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Hi Doug, 

The short answer is to use a water immersion lens on an upright scope.
However, Leica makes a 20x NA 0.7 multi-immersion objective that you
should check out (no commercial interest).  I have used both the Nikon and
Zeiss equivalents on inverted scopes when we needed max imaging depth.

The deeper you image the more you will lose to spherical aberration unless
you match the refractive index of lens immersion with your sample medium.
A multi-immersion lens lets you do that better than most alternatives.  On
the other hand flexibility always necessitates optical trade-offs; others
here can describe the considerations there better than I can.

All the best, 


TF

Timothy Feinstein, Ph.D. | Confocal Manager
Van Andel Research Institute
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: 616-234-5819 | Email: [log in to unmask]







On 4/10/14, 11:38 AM, "Cromey, Douglas W - (dcromey)"
<[log in to unmask]> wrote:

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>
>I am working with a lab that is interested in doing fluorescence
>microscopy on spheroid assays (clumps of cancer cells seeded and growing
>inside a moderately thick collagen matrix).  They are looking at a number
>of different microscopy techniques on campus.  Because our Leica confocal
>was mostly configured for 2D cultured cell and tissue sections, we have
>quickly discovered the working distance limitations of our existing
>objectives.  Our local Leica technical representative will be loaning us
>Leica's fabulous 25x/0.95 water immersion objective (2.5mm WD) to try
>out.  This should help with WD issues, as well as spherical aberration in
>the sample (their dishes do at least have #1.5 thickness glass coverslip
>bottoms).
>
>Has anyone else worked with these types of assays before?  Any
>suggestions on the sample prep side or the imaging side to end up with
>better image data?
>
>Thanks,
>Doug
>
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>Douglas W. Cromey, M.S. - Associate Scientific Investigator
>Dept. of Cellular & Molecular Medicine, University of Arizona
>1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
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