long working distance objectives to image transwell cultures

[daj1u06]

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Hi,

can anyone recommend a long working distance objective (x40 - x63 range) to
use on a Leica SP5 inverted frame confocal to image cells growing on top of a
transwell membrane where the membrane sits about 1mm above the top of the
multiwell plate bottom (plastic 24 well plates). We will be imaging through the
plastic well bottom, medium and membrane - the working distance is going to
need to be 2.3-2.5mm. It is certainly a far from ideal situation but it is all my
user can acheive with his system.

We already have a Leica dry x40 HCX PL Fluotar (0.6 NA) but wondered if
there is anything much superior for the task in your experiences.

Thanks in advance,

Dave Johnston,
Biomedical Imaging Unit,
Southampton.

[Pascal Weber]

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Hello David,

I have used and made ​​images of such boxes. In fact, the best solution is not to
keep the membrane inside the box and after that you can use any water
immersion objective. If you want to know more contact me on my mail.
Best regards Pascal

[Chere Petty]

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Pascal, David has an  inverted scope so your objective will not really be of much help?. I have a N Plan L 40x  0.55 dry objective with a working distance of 3300 um. may not work for you but we use it to imagine bio films and I have an inverted SP5.
On Jun 10, 2013, at 12:57 PM, Pascal Weber wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello David,
>
> I have used and made ​​images of such boxes. In fact, the best solution is not to
> keep the membrane inside the box and after that you can use any water
> immersion objective. If you want to know more contact me on my mail.
> Best regards Pascal
>

Chere Petty M.S.
Manager of UMBC Keith R. Porter Core Imaging Facility
Department  of Biological Sciences
University of Maryland Baltimore County
(UMBC)
1000 Hilltop Circle
Baltimore Maryland 21250
301-367-8408
[hidden email]
emumbc.com

[Tim Feinstein-2]

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Hi David,

Even taking into account the plastic bottom and very long working distance, the optical properties of transwell membranes are pretty suboptimal.  In facing the same problem we tried some new slides from Simpore designed to work with inverted objectives of reasonable working distance.  The were a bit tetchy to work with, in particular getting a medium miniscus under the membrane called for fine gel tips and a lot of patience, but the optically clear membrane and coverslip bottom made for nice imaging.  No commercial interest.  

All the best,


Tim Feinstein

Sent from my iPhone

On Jun 10, 2013, at 12:06 PM, David Johnston <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> can anyone recommend a long working distance objective (x40 - x63 range) to
> use on a Leica SP5 inverted frame confocal to image cells growing on top of a
> transwell membrane where the membrane sits about 1mm above the top of the
> multiwell plate bottom (plastic 24 well plates). We will be imaging through the
> plastic well bottom, medium and membrane - the working distance is going to
> need to be 2.3-2.5mm. It is certainly a far from ideal situation but it is all my
> user can acheive with his system.
>
> We already have a Leica dry x40 HCX PL Fluotar (0.6 NA) but wondered if
> there is anything much superior for the task in your experiences.
>
> Thanks in advance,
>
> Dave Johnston,
> Biomedical Imaging Unit,
> Southampton.

[Chere Petty]

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Pitt,
 We need wells and Number 1.5 coverslips. Hummm I will look into this company. Thanks!
On Jun 10, 2013, at 3:52 PM, Pitt wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi David,
>
> Even taking into account the plastic bottom and very long working distance, the optical properties of transwell membranes are pretty suboptimal.  In facing the same problem we tried some new slides from Simpore designed to work with inverted objectives of reasonable working distance.  The were a bit tetchy to work with, in particular getting a medium miniscus under the membrane called for fine gel tips and a lot of patience, but the optically clear membrane and coverslip bottom made for nice imaging.  No commercial interest.  
>
> All the best,
>
>
> Tim Feinstein
>
> Sent from my iPhone
>
> On Jun 10, 2013, at 12:06 PM, David Johnston <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi,
>>
>> can anyone recommend a long working distance objective (x40 - x63 range) to
>> use on a Leica SP5 inverted frame confocal to image cells growing on top of a
>> transwell membrane where the membrane sits about 1mm above the top of the
>> multiwell plate bottom (plastic 24 well plates). We will be imaging through the
>> plastic well bottom, medium and membrane - the working distance is going to
>> need to be 2.3-2.5mm. It is certainly a far from ideal situation but it is all my
>> user can acheive with his system.
>>
>> We already have a Leica dry x40 HCX PL Fluotar (0.6 NA) but wondered if
>> there is anything much superior for the task in your experiences.
>>
>> Thanks in advance,
>>
>> Dave Johnston,
>> Biomedical Imaging Unit,
>> Southampton.
>

Chere Petty M.S.
Manager of UMBC Keith R. Porter Core Imaging Facility
Department  of Biological Sciences
University of Maryland Baltimore County
(UMBC)
1000 Hilltop Circle
Baltimore Maryland 21250
301-367-8408
[hidden email]
emumbc.com

[George McNamara]

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Hi David,

Use whatever objective lens enables you to answer your research
questions, after you put the cells on the other side - see

Histochem Cell Biol. 2007 May;127(5):463-72. Epub 2007 Feb 17.

Four-dimensional imaging of filter-grown polarized epithelial cells.

Wakabayashi Y, Chua J, Larkin JM, Lippincott-Schwartz J, Arias IM.

Cell Biology and Metabolism Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD 20892, USA.
[hidden email]

Understanding how epithelial cells generate and maintain polarity and function
requires live cell imaging. In order for cells to become fully polarized, it is
necessary to grow them on a permeable membrane filter; however, the translucent
filter obstructs the microscope light path required for quantitative live cell
imaging. Alternatively, the membrane filter may be excised but this eliminates
selective access to apical and basolateral surfaces. Conversely, epithelial cells
cultured directly on glass exhibit different phenotypes and functions from filter
grown cells. Here, we describe a new method for culturing polarized epithelial
cells on a Transwell filter insert that allows superior live cell imaging with
spatial and temporal image resolution previously unachievable using conventional
methods. Cells were cultured on the underside of a filter support. Epithelial
cells grown in this inverted configuration exhibit a fully polarized
architecture, including the presence of functional tight junctions. This new
culturing system permits four-dimensional (three spatial dimension over time)
imaging of endosome and Golgi apparatus dynamics, and permits selective
manipulation of the apical and basolateral surfaces. This new technique has wide
applicability for visualization and manipulation of polarized epithelial cells.

PMID: 17308935
PMCID:   PMC3541541
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541541/ 



On 6/10/2013 11:06 AM, David Johnston wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> can anyone recommend a long working distance objective (x40 - x63 range) to
> use on a Leica SP5 inverted frame confocal to image cells growing on top of a
> transwell membrane where the membrane sits about 1mm above the top of the
> multiwell plate bottom (plastic 24 well plates). We will be imaging through the
> plastic well bottom, medium and membrane - the working distance is going to
> need to be 2.3-2.5mm. It is certainly a far from ideal situation but it is all my
> user can acheive with his system.
>
> We already have a Leica dry x40 HCX PL Fluotar (0.6 NA) but wondered if
> there is anything much superior for the task in your experiences.
>
> Thanks in advance,
>
> Dave Johnston,
> Biomedical Imaging Unit,
> Southampton.
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054

[Dan Focht]

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Dave

Cells can be imaged in Transwells on an inverted scope with standard
objectives in the following manner:
Plate the cells on the basil side of the membrane by inverting the
Transwell and placing a short piece of tubing around the membrane
forming a temporary well.
Apply the cells in this well and incubate for a few hours or until they
attach. Depending on the porosity of the membrane it is sometimes
necessary to plug the tubular underside of the membrane.  We make an
autoclavable o-ring device for this purpose.
After a few hours, remove the Transwell from the incubator, remove the
temporary tubing and return the Transwell in its proper orientation into
the original SBS tray with media and allow the cells to grow to confluency.

When plating is complete you take the Transwell from the SBS tray and
place it in a Bioptechs Transwell adapter that goes into a Bioptechs
Delta T dish.  You can adjust the proximity of the cells on the
transwell membrane to the top of the coverslip by rotating the outer
ring on the Transwell Adapter.  In this manner you can directly image
the cells with standard objectives through a coverslip and not worry
about the membrane.
The coverslip will provide much better resolution than plastic, the
Delta T system provides temperature control and media containment.
You also have the option of basil or apical perfusion depending on what
accessories you use with it.

Link:
http://www.bioptechs.com/Products/Delta_T/Options/options.html#membraneadapter

Dan



On 6/10/2013 12:06 PM, David Johnston wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> can anyone recommend a long working distance objective (x40 - x63 range) to
> use on a Leica SP5 inverted frame confocal to image cells growing on top of a
> transwell membrane where the membrane sits about 1mm above the top of the
> multiwell plate bottom (plastic 24 well plates). We will be imaging through the
> plastic well bottom, medium and membrane - the working distance is going to
> need to be 2.3-2.5mm. It is certainly a far from ideal situation but it is all my
> user can acheive with his system.
>
> We already have a Leica dry x40 HCX PL Fluotar (0.6 NA) but wondered if
> there is anything much superior for the task in your experiences.
>
> Thanks in advance,
>
> Dave Johnston,
> Biomedical Imaging Unit,
> Southampton.
>
>

--
Dan Focht Bioptechs Inc. V: [phone] www.bioptechs.com

[Pascal Weber]

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I did it with an water immersion objectiv ! It was the Zeiss 20X na= 2.0 on an
inverted microscope ( Zeiss two photon microscope). This objectiv has 1.9mm wd.
I put on a silicon collar and enought water for immersion (the limit is the collar). I
made a special plate who accept the internal well (with only the membrane).
I can send pdf.
  Regards