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60X Silicone Objective on BD Pathway 855

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Dharshini

60X Silicone Objective on BD Pathway 855

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Hi,

To be able to include a new objective in the BD PW-855 system,
the "Geometry setup" needs to be changed to get the required resolution.

Could someone please let me know, what are the key factors that should be
kept in mind before changing the "Measurement Reference" for the above
mentioned Olympus x60 objective?

I am trying to use this objective to image the mitochondrial changes to
different drug treatments. Are there any other minor changes that will help
improve the resolution of the final images in BD PW-855?

Appreciate your comments or suggestions.

Thank you.

Dharshini

Vitaly Boyko

nuclear membrane marker

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Dear All,

one of our users is looking for a relatively abundant marker that is homogeneously distributed over the nuclear membrane in cultured human cells (it could be a well characterized Ab against the nuclear receptor, or simply specific (nuclear) membrane stain).

Many thanks in advance,

Vitaly

Josef Gotzmann

Re: nuclear membrane marker

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Dear Vitaly,

Frankly, there is no really specific nuclear membrane dye. I remember a
paper from Zal et al., (Traffic 2006; 7: 16071613), which says the dye
FM4-64 is specific for the nuclear membrane, though in our tests the results
were inconsistent and it also stained other membrane entities.
I would suggest to use anti-NPC antibodies - these should specifically stain
the nuclear membrane (in interphase cells) - famous in the field is mAb414.

hope this helps,
Josef

On Tue, 11 Sep 2012 14:57:00 -0700, Vitaly Boyko <[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,
>
>one of our users is looking for a relatively abundant marker that is
homogeneously distributed over the nuclear membrane in cultured human cells
(it could be a well characterized Ab against the nuclear receptor, or simply
specific (nuclear) membrane stain).
>
>Many thanks in advance,
>
>Vitaly

jmvdwin

Re: nuclear membrane marker

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Dear,

You may consider LaminA/C:

fluorescent constructs for live imaging
e.g. http://www.ncbi.nlm.nih.gov/pubmed?term=20079404
and Lamin Abs work usually fine on fixed material...

With best wishes.

JM

At 09:35 12/09/2012, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Vitaly,
>
>Frankly, there is no really specific nuclear membrane dye. I remember a
>paper from Zal et al., (Traffic 2006; 7: 16071613), which says the dye
>FM4-64 is specific for the nuclear membrane, though in our tests the results
>were inconsistent and it also stained other membrane entities.
>I would suggest to use anti-NPC antibodies - these should specifically stain
>the nuclear membrane (in interphase cells) - famous in the field is mAb414.
>
>hope this helps,
>Josef
>
>On Tue, 11 Sep 2012 14:57:00 -0700, Vitaly Boyko
><[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,
> >
> >one of our users is looking for a relatively abundant marker that is
>homogeneously distributed over the nuclear membrane in cultured human cells
>(it could be a well characterized Ab against the nuclear receptor, or simply
>specific (nuclear) membrane stain).
> >
> >Many thanks in advance,
> >
> >Vitaly

Jean-Marie.

Jean-Marie Vanderwinden, M.D., Ph.D.
Directeur de Recherche F.N.R.S. / Research
Director, National Fund for Scientific Research (Belgium)
Neurophysiology lab http://www.ulb.ac.be/medecine/neurophy/
Light Microscopy Facility http://limif.ulb.ac.be/

Postal address:
Laboratoire de Neurophysiologie (Prof. S.N. Schiffmann),
Faculté de Médecine, Campus Erasme, CP 601,
Université Libre de Bruxelles,
808 route de Lennik, B-1070 Brussels, Belgium.

(: Secretary: +(32).2.555.42.30, Direct: +(32).2.555.69.88
7: +(32).2.555.41.21,
. [hidden email]

Skype: Jean Marie Vanderwinden
12voip / netappel: jeanmarievanderwinden

Ian Dobbie

Re: 60X Silicone Objective on BD Pathway 855

In reply to this post by Dharshini

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Dharshini <[hidden email]> writes:

> Hi,
>
> To be able to include a new objective in the BD PW-855 system,
> the "Geometry setup" needs to be changed to get the required resolution.
>
> Could someone please let me know, what are the key factors that should be
> kept in mind before changing the "Measurement Reference" for the above
> mentioned Olympus x60 objective?
>
> I am trying to use this objective to image the mitochondrial changes to
> different drug treatments. Are there any other minor changes that will help
> improve the resolution of the final images in BD PW-855?

We use this lens extensively on widefield and spinning disk systems
mostly imaging drosophila embryos in halocarbon oil. It
is a brilliant lens for this. The one thing I would say is that getting
the correction collar right makes a big difference.

Ian

Sylvie Le Guyader

Re: 60X Silicone Objective on BD Pathway 855

In reply to this post by Dharshini

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Hi Dharshini

You need to find first what currently limits your resolution: it is the objective or the camera? Which objective are you currently using? Do you bin your camera? If the camera is the limitation, changing objective will not help.

You can find some bits of information about the BD Pathway here: http://www.bdbiosciences.com/instruments/pathway/faqs/camera.jsp
I am not sure why they mention the pixel size at 0.16um without bin. That doesn't sound right to me but I might be missing something. According to Hamamastu, the ORCA-AG pixels are 6.45um.
I assume that you want to use the Olympus 60x NA 1.3 silicone immersion.

If there is no extra magnification in the system and if we consider the Nyquist sampling requirement to be >2, 0.61*520*60/2*1000*1.3= 7.32 so your camera pixels should be smaller than 7.32 um to image green fluorophores with the best resolution allowed by your objective. 6.45 um pixels are small enough so using the 60x NA1.3 will improved the resolution compared to the other objectives on the BD Pathway.

If you bin, the pixels will be too large and the camera becomes the limiting factor for all objectives so changing objective will not help.

If you are undersampling (if you bin), increasing the contrast (by e.g. reducing the gain or averaging to increase the signal to noise ratio) might help a bit but I am not certain about that.

Other considerations to keep in mind that might prevent you from using this objective: you will not be able to image more than 2 wells reliably (loss of focus) if you image more than once (i.e. no time lapse) and do not use some sort of silicone dispenser. You could build one. It is messy but it works.

Please dear list members, let me know if anything I wrote is incorrect.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14183 Huddinge
Sweden
office: +46 (0) 8 5248 1107
LCI room: +46 (0) 8 5248 1172
mobile: +46 (0) 73 733 5008

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Dharshini
> Sent: 11 September 2012 17:41
> To: [hidden email]
> Subject: 60X Silicone Objective on BD Pathway 855
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> To be able to include a new objective in the BD PW-855 system, the "Geometry
> setup" needs to be changed to get the required resolution.
>
> Could someone please let me know, what are the key factors that should be kept in
> mind before changing the "Measurement Reference" for the above mentioned
> Olympus x60 objective?
>
> I am trying to use this objective to image the mitochondrial changes to different
> drug treatments. Are there any other minor changes that will help improve the
> resolution of the final images in BD PW-855?
>
> Appreciate your comments or suggestions.
>
> Thank you.
>
> Dharshini

Cameron Nowell

Re: nuclear membrane marker

In reply to this post by jmvdwin

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Howdy,

I would second the suggestion of Lamin. We have had some spectacular images generated using it.

Cheers

Cam

Cameron J. Nowell
Microscpy Manager
Centre for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm

________________________________

From: Confocal Microscopy List on behalf of Jean-Marie Vanderwinden
Sent: Wed 12/09/2012 6:49 PM
To: [hidden email]
Subject: Re: nuclear membrane marker

*****
To join, leave or search the confocal microscopy listserv, go to:
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Dear,

You may consider LaminA/C:

fluorescent constructs for live imaging
e.g. http://www.ncbi.nlm.nih.gov/pubmed?term=20079404
and Lamin Abs work usually fine on fixed material...

With best wishes.

JM

At 09:35 12/09/2012, you wrote:

Steffen Dietzel

Re: nuclear membrane marker

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By definition, Lamin is not in the nuclear membrane but underneath. So
depending on what your question and your planed resolution is, this may
or may not be a problem.

Have a look at this comparison of anti-nuclear pore and anti-lamin with
confocal and 3D-SIM by Schermelleh et al.

https://commons.wikimedia.org/wiki/File:3D-SIM-1_NPC_Confocal_vs_3D-SIM.jpg

(make sure you don't loose the part of the URL in the second line, link
to the publication is in the figure legend)

Note that anti-NPC is rather homogeneous in confocal but not in 3D-SIM.
Good luck

Steffen

On 12.09.2012 12:56, Cameron Nowell wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Howdy,
>
> I would second the suggestion of Lamin. We have had some spectacular images generated using it.
>
> Cheers
>
> Cam
>
>
> Cameron J. Nowell
> Microscpy Manager
> Centre for Advanced Microscopy
> Ludwig Insttue for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> Office: +61 3 9341 3155
> Mobile: +61422882700
> Fax: +61 3 9341 3104
>
> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Jean-Marie Vanderwinden
> Sent: Wed 12/09/2012 6:49 PM
> To: [hidden email]
> Subject: Re: nuclear membrane marker
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear,
>
> You may consider LaminA/C:
>
> fluorescent constructs for live imaging
> e.g. http://www.ncbi.nlm.nih.gov/pubmed?term=20079404
> and Lamin Abs work usually fine on fixed material...
>
> With best wishes.
>
> JM
>
> At 09:35 12/09/2012, you wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Vitaly,
>>
>> Frankly, there is no really specific nuclear membrane dye. I remember a
>> paper from Zal et al., (Traffic 2006; 7: 16071613), which says the dye
>> FM4-64 is specific for the nuclear membrane, though in our tests the results
>> were inconsistent and it also stained other membrane entities.
>> I would suggest to use anti-NPC antibodies - these should specifically stain
>> the nuclear membrane (in interphase cells) - famous in the field is mAb414.
>>
>> hope this helps,
>> Josef
>>
>> On Tue, 11 Sep 2012 14:57:00 -0700, Vitaly Boyko
>> <[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,
>>>
>>> one of our users is looking for a relatively abundant marker that is
>> homogeneously distributed over the nuclear membrane in cultured human cells
>> (it could be a well characterized Ab against the nuclear receptor, or simply
>> specific (nuclear) membrane stain).
>>>
>>> Many thanks in advance,
>>>
>>> Vitaly
>
> Jean-Marie.
>
> Jean-Marie Vanderwinden, M.D., Ph.D.
> Directeur de Recherche F.N.R.S. / Research
> Director, National Fund for Scientific Research (Belgium)
> Neurophysiology lab http://www.ulb.ac.be/medecine/neurophy/
> Light Microscopy Facility http://limif.ulb.ac.be/
>
> Postal address:
> Laboratoire de Neurophysiologie (Prof. S.N. Schiffmann),
> Faculté de Médecine, Campus Erasme, CP 601,
> Université Libre de Bruxelles,
> 808 route de Lennik, B-1070 Brussels, Belgium.
>
> (: Secretary: +(32).2.555.42.30, Direct: +(32).2.555.69.88
> 7: +(32).2.555.41.21,
> . [hidden email]
>
> Skype: Jean Marie Vanderwinden
> 12voip / netappel: jeanmarievanderwinden
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

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