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Hi Jerry

There is no reason why resolution should change except that the rear 
aperture is not filled and/or the light does not have the correct degree 
of collimation so that sperical aberration may be present. You must 
overfill the rear aperture to get good z-resolution -don't focus the 
light through the rear aperture. I note that your original figure of  z 
resultion of 0.2 um for reflected mode sounds very unlikely -that's way 
above the diffraction limit.

Cheers Mark

. Jerry Sedgewick wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi All,
>
> I am working with a graduate student on a home built 2 photon. In the 
> past he had routinely used 750nm on a Tsunami (5 watt) and was able to 
> see a reflection off the surface of a silicon wafer in order to 
> determine the bottom of his sample (the sample itself is polymer beads 
> which do not fluoresce at non-polymerizing wavelengths). Now we have 
> gone to another laser system (the 10 watt MaiTai)and he is not able to 
> see the reflection with the same precision (this probably has nothing 
> to do with the new laser, but with spot size in the back aperture or 
> something else: it's very well aligned).
>
> Before the new laser (and shorter laser path) he could see the 
> reflection with .2 micron accuracy (the reflection fades rapidly with 
> .2 micron z-resolution). Now he sees the reflection at about a 2 
> micron accuracy.
>
> I have been noticing that the z-resolution in general seems not quite 
> up to par with thin focal volumes expected with a multiphoton. This 
> new setup seems to have about the same z-resolution as a single 
> photon, though I have never measured the actual z-resolution. I don't 
> generally use a lens in the light path to converge the beam: when I 
> do, it's a 1000mm focal length plano-concave lens which creates a 
> narrower diameter spot on the back aperture of the lens. I do use a 
> lens to narrow the diameter of the back projected image onto the PMTs.
>
> Any thoughts?
>
> Jerry Sedgewick
> University of Minnesota