FW: labelling cell nuclei

[Keith Morris]

Actually all I am saying is that molecular cytogenetists often go to a great
deal of trouble to get their nuclear material into 2D [flat] on the slide,
in which case the confocal loses many of its advantages over the high power
wide-field upright microscope and indeed the confocal images can look a tad
'grainy' in comparison [we need high contrast as much as resolution]. Also a
standard confocal microscope would be pushed to easily carry out a multiplex
FISH karyotyping on a chromosome metaphase spread which requires the rapid
imaging of six fluorochrome chromosome paints sequentially [Spectrum Gold,
Red, Far Red, Green, Aqua/DEAC and DAPI] and the software to karyotype the
images. Plus the confocal often takes a tad longer to acquire images, and
this can be significant as cytogenetics spend half their existence locating
and imaging metaphase spreads, chromatin fibres, interphase nuclei, swapping
objects etc. We also use transmitted light for Giesma banding stains, which
isn't the confocals strong point.

It totally depends really on what you want to look at though. If you want to
look at fluorescent in-situ hybridisation [FISH], or some comparable
chromatin imaging technique, in 3D within say the interphase nucleus, then
naturally something like our Zeiss 510 MetaHead confocal would generally be
our first choice [I expect a wide-field microscope with a z motor and 3D
de-convolution software might also produce images of interest, although I
would still use the confocal as well with the sample]. Plus we have
Improvision's Volocity 4D software. Our confocal does lack a high NA 100x
objective though, which would be useful despite the '10x optical zoom' we
have with our 63x plan apochromat. So for confocal microscope reviews
relating to the nucleus I'd search for articles with titles like '3D
Fluorescence[or Fluorescent] In Situ Hybridization for Imaging Interphase
Chromosomes/nuclei' and 'chromatin 3D spatial organization in the nucleus'.
Although 3D FISH represents a small amount of our cytogenetics work, it has
become an important aspect of it. I don't know of any reviews of 3D FISH as
such [I'm relatively new to Molecular Cytogenetics so I'll ask the
cytogeneticists here].

As I mentioned our group head is co-editing a large tome on all aspects of
modern FISH imaging, but that won't be published until
next year. My big book of FISH ['Introduction to flourescence in situ
hybridization. Principles and Clinical Applications', 1999: M Andreeff & D
Pinkel, editors], makes no mention at all of 3D FISH applications, probably
owing to its age.


Keith

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Dr Keith J Morris
Molecular Cytogenetics and Microscopy Core
The Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford
OX3 7BN
United Kingdom

Tel:  +44   ( 0 ) 1865  287568
Email:   [hidden email]
HomePage:  http://www.well.ox.ac.uk/cytogenetics