Jennifer Kramer wrote:
 
>One important point here not mentioned is the presence of the pinhole or
>slit in the back focal plane of the objective lens.
>This can exclude up to 99% of all emitted light, requiring use of high
>probe concentrations (which can be potentially toxic).
 
This sort of statement always mmakes me see red!  The only light the
confocal pinhole excludes is the *out of focus* light!!  The Airy
disk at the point of focus more or less all goes into the final
image (depending on the pinhole setting).
 
The reason for the better light budget of deconvolution systems has
nothing to do with the pinhole and everything to do with serial vs
parallel collection of images.  In terms of light budget, a one
second scan of a confocal image (collecting 256K pixels) is equivalent
to a 4 microsecond exposure of a widefield image with the same
light intensity.  Looking at it the other way round, a single
widefield video frame (1/25 sec) is equivalent to scanning a
confocal image for 10,000 seconds!!
 
Of course in confocal we try to overcome this by using brighter
light (a laser) but because fluorochromes saturate it isn't a total
solution.  And if we crank up the light beyond the saturation point
we get severe bleaching, as Jennifer points out.
 
So, yes, deconvolution does have a much better light budget, but
NOT because "we are throwing away 99% of the light" in confocal.
On the other side of the coin, confocal is (to a very rough
approximation) object independent in its 3D imaging capability.
Deconvolution varies from quite good (isolated point objects)
to completely ineffective (very extended objects).  As with
everything else in this game, we have to trade off one desirable
property to do better on another!
 
                                        Guy Cox