Posted by
Zdenek Svindrych-2 on
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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
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Od: MODEL, MICHAEL <
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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
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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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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
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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
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> On 4 Aug 2018, at 13:36, MODEL, MICHAEL <
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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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> Sent from my phone
>
>> On 3 Aug 2018, at 22:05, Kilgore, Jason A. <Jason.Kilgore@THERMOFISHER.
COM> 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:
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On Behalf Of Jean Ross
>> Sent: Friday, August 03, 2018 12:49 PM
>> To:
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>> 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
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