Advice please

[Tom Smulders]

Dear ImageJ users,

I'm a long-time user of NIH Image, but only a recent member of the
ImageJ list.  I was hoping to solicit some advice on setting up the
microscopy part of my laboratory, as we have just started a new
project and I need to get the microscopic quantification going soon.

The project I am working on requires mostly counting of cells and
measurement of brain structure volumes.  We are looking at seasonal
patterns in adult neurogenesis, total neuron numbers and hippocampal
volume in the brains of birds (great tits and willow tits to be
exact). Our histological strategy will be the following:

We will cut/are cutting 10 micron coronal serial sections from these
brains (fresh frozen).  We are cutting these on a cryostat (so
directly onto the slides) and splitting the sections into 10 parallel
series, so that each series contains sections from throughout the
brain, spaced by 100 microns.  These sections will be stained with
fluorescent immunocytochemistry to look for the expression of
different markers of neuronal differentiation in BrdU-labeled
cells.  In other words: we need to count single- and double-labeled
fluorescent cells, as well as count total numbers of cells (probably
using DAPI in the same sections, or counting from a separate series
stained with Nissl stain).

My equipment situation is as follows: I have a fluorescent
microscope, currently without an imaging system on it.  I need to
purchase a system that can acquire images from the fluorescent as
well as regular light microscopic sections, and then I need to do the
counts.  And I would like advice on the following issues:

1) Would ImageJ be able to do all the quantification I need?
2) a) Can ImageJ actually be used as capturing software, or is it
better to get some software package that comes with whichever camera I buy?
     b) Any recommendations of a good quality, good value digital
camera for low light conditions?
3) a) What about stereology?
     b) Is there a Stereology module/plug-in for ImageJ?
     c) Can stereological techniques even be applied to the tissue as
we have sectioned it?
     d) If I were intent on using stereological methods to count
total neuron numbers in the hippocampus, am I correct in assuming
that I would need to cut thicker sections in order to be able to use
the optical dissector method?
     e) Can that be done if the sections I take are 100-150microns
apart throughout the nucleus (doesn't this violate the dictum that
each particle needs to have the same chance of being counted?)?
     f) The hippocampus is also rather heterogeneous in its cell
density (i.e. it is structured, be it not as structured as the
mammalian hippocampus): are there random sampling schemes that will
still estimate the total cell numbers accurately?

Additionally, I want the system to be able to measure optical
densities and count silver grains, but I understand that ImageJ can
do that without any problems...

Feel free to e-mail me back directly, if it is inappropriate to get
all these replies to the list.  And I apologize if you get requests
like this often.

Sincerely,

Tom Smulders
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Tom Smulders, Ph.D., Lecturer
School of Biology and Psychology (Division of Psychology)

The Henry Wellcome Building for Neuroecology, University of Newcastle
Newcastle upon Tyne, NE1 7RU; Tel: ..[phone]; Fax:
..44-(0)191-222-5622

http://www.staff.ncl.ac.uk/tom.smulders 

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Any opinions expressed in this e-mail are mine, and mine only.
They are not those of the University of Newcastle, nor of any of its parts.