&m=F8IDbXdJNbnAT6ffW_HjXIuMlAG3s16jAZb0eldCiQU&s=_ngqUkuraSCI6zAmWz
>>>>> z6NEeoPtsEIhu0PMk_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:  [phone]
>>>>> Fax:  [phone]
>> --
>> ------------------------------------------------------------
>> 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