>,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.

I cannot see why there is a difference between confocal & 2-photon
here.  The rejection of out-of-focus light is just a statistical matter
in either case, and the psf is essentially the same shape (but the
volume is larger in 2-photon because the exciting wavelength is longer).
But what one is imaging is not a sharp-edged slice like a slice of
bread but a volume in which the centre line contributes most, and the
contribution falls off (according to the axial response function) as
you move away from the line.

The more relevant point for ratio vs. non ratio imaging is that the
area above the slice being sampled will still absorb light (both
exciting and emitting) since, after all, the light has to travel
through it!  So ratio imaging is just as important in confocal (or
2p) if we are going to image at different or unknown depths
within our sample.

>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.

Whether confocal or not the key thing is that
1. The background must not be 0 anywhere in any image.
2. The peaks must not saturate anywhere in any image
since otherwise ratios would be meaningless.  But provided
these criteria are met ratioing should give a reasonably
accurate result.  I can't see why regions of varying thickness
make any difference unless, of course, they also vary in Ca
content or pH or whatever you are measuring.

                                                Guy

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