,In reply to my first posting,

John J. Lemasters, MD, PhD wrote

>You can't have it both ways.  If it is confocal microscopy, then excited light
>from out of focus planes is being excluded from the observed image.  If
>it is a well designed confocal system, this stray light is small and
>distributed evenly throughout the image.
> To posit that confocal microscopy
>collects a lot of light outside its "collection" volume seems a contradiction
>of terms.

In confocal microscopy, as opposed to two-photon microsopy, I believe that
some light that is emitted from fluorophores outside the effective
collection volume  must pass through the collection volume and be measured
along with light emitted from fluorophores within the collection volume. So
to my mind this does not involve a confocal "contradiction".
However, my intuition and the experiment I mentioned in the first message,
make me agree with you that it is not a great contribution and i doubt that
its elimination by 2-photon microscopy is something to be excited about.

My concern is not really with confocal microscopy (although I still think
the above problem is an interesting teaser). My concern is with
non-confocal "normal" microscopy. I may not have made myself clear enough
in the original message, but it is in this approach where the contribution
of light from fluorophores outside the region of interest is definitely not
insignificant and when a cell has regions of varying thickness, it can
cause the problems with ratioing first described by Silver et al, and
summarised in my message.

>
>Because confocal microscopy collects a optical slice of uniform thickness, the
>importance of ratiometric measurement is diminished since the fluorescence
>intensity becomes mostly independent of cell shape and volume. Ratioing
>helps to calibrate concentration but there are also other ways to do this
>without ratioing.

One can still run into problems with variations in organelle density within
the cell, especially if the organelles cant be distinguished at the LM
level.
>
>John J. Lemasters, MD, PhD
>Director of Confocal Microscopy
>Department of Cell Biology & Anatomy
>University of North Carolina at Chapel Hill
>CB# 7090, 236 Taylor Hall
>Chapel Hill, NC 27599-7090 USA
>Tel: 919-966-5507
>FAX: 919-966-1856
>E-mail: [log in to unmask]


Dr Geoff Hyde
Lecturer
School of Biological Science
University of New South Wales
Fax: 612 9385 1558
Ph:  612 9385 1648
Email: [log in to unmask]