Aryeh Weiss wrote:

> Even when comparing emission between two dyes within one sample, you have to
> check your field flatness. Not only might the field not be flat, but the
> response to a uniform dye concentration can vary between channels.
> This may be caused by chromatic aberations. In any case, it means that if you
> are comparing the relative responses between two channels (in effect, a ratio
> method), you may get spurious results due to having a nonflat and chromatically
> varying field.


Aryeh - I couldn't agree more. I was only talking about comparing
results with a single label. I would reckon that to compare absolute
values between different dyes, even in the same section, is next to
impossible! However, we do sometimes use corelations between the
intensity of two or three labels in the same section. For each channel,
we have to ensure that  all the labelling and collection criteria are as
stringent as possible.

Two other points about the types of things we do...

First, as suggested by Aryeh, we always use only the central portion of
the lens - usually a 3x zoom factor, left in the central axis (ie we
don't move the beam off centre) - this minimises abberation effects (we
hope!!)

Second, we usually do this sort of analysis on nerve terminals that are
double or triple labelled. These structures are from 0.1 - about 3
microns diameter, and usually are in material that has been sectioned at
10 - 20 microns. The tissue is embedded and sectioned in polyethylene
glycol and mounted in buffered glycerol - this means that the tissue is
fully hydrated but not very thick. Optically it seems to be pretty good
- ie uniform penetration of antibodies and light, and there is virutally
no shrinkage in our preps. I would not like to do any sort of
quantitative analysis of fluorescent intensity on larger objects which
either are not fully contained within the section thickness or which
have to be observed in thicker sections...

Hope that helps,

IAN


--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

Phone:  +61-8-8204 5271
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