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Dardo Ferrera wrote:
> "Large-frame, high-power argon ion lasers can provide UV lines but are
> rather expensive. Also, the axial chromatic aberration present in many
> objectives between the UV and visible wavelengths often gives rise to
> disappointing results"
>
> I wonder whether somebody may provide an explanation for this chromatic
> aberration if in fact is a true phenomenon.

It certainly is a true phenomenon.  The refractive index of
any glass changes with wavelength (dispersion) and lens
manufacturers correct for this at two wavelengths (achromats)
or 3 wavelengths (apochromats) by combining glasses of diferent
dispersion.  These wavelengths are normally blue and red, with
green added in apochromats.  Recently some manufacturers have
moved their shortest correction point into the violet because of
the popularity of 405nm lasers (these are marked VC or similar).
None of these lenses make any attempt to correct chromatic
aberration in the UV though you could probably hope to do
better with a VC lens than a standard one.

In the days when some confocal manufacturers offered argon
UV lasers as options they included correction lenses matched
to individual objectives to bring the spot to the correct focus.
My personal experience was that it was never very satisfactory.
You would have to do something similar.  Alignment is a problem
since the beam is invisible and safety issues are also a big
problem.   (Bio-Rad used to put the entire scope in an orange
plexiglass box).

My advice is don't but .... if you do have some pressing reason
to use UV rather than 405 buy a cheap, small UV diode laser
rather than a huge, hugely expensive, water cooled argon!


> At the same time, I would like to ask if somebody has ever compared the
> tissue autofluorescence( in different channels and percent transmission of
> laser power) generated with single photon excitation with UV light vs 2
> photon excitation at 700-750 nm.

The question of percent transmission of laser power is rather
hard to answer since fluorescence intensity in single photon
mode varies linearly with excitation but in 2P it's as the
square of the intensity.  The spectral makeup isn't so hard -
I have the gear to take a spectrum in either 2P or 1P albeit
on different scopes so if you tell me what tissue are interested
in I could probably try it for you.

                                                Guy Cox


--
Associate Professor Guy Cox
Electron Microscope Unit,
University of Sydney,
NSW 2006, Australia

Phone:+61 2 9351 3176    Fax:+61 2 9351 7682
http://www.guycox.net