> Paul, > > How does deconvolution solve the lensing problem? Surely by assuming a > fixed PSF, they would also produce incorrect results as well. I never claimed that it does, but rather in deconvolution it seems less severe for the case of multiple beads on top of each other and I mention it because people get the idea that since it is a confocal, it must be revealing truth (a confocal wouldn't lie, would it?) The truth of the matter is that bad optics and non-uniform optical paths through the specimen effect all imaging. If one thinks that confocal is immune, one is uninformed. I thought that that was one of Wilson's messages at Scanning. > And I thought that you and I had agreed at the Scanning meeting last month > that it was unfair to use the term "resolution" as a measure ot the spatial > accuracy of an imaging process once deconvolution was involved? I attach > an old message that outlines the argument to the end of this note. > > I would add to it only the fact that Hans VanderVoort ([log in to unmask], J. > Geradtsweg 181, 1222 PS Hilversum, The Netherlands. URL: > http://www.svi.nl/tel: (0)35-6859405 or (0)653-345445. fax: (0)35-6837971) > has recently had great success in deconvolving confocal data*. > > This software not only seems to "improve the resolution" but also reduces > the apparent noise by taking advantage of the fact that Nyquist Sampling of > the image of a point object (i.e. a 3D Airy Disk) with a pixel size > suitable to recording data out to the Rayleigh Criterion (i.e. two pixels > between the center and the first dark ring in all directions) requires > collecting data from 64 voxels. Knowing the PSF can effectively allow you > to average the signal collected in these 64 voxels (or more). > > From now on, if one must try to compare the "resolution" of the two > techniques, let's compare deconvolved WF with deconvolved Confocal. > > Jim Pawley First, by resolution I mean the ability to dissociate two close objects or the ability to extract spatial information that is consitent with otherwise confirmed reality at a spatial frequency that is higher than the method that one is comparing to. If one system gives me a better image, i.e., that better agrees with reality as measured by an independent method, then I would be inclined to call that higher resolution. The tests that we have used would be consistent with this definition. I can see the value of deconvolving confocal and we are working frantically on a method to do so. I am sure that we too will find a method that works. The power spectra of the confocal images shows that there is plenty of room for resolution extension. I am just trying to find the best way to get that data out. However, since our starting data for the case of a couple of cell layers has our starting data with better signal to noise, more linearity, and at least as high of spatial resolution, then I would not expect that we could get even better resolution with decon/confocal for this case. I may be wrong and I hope to be able to confirm it this summer. In the meantime I will look further at VanderVoot's method.