Silicone oil immersion lenses for live cell imaging

[Jacqueline Ross]

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Dear All,

I'm currently trialling 2 silicone oil immersion lenses from Olympus (30x/1.05NA and 60x/1.3NA) for live cell imaging on our Olympus FV1000 confocal, which is on an upright fixed stage microscope. So far, they both seem excellent and offer the higher level of resolution I'm looking for.

I am particularly interested in how these lenses compare with high NA water-immersion objective lenses. At the moment, we have long working distance water-immersion lenses or standard oil immersion so I don't have a high NA water-immersion objective lens to do my own comparison.

If anyone has personal experience with both types of lenses, I would be keen to hear their view.

I'm particularly interested in how the 60x/1.2NA water-immersion lens compares to the silicone oil immersion 60x/1.3NA, since I would like to buy one or the other.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/

[Pascal Weber]

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I have no experience with these two objetives. A long time ago but I had done
this compraison between two objectives same magnification water and oil. The
conclusion was that oil was higher yielding than water and had a better
resolution for the water immersion. Just one question how do you proceed to
do cells with an upright microscope and immersion objectives ?

In my previous lab i do this : on an inverted microscope (Zeiss) i use the
water 20X LD obj (75mm) to get a wilde FOV with the best resolution.

[Stefan Sokoll]

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Hello,

I am doing single particle tracking using an upright confocal spinning disk setup. The setup
basically consists of an Olympus TIRF100x oil objective, an Olympus microscope and on top a Yokogawa
spinning disk CSU-X1 with an Andor EMCCD camera.

Analyzing the 3D PSF of latex beats I discover that the PSF is not symmetrical in xy direction,
instead the x expansion is roughly 50nm larger than in y direction.

I wonder what might be the main cause for this effect? Tilted cover slip, adjustment of the devices,
the CSU itself or ...? So far I just put the devices on top of each other but due to the upright
setup I cannot check the symmetry of the laser beam e.g. over long distance at a wall.

Any ideas on how to best start calibrating the setup are very welcome. Thanks in advance!

Best,
Stefan

--
---------
Dipl. Ing.-Inf. Stefan Sokoll
Lab. Molecular Physiology
Dept. Neurochemistry & Molecular Biology
Leibniz Institute for Neurobiology (LIN)
Brenneckestrasse 6
39118 Magdeburg
Germany

Mail: [hidden email]
Tel.: +49 - 391 - 626393171

[George McNamara]

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Hi Jacqui,

The Olympus press release,

    Olympus has introduced new 30x and 60x silicone oil objectives, its
    latest innovation in silicone-immersion microscope optics. The new
    UPLSAPO 30x and
    UPLSAPO 60x objectives can markedly improve optical performance for
    live cell confocal, widefield fluorescence, multiphoton,
    differential interference contrast
    (DIC) and other applications. The new silicone oil objectives have
    distinct advantages over both traditional oil- and water-immersion
    objectives for deep and long
    -term imaging. They provide higher resolution and brighter images
    when imaging within deep samples when compared with oil-immersion
    objectives, and are
    useful in long-term imaging experiments where, in the past, water
    evaporation has been an issue.
    The new 30x, 1.05 NA, 0.8 mm working distance and 60x, 1.3 NA, 0.3
    mm working distance objectives reduce loss of
    contrast due to spherical aberration when compared to conventional
    oil objectives. By reducing the mismatch between the
    refractive index of the specimen and that of the immersion medium
    into which the objective is dipped, they provide higher
    resolution and brightness when imaging into thick samples.
    While water-immersion lenses are traditionally used to reduce
    refraction index mismatch, they are not practical for longterm,
    time-lapse imaging because of evaporation and low viscosity.
    Glycerol-immersion optics have been used as a
    substitute for water, but glycerol is not ideal because it tends to
    draw moisture from the air, resulting in changes in
    refractive index over time. Silicone oil is very stable and does not
    have these issues, and more closely matches the
    refractive index (N=1.404) of intracellular components, making these
    new objectives useful for imaging into cells and also
    for long-term, time-lapse experiments.
    The new objectives also feature a correction collar, enabling the
    user to correct for the spherical aberration that is present when
    imaging into a specimen
    beyond the coverslip. Image resolution and contrast, along with
    fluorescence performance, are optimized and maintained by adjusting
    this collar.


states the RI = 1.404 better matches "intracellular components". So, if
you are imaging entirely through homogeneous cells, the silicone lens
should win, no matter how far into the specimen you image in Z. If you
are imaging a cell-sparse specimen, that is, imaging mostly through
saline or saline + some percent serum, an RI=1.333 water immersion lens
will likely win at depth -- I hope none of your users just want to image
a volume of 50 um of just H2O.

The brightness curves with depth should behave similar - in the sense of
RI matching should be best - to the Staudt ... Hell Microscopy Res Tech
paper's graph.

The Olympus p.r. does not mention an interesting (but possibly labor
intensive for you to do) solution: increase the R.I. of the cell culture
medium to close to 1.404.

On page 621 of my mpmicro.doc is a table with R.I'.s of cell components
(actually, several tables from different sources, none of which agree
perfectly, below is the table from one source ... if anyone has a
different version of mpmicro from the web, just search for Widder):

Johnsen and Widder (1999) (their table 1) list the following values for
different cell components:
Cell Compartment Absolute refractive index Reference
Cell membrane            1.46-1.60 Quinby-Hunt & Hunt (1988)
Cell membrane            1.46-1.54 Meyer (1979)
Collagen                      1.55 Chapman (1976)
Cytoplasm                   1.35 Charney & Brackett (1961)
Lipids                          1.48 Beuthan et al. (1996)
Mitochondria               1.40 Beuthan et al. (1996)

S. Johnsen, E. A. Widder (1999) The physical basis of transparency in
biological tissue: ultrastructure and the minimization of light
scattering. J Theor Biol 199:181--198.
(ok, maybe picking values from a theoretical journal is not the best
possible choice).

One upshot of the table above is that cells are not homogeneous. Other
sources give different values for "cytoplasm", including:

Cytoplasm             1.374
Cytoplasm (range) 1.358-1.374
Cytoplasm (range) 1.353-1.368 Relative dry mass of 11% to 19%, for
regions of cytoplasm outside focal and close contacts.

I recall calculating that a whole lot of BSA or HSA (for examples) could
be used to increase the R.I. to get closer to ~1.4 - however, I don't
know what that will do to scattering properties or viscosity.


So, I suggest testing brightness, resolution, and performance with
different fluorophores, in Z-series. Numerically correct deconvolution
of all your confocal data sets would be useful. If possible, measure the
thickness of each coverglass (and make sure each is on flat).

Sincerely,

George
p.s. may everyone's light rays be as straight as possible.



On 6/28/2012 2:47 AM, Jacqui Ross wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I'm currently trialling 2 silicone oil immersion lenses from Olympus (30x/1.05NA and 60x/1.3NA) for live cell imaging on our Olympus FV1000 confocal, which is on an upright fixed stage microscope. So far, they both seem excellent and offer the higher level of resolution I'm looking for.
>
> I am particularly interested in how these lenses compare with high NA water-immersion objective lenses. At the moment, we have long working distance water-immersion lenses or standard oil immersion so I don't have a high NA water-immersion objective lens to do my own comparison.
>
> If anyone has personal experience with both types of lenses, I would be keen to hear their view.
>
> I'm particularly interested in how the 60x/1.2NA water-immersion lens compares to the silicone oil immersion 60x/1.3NA, since I would like to buy one or the other.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit
> School of Medical Sciences
> Faculty of Medical&  Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland, NEW ZEALAND
>
> Tel: [phone] Ext 87438
> Fax: [phone]
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>
>    

[Sebastian Rhode]

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Hi Stefan,

from my experience so far, there can be a couple of things go wrong. When I
found asymmetric PSF, the most common problem were caused by:

- the objective itself - Did you check the PSF of those beads using the same
objective on different setups?

- Do you use a CMount with built-in optics (Aftermagnification) since use a
CCD with rather large pixels?

- Dichroic - especially the CSU-X1 use very thin (0.5mm) dichroics, which
will not be very flat (for this reason we use 5mm substrates for our
dicroics) - so this might induce a curvature in one direction and finally
lead to astigmatism

By the way, what are the values you calculated?

By the way, no commercial interests here.

Cheers,  Sebi

Dr. Sebastian Rhode
Project Manager Research & Development      

TILL Photonics GmbH
an FEI Company



On Thu, 28 Jun 2012 12:08:02 +0200, Stefan Sokoll <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello,
>
>I am doing single particle tracking using an upright confocal spinning disk
setup. The setup
>basically consists of an Olympus TIRF100x oil objective, an Olympus
microscope and on top a Yokogawa
>spinning disk CSU-X1 with an Andor EMCCD camera.
>
>Analyzing the 3D PSF of latex beats I discover that the PSF is not
symmetrical in xy direction,
>instead the x expansion is roughly 50nm larger than in y direction.
>
>I wonder what might be the main cause for this effect? Tilted cover slip,
adjustment of the devices,
>the CSU itself or ...? So far I just put the devices on top of each other
but due to the upright
>setup I cannot check the symmetry of the laser beam e.g. over long distance
at a wall.
>
>Any ideas on how to best start calibrating the setup are very welcome.
Thanks in advance!

>
>Best,
>Stefan
>
>--
>---------
>Dipl. Ing.-Inf. Stefan Sokoll
>Lab. Molecular Physiology
>Dept. Neurochemistry & Molecular Biology
>Leibniz Institute for Neurobiology (LIN)
>Brenneckestrasse 6
>39118 Magdeburg
>Germany
>
>Mail: [hidden email]
>Tel.: +49 - 391 - 626393171

[Stefan Sokoll]

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Hi Sebastian,

I am simply calculating the x/y fwhm of each particle. I already used different objectives, still
giving the same results, thus it's probably not a matter of the objective. Also we use no
aftermagnification.

Unfortunately, I don't understand what you mean with using 5mm substrates on the dicroics. Maybe you
can explain this in more detail?

Thanks!
Stefan

Sebastian Rhode wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Stefan,
>
> from my experience so far, there can be a couple of things go wrong. When I
> found asymmetric PSF, the most common problem were caused by:
>
> - the objective itself - Did you check the PSF of those beads using the same
> objective on different setups?
>
> - Do you use a CMount with built-in optics (Aftermagnification) since use a
> CCD with rather large pixels?
>
> - Dichroic - especially the CSU-X1 use very thin (0.5mm) dichroics, which
> will not be very flat (for this reason we use 5mm substrates for our
> dicroics) - so this might induce a curvature in one direction and finally
> lead to astigmatism
>
> By the way, what are the values you calculated?
>
> By the way, no commercial interests here.
>
> Cheers,  Sebi
>
> Dr. Sebastian Rhode
> Project Manager Research & Development      
>
> TILL Photonics GmbH
> an FEI Company
>
>
>
> On Thu, 28 Jun 2012 12:08:02 +0200, Stefan Sokoll <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I am doing single particle tracking using an upright confocal spinning disk
> setup. The setup
>> basically consists of an Olympus TIRF100x oil objective, an Olympus
> microscope and on top a Yokogawa
>> spinning disk CSU-X1 with an Andor EMCCD camera.
>>
>> Analyzing the 3D PSF of latex beats I discover that the PSF is not
> symmetrical in xy direction,
>> instead the x expansion is roughly 50nm larger than in y direction.
>>
>> I wonder what might be the main cause for this effect? Tilted cover slip,
> adjustment of the devices,
>> the CSU itself or ...? So far I just put the devices on top of each other
> but due to the upright
>> setup I cannot check the symmetry of the laser beam e.g. over long distance
> at a wall.
>> Any ideas on how to best start calibrating the setup are very welcome.
> Thanks in advance!
>> Best,
>> Stefan
>>
>> --
>> ---------
>> Dipl. Ing.-Inf. Stefan Sokoll
>> Lab. Molecular Physiology
>> Dept. Neurochemistry & Molecular Biology
>> Leibniz Institute for Neurobiology (LIN)
>> Brenneckestrasse 6
>> 39118 Magdeburg
>> Germany
>>
>> Mail: [hidden email]
>> Tel.: +49 - 391 - 626393171

--
---------
Dipl. Ing.-Inf. Stefan Sokoll
Lab. Molecular Physiology
Dept. Neurochemistry & Molecular Biology
Leibniz Institute for Neurobiology (LIN)
Brenneckestrasse 6
39118 Magdeburg
Germany

Mail: [hidden email]
Tel.: +49 - 391 - 626393171

[John Oreopoulos]

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Stefan,

Would you be able to post to the web somewhere a few example images so we can see as well? Maybe even a XZ and XY cross-section reconstructions through a few beads? Is the effect the same all across the field of view? The shape of the PSF can sometimes tell you if there are any specific aberrations in play.

The best way to isolate the problem would be to move the objective over to another scope (without a CSU) and try collecting the same data on the same type of sample. What happens if you use a similar objective on the original scope system? Then you could also try removing the CSU on your first scope and connect the camera directly to the upright port, and again collect the data with the objective in question (use the arc lamp for a light source and scope filter cube). This will determine if it's the objective, the scope, or maybe the CSU.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2012-06-28, at 6:08 AM, Stefan Sokoll wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I am doing single particle tracking using an upright confocal spinning disk setup. The setup
> basically consists of an Olympus TIRF100x oil objective, an Olympus microscope and on top a Yokogawa
> spinning disk CSU-X1 with an Andor EMCCD camera.
>
> Analyzing the 3D PSF of latex beats I discover that the PSF is not symmetrical in xy direction,
> instead the x expansion is roughly 50nm larger than in y direction.
>
> I wonder what might be the main cause for this effect? Tilted cover slip, adjustment of the devices,
> the CSU itself or ...? So far I just put the devices on top of each other but due to the upright
> setup I cannot check the symmetry of the laser beam e.g. over long distance at a wall.
>
> Any ideas on how to best start calibrating the setup are very welcome. Thanks in advance!
>
> Best,
> Stefan
>
> --
> ---------
> Dipl. Ing.-Inf. Stefan Sokoll
> Lab. Molecular Physiology
> Dept. Neurochemistry & Molecular Biology
> Leibniz Institute for Neurobiology (LIN)
> Brenneckestrasse 6
> 39118 Magdeburg
> Germany
>
> Mail: [hidden email]
> Tel.: +49 - 391 - 626393171

[Sebastian Rhode]

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Hi Stefan,

I might be wrong in your case, but I have seen quite a lot of systems, where
a rather thin dichroic introduces optical problems. Imagine the dichroic is
bend a bit due to its mounting along on axis. So this can lead to a curved
surface along one axis of the dichroic. But this curvature introduces a
focal length along on axis.

And finally those kind of things will "mess up" your otherwise perfect PSF.
To avoid we use 2mm substrates (TITF, FRAP, WF) inside the microscope or
even 5mm substrates for our dichroics inside our spinning disc system.

Cheers,  Sebi

[Stefan Sokoll]

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Ok, now I got it. Fortunately, we have different dicroics so I will exchange them and check for
changes on the PSF.

I analyze the aberrations not directly on the plot of the PSF instead I analyze the changes of the
2D fwhm over z (separated for x and y direction), which e.g. leads to the graphs on the left in the
attached image. There is an offset between the x and y fwhm and both curves also seem a little
shifted over z.

Choosing a particle that is not centered (here I choose one that is on the outer right of the field
of view but same y coordinate) changes the offset/shifting and in this example even leads to a
perfect match of the curves (graph on the right). Maybe this also points to a specific aberration?

I recognized that attaching files is not allowed on that list so I will send the image directly.

Best,
Stefan

Sebastian Rhode wrote:

> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Stefan,
> >
> > I might be wrong in your case, but I have seen quite a lot of systems, where
> > a rather thin dichroic introduces optical problems. Imagine the dichroic is
> > bend a bit due to its mounting along on axis. So this can lead to a curved
> > surface along one axis of the dichroic. But this curvature introduces a
> > focal length along on axis.
> >
> > And finally those kind of things will "mess up" your otherwise perfect PSF.
> > To avoid we use 2mm substrates (TITF, FRAP, WF) inside the microscope or
> > even 5mm substrates for our dichroics inside our spinning disc system.
> >
> > Cheers,  Sebi
> >

Stefan,

Would you be able to post to the web somewhere a few example images so we can see as well? Maybe
even a XZ and XY cross-section reconstructions through a few beads? Is the effect the same all
across the field of view? The shape of the PSF can sometimes tell you if there are any specific
aberrations in play.

The best way to isolate the problem would be to move the objective over to another scope (without a
CSU) and try collecting the same data on the same type of sample. What happens if you use a similar
objective on the original scope system? Then you could also try removing the CSU on your first scope
and connect the camera directly to the upright port, and again collect the data with the objective
in question (use the arc lamp for a light source and scope filter cube). This will determine if it's
the objective, the scope, or maybe the CSU.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca
--
---------
Dipl. Ing.-Inf. Stefan Sokoll
Lab. Molecular Physiology
Dept. Neurochemistry & Molecular Biology
Leibniz Institute for Neurobiology (LIN)
Brenneckestrasse 6
39118 Magdeburg
Germany

Mail: [hidden email]
Tel.: +49 - 391 - 626393171

[Andrew York]

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 Sadly I haven't done head-to-head comparisons, but we've been happy using
the 60x silicone objective you mention, and we've also been happy with our
Olympus 60x 1.2 water objective on a different system. We've used the
silicone for single cells, tissue samples, and embryos, and gotten
excellent results >50 microns deep. We've used the water objective
primarily in single cells 10 microns thick or less.

 For both objectives I've found the correction collar to be fairly
important, and the setting that gives best results is not always what you
would expect from the specified coverslip thickness. For the water
objective, the tilt of the coverslip is extremely important (as described
here:  http://www.ncbi.nlm.nih.gov/pubmed/15369482 ).

 If you really care about high resolution, demo several of each objective,
and pick the one that has the best PSF. We've noticed substantial
performance variation for supposedly identical objectives. I think you can
also ask Olympus for a "PSF-grade" objective. I think this just means they
pick out a good one for you. I'm not sure there's enough silicone
objectives to be choosy, but it's worth a shot. Neither of our objectives
is "PSF-grade", so I can't say how much better they are.

On Thu, Jun 28, 2012 at 2:47 AM, Jacqui Ross <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I'm currently trialling 2 silicone oil immersion lenses from Olympus
> (30x/1.05NA and 60x/1.3NA) for live cell imaging on our Olympus FV1000
> confocal, which is on an upright fixed stage microscope. So far, they both
> seem excellent and offer the higher level of resolution I'm looking for.
>
> I am particularly interested in how these lenses compare with high NA
> water-immersion objective lenses. At the moment, we have long working
> distance water-immersion lenses or standard oil immersion so I don't have a
> high NA water-immersion objective lens to do my own comparison.
>
> If anyone has personal experience with both types of lenses, I would be
> keen to hear their view.
>
> I'm particularly interested in how the 60x/1.2NA water-immersion lens
> compares to the silicone oil immersion 60x/1.3NA, since I would like to buy
> one or the other.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland, NEW ZEALAND
>
> Tel: [phone] Ext 87438
> Fax: [phone]
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>

[Lloyd Donaldson]

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Hi Jacqui

We have a 63x 1.2NA water, a 63x 1.4NA oil and a 63x 1.3NA glycerol objectives. I would rank them in that order in terms of image quality - the glycerol lens is the best although the oil is close behind. The water lens is not so good but that may relate to our samples which have a high refractive index and hence water is not ideal. If you want to try them out with your samples let me know - your samples are quite different from ours so could behave quite differently.

Regards - Lloyd


Dr Lloyd Donaldson
Project Leader - Microscopy & Wood Identification
Manufacturing and Biomaterials
Scion - Forests, Products, Innovation
49 Sala Street, Rotorua 3010
Ph 07 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
Sent: Thursday, 28 June 2012 6:47 p.m.
To: [hidden email]
Subject: Silicone oil immersion lenses for live cell imaging

*****
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Dear All,

I'm currently trialling 2 silicone oil immersion lenses from Olympus (30x/1.05NA and 60x/1.3NA) for live cell imaging on our Olympus FV1000 confocal, which is on an upright fixed stage microscope. So far, they both seem excellent and offer the higher level of resolution I'm looking for.

I am particularly interested in how these lenses compare with high NA water-immersion objective lenses. At the moment, we have long working distance water-immersion lenses or standard oil immersion so I don't have a high NA water-immersion objective lens to do my own comparison.

If anyone has personal experience with both types of lenses, I would be keen to hear their view.

I'm particularly interested in how the 60x/1.2NA water-immersion lens compares to the silicone oil immersion 60x/1.3NA, since I would like to buy one or the other.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/



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[Zac Arrac Atelaz]

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Stefan: I am almost sure that Sebastian is talking about a 5mm thick crystal to built the dichroic on to, so all the dichroics will have this thickness. Best regards
 > Date: Thu, 28 Jun 2012 15:04:44 +0200

> From: [hidden email]
> Subject: Re: calibration of confocal spinning disk setup (yokogawa)
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Sebastian,
>
> I am simply calculating the x/y fwhm of each particle. I already used different objectives, still
> giving the same results, thus it's probably not a matter of the objective. Also we use no
> aftermagnification.
>
> Unfortunately, I don't understand what you mean with using 5mm substrates on the dicroics. Maybe you
> can explain this in more detail?
>
> Thanks!
> Stefan
>
> Sebastian Rhode wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Stefan,
> >
> > from my experience so far, there can be a couple of things go wrong. When I
> > found asymmetric PSF, the most common problem were caused by:
> >
> > - the objective itself - Did you check the PSF of those beads using the same
> > objective on different setups?
> >
> > - Do you use a CMount with built-in optics (Aftermagnification) since use a
> > CCD with rather large pixels?
> >
> > - Dichroic - especially the CSU-X1 use very thin (0.5mm) dichroics, which
> > will not be very flat (for this reason we use 5mm substrates for our
> > dicroics) - so this might induce a curvature in one direction and finally
> > lead to astigmatism
> >
> > By the way, what are the values you calculated?
> >
> > By the way, no commercial interests here.
> >
> > Cheers,  Sebi
> >
> > Dr. Sebastian Rhode
> > Project Manager Research & Development      
> >
> > TILL Photonics GmbH
> > an FEI Company
> >
> >
> >
> > On Thu, 28 Jun 2012 12:08:02 +0200, Stefan Sokoll <[hidden email]>
> > wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello,
> >>
> >> I am doing single particle tracking using an upright confocal spinning disk
> > setup. The setup
> >> basically consists of an Olympus TIRF100x oil objective, an Olympus
> > microscope and on top a Yokogawa
> >> spinning disk CSU-X1 with an Andor EMCCD camera.
> >>
> >> Analyzing the 3D PSF of latex beats I discover that the PSF is not
> > symmetrical in xy direction,
> >> instead the x expansion is roughly 50nm larger than in y direction.
> >>
> >> I wonder what might be the main cause for this effect? Tilted cover slip,
> > adjustment of the devices,
> >> the CSU itself or ...? So far I just put the devices on top of each other
> > but due to the upright
> >> setup I cannot check the symmetry of the laser beam e.g. over long distance
> > at a wall.
> >> Any ideas on how to best start calibrating the setup are very welcome.
> > Thanks in advance!
> >> Best,
> >> Stefan
> >>
> >> --
> >> ---------
> >> Dipl. Ing.-Inf. Stefan Sokoll
> >> Lab. Molecular Physiology
> >> Dept. Neurochemistry & Molecular Biology
> >> Leibniz Institute for Neurobiology (LIN)
> >> Brenneckestrasse 6
> >> 39118 Magdeburg
> >> Germany
> >>
> >> Mail: [hidden email]
> >> Tel.: +49 - 391 - 626393171
>
> --
> ---------
> Dipl. Ing.-Inf. Stefan Sokoll
> Lab. Molecular Physiology
> Dept. Neurochemistry & Molecular Biology
> Leibniz Institute for Neurobiology (LIN)
> Brenneckestrasse 6
> 39118 Magdeburg
> Germany
>
> Mail: [hidden email]
> Tel.: +49 - 391 - 626393171

     

[Jacqueline Ross]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Lloyd, Andrew, George,

Thanks very much for your replies, which are very helpful. I also got some other comments off-list which were also much appreciated.

Thanks Lloyd for the offer to test your lenses. I'll keep it in mind but I really want the comparison against the silicone oil immersion lens. Interesting to hear that the glycerol lens is the best for your specimens.

Thanks Andrew for your comments and comparison of the two lenses. We have a large variety of specimens imaged on our systems including zebrafish so it would be good to offer increased resolution as well as improved depth. I wasn't aware of the "PSF-grade" lenses so I'll be contacting my local representative to ask about that!

With regard to George's information, the information from Olympus on the refractive index matching is also the reason that I am considering this lens. Otherwise, I would have just purchased the water lens.

Finally, in answer to your question Pascal, it is a bit trickier using these immersion lenses for live cell work on an upright microscope. We have to use a chamber with coverglass, i.e. you culture the cells on coverslips and then use a small chamber. There are a number of these available in the market currently.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lloyd Donaldson
Sent: Friday, 29 June 2012 8:07 a.m.
To: [hidden email]
Subject: Re: Silicone oil immersion lenses for live cell imaging

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Jacqui

We have a 63x 1.2NA water, a 63x 1.4NA oil and a 63x 1.3NA glycerol objectives. I would rank them in that order in terms of image quality - the glycerol lens is the best although the oil is close behind. The water lens is not so good but that may relate to our samples which have a high refractive index and hence water is not ideal. If you want to try them out with your samples let me know - your samples are quite different from ours so could behave quite differently.

Regards - Lloyd


Dr Lloyd Donaldson
Project Leader - Microscopy & Wood Identification
Manufacturing and Biomaterials
Scion - Forests, Products, Innovation
49 Sala Street, Rotorua 3010
Ph 07 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
Sent: Thursday, 28 June 2012 6:47 p.m.
To: [hidden email]
Subject: Silicone oil immersion lenses for live cell imaging

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear All,

I'm currently trialling 2 silicone oil immersion lenses from Olympus (30x/1.05NA and 60x/1.3NA) for live cell imaging on our Olympus FV1000 confocal, which is on an upright fixed stage microscope. So far, they both seem excellent and offer the higher level of resolution I'm looking for.

I am particularly interested in how these lenses compare with high NA water-immersion objective lenses. At the moment, we have long working distance water-immersion lenses or standard oil immersion so I don't have a high NA water-immersion objective lens to do my own comparison.

If anyone has personal experience with both types of lenses, I would be keen to hear their view.

I'm particularly interested in how the 60x/1.2NA water-immersion lens compares to the silicone oil immersion 60x/1.3NA, since I would like to buy one or the other.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/



This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
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[Robin Battye-2]

Sebastian is talking about the TILL spinning disc, not the Yokogawa spinning disc. The dichroics will be different between these two devices.  With Yokogawa the dichroics  is placed between the pinhole disc and the microlens  disc and needs to meet specific dimensions, one of those includes a thin substrate on which to sputter you coatings.  This thin substrate can be prone to bending, although steps can be taken to minimize this. The TILL system has a single disc and the dichroics is in free space so that you can make it as thick as you choose.  

If you would like to discuss further details please contact me offline.

Cheers Robin


Robin Battye, M.Sc., Ph. D.
Confocal Product Manager/
Technical Sales Specialist
Mobile: [phone]

Spectral Applied Research
9078 Leslie St., Unit 11
Richmond Hill, Ontario
L4B 3L8

Office: [phone] ext. 406
Fax: [phone]
www.spectral.ca

 We believe that performance comes before Compromise.

 



-------- Original message --------
Subject: Re: calibration of confocal spinning disk setup (yokogawa)
From: Zac Arrac Atelaz <[hidden email]>
To: [hidden email]
CC: Re: calibration of confocal spinning disk setup (yokogawa)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****


Stefan: I am almost sure that Sebastian is talking about a 5mm thick crystal to built the dichroic on to, so all the dichroics will have this thickness. Best regards

> Date: Thu, 28 Jun 2012 15:04:44 +0200
> From: [hidden email]
> Subject: Re: calibration of confocal spinning disk setup (yokogawa)
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Sebastian,
>
> I am simply calculating the x/y fwhm of each particle. I already used different objectives, still
> giving the same results, thus it's probably not a matter of the objective. Also we use no
> aftermagnification.
>
> Unfortunately, I don't understand what you mean with using 5mm substrates on the dicroics. Maybe you
> can explain this in more detail?
>
> Thanks!
> Stefan
>
> Sebastian Rhode wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Stefan,
> >
> > from my experience so far, there can be a couple of things go wrong. When I
> > found asymmetric PSF, the most common problem were caused by:
> >
> > - the objective itself - Did you check the PSF of those beads using the same
> > objective on different setups?
> >
> > - Do you use a CMount with built-in optics (Aftermagnification) since use a
> > CCD with rather large pixels?
> >
> > - Dichroic - especially the CSU-X1 use very thin (0.5mm) dichroics, which
> > will not be very flat (for this reason we use 5mm substrates for our
> > dicroics) - so this might induce a curvature in one direction and finally
> > lead to astigmatism
> >
> > By the way, what are the values you calculated?
> >
> > By the way, no commercial interests here.
> >
> > Cheers,  Sebi
> >
> > Dr. Sebastian Rhode
> > Project Manager Research & Development     
> >
> > TILL Photonics GmbH
> > an FEI Company
> >
> >
> >
> > On Thu, 28 Jun 2012 12:08:02 +0200, Stefan Sokoll <[hidden email]>
> > wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello,
> >>
> >> I am doing single particle tracking using an upright confocal spinning disk
> > setup. The setup
> >> basically consists of an Olympus TIRF100x oil objective, an Olympus
> > microscope and on top a Yokogawa
> >> spinning disk CSU-X1 with an Andor EMCCD camera.
> >>
> >> Analyzing the 3D PSF of latex beats I discover that the PSF is not
> > symmetrical in xy direction,
> >> instead the x expansion is roughly 50nm larger than in y direction.
> >>
> >> I wonder what might be the main cause for this effect? Tilted cover slip,
> > adjustment of the devices,
> >> the CSU itself or ...? So far I just put the devices on top of each other
> > but due to the upright
> >> setup I cannot check the symmetry of the laser beam e.g. over long distance
> > at a wall.
> >> Any ideas on how to best start calibrating the setup are very welcome.
> > Thanks in advance!
> >> Best,
> >> Stefan
> >>
> >> --
> >> ---------
> >> Dipl. Ing.-Inf. Stefan Sokoll
> >> Lab. Molecular Physiology
> >> Dept. Neurochemistry & Molecular Biology
> >> Leibniz Institute for Neurobiology (LIN)
> >> Brenneckestrasse 6
> >> 39118 Magdeburg
> >> Germany
> >>
> >> Mail: [hidden email]
> >> Tel.: +49 - 391 - 626393171
>
> --
> ---------
> Dipl. Ing.-Inf. Stefan Sokoll
> Lab. Molecular Physiology
> Dept. Neurochemistry & Molecular Biology
> Leibniz Institute for Neurobiology (LIN)
> Brenneckestrasse 6
> 39118 Magdeburg
> Germany
>
> Mail: [hidden email]
> Tel.: +49 - 391 - 626393171

            

[Michal Opas]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello,

This is a totally non-confocal and likely trivial question. I got a
small USB microscope camera and while I see how it can be controlled by
the OS under Win XP, I am at a total loss with Win 7 Pro.
a) is there a build in webcam viewer like in XP
b) is there a way to link such a camera to ImageJ of Fiji or maybe there
is a freeware standalone program controlling such a camera.

Thanks in advance,

Michal

--

* Dr. Michal Opas
      Professor
      Department of Laboratory Medicine and Pathobiology
      University of Toronto
      1 King's College Circle
      Medical Sciences Building, room 6326
      Toronto, Ontario, M5S 1A8 Canada

**°°°°°°°°°°°°°**
  phone: [phone] (laboratory)
         [phone] (office)
    fax: [phone]
e*-*mail: **[hidden email] <mailto:[hidden email]>***

*WWW**:** **http://www.utoronto.ca/mocell
*

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Michal,

You can setup "XP mode" inside Windows 7 ... or download and setup
VMware Player and install XP in that.

ImageJ ... Looking at the Micro-Manager.org list, there are TWAIN and
"OpenCVgrabber" in the camera list
http://valelab.ucsf.edu/~MM/MMwiki/index.php/Device_Support

George
p.s. suggestion for the micro-manager folks ... how about interfacing
with the Canon Hack Development kit, recently mentioned on the listserv
http://chdk.wikia.com/wiki/CHDK
I see it CHDK was written up in IEEE Spectrum
http://spectrum.ieee.org/geek-life/hands-on/using-the-canon-hack-development-kit/0/
You (mm folks( should consider writing an article for IEEE Spectrum.
Also for Journal of Biomolecular Technologies.


On 7/2/2012 8:48 PM, Michal Opas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> This is a totally non-confocal and likely trivial question. I got a
> small USB microscope camera and while I see how it can be controlled
> by the OS under Win XP, I am at a total loss with Win 7 Pro.
> a) is there a build in webcam viewer like in XP
> b) is there a way to link such a camera to ImageJ of Fiji or maybe
> there is a freeware standalone program controlling such a camera.
>
> Thanks in advance,
>
> Michal
>

[Michal Opas]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Listers,

Forgive my ignorance but did anyone used plastic coverslips for
immunofluorescence? If so would standard immerion oil work?
Thanks in advance!

Michal *
<http://www.utoronto.ca/mocell>*

[leoncio vergara]

*****
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I havent use plastic coverslips for anything in a long time, but working with IBIDI chambers, it seems not immersion oils are compatible. The company provide a list of brands and products that are compatible with their slides. So Iguess the answer would be, not all immersion oils are the same, some may not be compatible.

Leoncio

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Michal Opas [[hidden email]]
Sent: Wednesday, January 16, 2013 8:58 AM
To: [hidden email]
Subject: plastic coverslips & immersion imaging

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Listers,

Forgive my ignorance but did anyone used plastic coverslips for
immunofluorescence? If so would standard immerion oil work?
Thanks in advance!

Michal *
<http://www.utoronto.ca/mocell>*

[Richard E. Edelmann]

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        Yes, for one series of projects we did use plastic coverslips (studying
biofilm formation on plastic substrate).  And yes normal cargill immersion oil
worked.

        The  biggest issue we had with the plastic coverslips was thickness -
thickness was terrible.  And often would exceed the 100um working distance
(WD) of the lens we were using.  (i.e. the 170um optical coverslip thickness +
100um WD)  and thus the samples could not be focused.  Took a bit to
discover this, then we took to using a micrometer to measure the coverslips
and moved to a 150um WD lens.



On 16 Jan 2013 at 9:58, Michal Opas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy *****
>
> Hello Listers,
>
> Forgive my ignorance but did anyone used plastic coverslips for
> immunofluorescence? If so would standard immerion oil work?
> Thanks in advance!
>
> Michal *
> <http://www.utoronto.ca/mocell>*

Richard E. Edelmann, Ph.D., Director
Center for Advanced Microscopy & Imaging
9C Upham Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712        Fax: [phone]
E-mail: [hidden email]
http://www.cami.muohio.edu