methanol immersion substitute

[Reto Fiolka]

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Dear all

We have a 16X NA 0.8 water immersion objective and it showed some spherical aberrations.

Since for our application we are not fixed in immersion fluid (non biological specimens), we
tried immersion fluids with slightly higher and lower refractive indices. Going slightly higher
in n was the wrong direction. The only thing to go lower in n available to us was Methanol
and Methanol-water mixtures.

With pure Methanol immersion (yes, this is a very bad idea), the spherical aberrations were
practically gone.

Does anyone know a fluid that has similar refractive index like Methanol (1.328) or lower
that could be mixed with water?  Methanol is kind of bad as an immersion fluid..


Thanks!

Reto

[Csúcs Gábor-3]

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Dear Reto,

If I am not mistaken, Zeiss sells an immersion oil that has a similar (identical) refractive index to water (but it is an oil). Could this work for you?

Greetings      Gabor


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Reto Fiolka
Sent: Saturday, December 6, 2014 11:05 PM
To: [hidden email]
Subject: methanol immersion substitute

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Dear all

We have a 16X NA 0.8 water immersion objective and it showed some spherical aberrations.

Since for our application we are not fixed in immersion fluid (non biological specimens), we tried immersion fluids with slightly higher and lower refractive indices. Going slightly higher in n was the wrong direction. The only thing to go lower in n available to us was Methanol and Methanol-water mixtures.

With pure Methanol immersion (yes, this is a very bad idea), the spherical aberrations were practically gone.

Does anyone know a fluid that has similar refractive index like Methanol (1.328) or lower that could be mixed with water?  Methanol is kind of bad as an immersion fluid..


Thanks!

Reto

[George McNamara]

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Hi reto,

I do not know if these are objective lens friendly:

http://www.focenter.com/script_0010_new.asp?ProductID=36025&cat=8&tid=128&navgid=8
AngstromLink AL-5229 Optical Coupling Fluid with a 1.29 index of
refraction in a 30cc bottle.

http://www.focenter.com/script_0010_new.asp?ProductID=9999&cat=1&tid=0
AngstromLink AL-2233 Optical Coating Diluent, 480cc Bottle with a 1.33
index of refraction. (Country of Use and Application must be specified
on purchase order. Cannot be used in Healthcare Applications)

These and methanol were the only R.I. < 1.33 entries in my Multi-Probe
Microscopy doc (pdf page 619)
http://works.bepress.com/gmcnamara/2/

//

As Gabor just mentioned, Zeiss Immersol W is available with the same
R.I. as water. Does not evaporate, though I doubt it is miscible with water.

You could use deconvolution to (try to) correct for spherical
aberration. I suggest you check with the 'decon' vendors, also the
various free ImageJ plugins, to see if they offer this.

George



On 12/6/2014 4:05 PM, Reto Fiolka wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all
>
> We have a 16X NA 0.8 water immersion objective and it showed some spherical aberrations.
>
> Since for our application we are not fixed in immersion fluid (non biological specimens), we
> tried immersion fluids with slightly higher and lower refractive indices. Going slightly higher
> in n was the wrong direction. The only thing to go lower in n available to us was Methanol
> and Methanol-water mixtures.
>
> With pure Methanol immersion (yes, this is a very bad idea), the spherical aberrations were
> practically gone.
>
> Does anyone know a fluid that has similar refractive index like Methanol (1.328) or lower
> that could be mixed with water?  Methanol is kind of bad as an immersion fluid..
>
>
> Thanks!
>
> Reto
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Richard E. Edelmann]

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Hello Reto:

Cargille makes a full line of indexing oils\fluids.  I would check with them

http://www.cargille.com

I have only used their immersion oils but they meet all manufacturers specs.


On Sat, Dec 6, 2014 at 5:05 PM, Reto Fiolka <[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.
> *****
>
> Dear all
>
> We have a 16X NA 0.8 water immersion objective and it showed some
> spherical aberrations.
>
> Since for our application we are not fixed in immersion fluid (non
> biological specimens), we
> tried immersion fluids with slightly higher and lower refractive indices.
> Going slightly higher
> in n was the wrong direction. The only thing to go lower in n available to
> us was Methanol
> and Methanol-water mixtures.
>
> With pure Methanol immersion (yes, this is a very bad idea), the spherical
> aberrations were
> practically gone.
>
> Does anyone know a fluid that has similar refractive index like Methanol
> (1.328) or lower
> that could be mixed with water?  Methanol is kind of bad as an immersion
> fluid..
>
>
> Thanks!
>
> Reto
>

--

Richard E. Edelmann, Ph.D.
Center for Advanced Microscopy & Imaging, Director
9C Upham Hall
Miami University
Oxford, OH 45056
Phone: [phone]
Email: [hidden email]
Web: www.cami.muohio.edu

[Zdenek Svindrych]

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Hi Reto,
fluorinated hydrocarbons usually have low refractive index, and some are
miscible with water,e.g. trifluoracetic acid
http://macro.lsu.edu/HowTo/solvents/Refractive%20Index.htm
(maybe its ammonium salt...)
trifluorethanol
http://www.halocarbon.com/halocarbon_media/Trifluoroethanol_208.pdf
tetrafluoropropanol
http://www.sigmaaldrich.com/catalog/product/aldrich/326275?lang=en&region=US

isopropyl fluoracetate
http://www.sigmaaldrich.com/catalog/product/aldrich/374083?lang=en&region=US


..etc.. But I'm not sure how healthy they are (for you and for your lenses).

Best,

zdenek


---------- Původní zpráva ----------
Od: Reto Fiolka <[hidden email]>
Komu: [hidden email]
Datum: 6. 12. 2014 17:07:00
Předmět: methanol immersion substitute

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Dear all

We have a 16X NA 0.8 water immersion objective and it showed some spherical
aberrations.

Since for our application we are not fixed in immersion fluid (non
biological specimens), we
tried immersion fluids with slightly higher and lower refractive indices.
Going slightly higher
in n was the wrong direction. The only thing to go lower in n available to
us was Methanol
and Methanol-water mixtures.

With pure Methanol immersion (yes, this is a very bad idea), the spherical
aberrations were
practically gone.

Does anyone know a fluid that has similar refractive index like Methanol
(1.328) or lower
that could be mixed with water? Methanol is kind of bad as an immersion
fluid..


Thanks!

Reto"

[Mark Cannell-2]

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Hi Reto

You don’t say if this objective is a dipping objective or one designed to use a coverslip. If the latter you need to use a different coverslip?

Cheers Mark

>
>
>
> Dear all
>
> We have a 16X NA 0.8 water immersion objective and it showed some spherical
> aberrations.
>
> Since for our application we are not fixed in immersion fluid (non
> biological specimens), we
> tried immersion fluids with slightly higher and lower refractive indices.
> Going slightly higher
> in n was the wrong direction. The only thing to go lower in n available to
> us was Methanol
> and Methanol-water mixtures.
>
> With pure Methanol immersion (yes, this is a very bad idea), the spherical
> aberrations were
> practically gone.
>
> Does anyone know a fluid that has similar refractive index like Methanol
> (1.328) or lower
> that could be mixed with water? Methanol is kind of bad as an immersion
> fluid..
>
>
> Thanks!
>
> Reto"

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Reto Fiolka]

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Hi Mark

It is a water dipping objective, so not designed to use a coverslip and it also has no
correction collar.
We operate it at 488nm, while it has
been designed for 2Photon imaging.
So it may not be well chromatically corrected in the vis.

But the spherical aberrations are mainly gone with Methanol immersion, hence the quest to
find a more friendly fluid with similar n.

We will look into the fluorocarbons and if there is a Zeiss oil as Gabor suggested.

Thanks,
Reto

[Reto Fiolka]

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Thanks also to Zdenek and George for the links.

The said objective is part of a Tony Wilson style perfect focusing system [1,2], thus it
does not face a sample, only a mirror and we can throw in almost anything that does not
hurt the optics.

As the name suggests, we would like to have it perfect (diffraction limited, highest strehl
ratio).

I have not done a simulation, but my intuition let me think that the immersion medium in
the remote objective could also pre-compensate some global refractive index mismatch
that the second objective may see that faces the sample. Would surely be fun to try if
one can remotely optimize the focus in the sample if it has some spherical aberrations
(presumably it will have, as it is the same type of objective).

Best,
Reto


PS: the remote focusing is a brilliant invention by the Wilson group and it is fun to build
one:
(1) Botcherby, Edward J., et al. "Aberration-free optical refocusing in high numerical
aperture microscopy." Optics letters 32.14 (2007).

(2) Botcherby, Edward J., et al. "Aberration-free three-dimensional multiphoton imaging
of neuronal activity at kHz rates." Proceedings of the National Academy of Sciences 109.8
(2012): 2919-2924.

[Mark Cannell-2]

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Hi Reto

I suggest you look at the system tube length. If incorrect you get spherical aberration…. Similarly adding or subtracting tune length will add or subtract spherical aberration. If you carefully measure objective magnification it will show if the tube length is correct.

Cheers Mark

On 8/12/2014, at 12:00 am, Reto Fiolka <[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.
> *****
>
> Thanks also to Zdenek and George for the links.
>
> The said objective is part of a Tony Wilson style perfect focusing system [1,2], thus it
> does not face a sample, only a mirror and we can throw in almost anything that does not
> hurt the optics.
>
> As the name suggests, we would like to have it perfect (diffraction limited, highest strehl
> ratio).
>
> I have not done a simulation, but my intuition let me think that the immersion medium in
> the remote objective could also pre-compensate some global refractive index mismatch
> that the second objective may see that faces the sample. Would surely be fun to try if
> one can remotely optimize the focus in the sample if it has some spherical aberrations
> (presumably it will have, as it is the same type of objective).
>
> Best,
> Reto
>
>
> PS: the remote focusing is a brilliant invention by the Wilson group and it is fun to build
> one:
> (1) Botcherby, Edward J., et al. "Aberration-free optical refocusing in high numerical
> aperture microscopy." Optics letters 32.14 (2007).
>
> (2) Botcherby, Edward J., et al. "Aberration-free three-dimensional multiphoton imaging
> of neuronal activity at kHz rates." Proceedings of the National Academy of Sciences 109.8
> (2012): 2919-2924.

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[jerie]

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Hi Reto,

not sure if I recall the concept correctly, but I remember having thought
in one of the pink pig presentations, if it was possible to use an
adaptable optical element instead of the mirror to correct for sample
aberrations. Would you think that would work?

Cheers, Jens



Visiting Scientist @ Center for Technological Development in Health (CDTS),
Oswaldo Cruz Foundation (Fiocruz), Ministry of Health, Rio de Janeiro,
Brazil.
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype jens.rietdorf

On Mon, Dec 8, 2014 at 6:21 AM, Mark Cannell <[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.
> *****
>
>
> Hi Reto
>
> I suggest you look at the system tube length. If incorrect you get
> spherical aberration.... Similarly adding or subtracting tune length will add
> or subtract spherical aberration. If you carefully measure objective
> magnification it will show if the tube length is correct.
>
> Cheers Mark
>
> On 8/12/2014, at 12:00 am, Reto Fiolka <[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.
> > *****
> >
> > Thanks also to Zdenek and George for the links.
> >
> > The said objective is part of a Tony Wilson style perfect focusing
> system [1,2], thus it
> > does not face a sample, only a mirror and we can throw in almost
> anything that does not
> > hurt the optics.
> >
> > As the name suggests, we would like to have it perfect (diffraction
> limited, highest strehl
> > ratio).
> >
> > I have not done a simulation, but my intuition let me think that the
> immersion medium in
> > the remote objective could also pre-compensate some global refractive
> index mismatch
> > that the second objective may see that faces the sample. Would surely be
> fun to try if
> > one can remotely optimize the focus in the sample if it has some
> spherical aberrations
> > (presumably it will have, as it is the same type of objective).
> >
> > Best,
> > Reto
> >
> >
> > PS: the remote focusing is a brilliant invention by the Wilson group and
> it is fun to build
> > one:
> > (1) Botcherby, Edward J., et al. "Aberration-free optical refocusing in
> high numerical
> > aperture microscopy." Optics letters 32.14 (2007).
> >
> > (2) Botcherby, Edward J., et al. "Aberration-free three-dimensional
> multiphoton imaging
> > of neuronal activity at kHz rates." Proceedings of the National Academy
> of Sciences 109.8
> > (2012): 2919-2924.
>
> Mark  B. Cannell Ph.D. FRSNZ
> Professor of Cardiac Cell Biology
> School of Physiology &  Pharmacology
> Medical Sciences Building
> University of Bristol
> Bristol
> BS8 1TD UK
>
> [hidden email]
>

[Vitaly Boyko]

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Dear List,
I am looking for fluorescent beads which do not form clusters (aggregates). Tetra-spec beads form clusters, ubnfortunately. Would someone have any suggestions?
Thank you.
Vitaly   

[Reto Fiolka]

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Hi Jens

Your idea would be challenging with current technology. In the focal plane of an objective,
the dimensions are microscopic and thus out of reach of current actuator technologies.

If one would want to try to correct local, sample induced aberrations (i.e. shift
variant aberrations), you can place an adaptable optical element (deformable mirror,
SLM) in an image plane, but its correction would be only correct locally. This scheme is
more realistic with current technology.

There are ideas to use multiple adaptive optical elements in different image planes to
correct locally varying, sample induced aberrations (multi-conjugate adaptive optics), but
the only working variants I am aware of are in astronomy and ophthalmology.

Na Ji (at Janelia) has once converted an water immersion objective into an air objective
[1] as a proof of principle for their AO system. I still wonder if one could use the Wilson
perfect focusing system to make the sample objective work with a slightly different
immersion medium than it was designed for via pre-compensation at the remote
objective (by using an immersion that has the same difference in refractive index, but
with opposite sign).

Kind of AO for poor people...

Best,
Reto

(1) Ji, Na, Daniel E. Milkie, and Eric Betzig. "Adaptive optics via pupil segmentation for
high-resolution imaging in biological tissues." nAture methods 7.2 (2009): 141-147.

[Myriam Gastard]

Wha about TDE? You can adjust it exactly to your application by pH and objective safe.
Just a thought.
Best,

Myriam Gastard, PhD
From my iPad.

> On Dec 7, 2014, at 7:02 PM, Reto Fiolka <[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.
> *****
>
> Thanks also to Zdenek and George for the links.
>
> The said objective is part of a Tony Wilson style perfect focusing system [1,2], thus it
> does not face a sample, only a mirror and we can throw in almost anything that does not
> hurt the optics.
>
> As the name suggests, we would like to have it perfect (diffraction limited, highest strehl
> ratio).
>
> I have not done a simulation, but my intuition let me think that the immersion medium in
> the remote objective could also pre-compensate some global refractive index mismatch
> that the second objective may see that faces the sample. Would surely be fun to try if
> one can remotely optimize the focus in the sample if it has some spherical aberrations
> (presumably it will have, as it is the same type of objective).
>
> Best,
> Reto
>
>
> PS: the remote focusing is a brilliant invention by the Wilson group and it is fun to build
> one:
> (1) Botcherby, Edward J., et al. "Aberration-free optical refocusing in high numerical
> aperture microscopy." Optics letters 32.14 (2007).
>
> (2) Botcherby, Edward J., et al. "Aberration-free three-dimensional multiphoton imaging
> of neuronal activity at kHz rates." Proceedings of the National Academy of Sciences 109.8
> (2012): 2919-2924.

[Reto Fiolka]

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Hi Myriam


Thank you for the suggestion!

According to this paper,

 Staudt, Thorsten, et al. "2, 2&#8242;&#8208;Thiodiethanol: A new water soluble mounting medium for
high resolution optical microscopy." Microscopy research and technique 70.1 (2007): 1-9.,

the tuning range is from water to oil.

However I would need a medium with refractive index slightly below water, so I think this
may not work.

But for many applications, TDE sounds very interesting to match the refractive index.

Best,
Reto