>>,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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Dr Geoff Hyde
Lecturer
School of Biological Science
University of New South Wales
Fax: 612 9385 1558
Ph:  612 9385 1648
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