Hi Everybody!
 
We have been given a DAPI filter cube to trial prior to purchase.  I have
two questions regarding its use :
 
1.   Different objective lenses give different images.  Why?
2.   General uses for a DAPI filter cube.  Can we justify the cost?
 
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1.   When we observe our specimen (macrophages within lung - cryostat
sections) using a 40x Pl Fluotar objective 1.00-.50 oil (LEICA lens), our
images are superb : clear, crisp, pretty.  This is the case both with oil
immersion and just dry viewing using this lens.
 
The problem occurs when we view our sample with our 60x Pl Apo objective 1.4
oil (LEICA lens).  The image becomes non-existent or ghost-like.  A pale
"shadow" only is seen, with no detail visible at all.  However, viewing with
green and blue excitation lines (fluorescence microscope) continues to give
excellent (although non-specific fluorescent) images on both the 40x and 60x
objectives.
 
I discussed our problem with LEICA.  At first thought, the explanation they
proposed was that the 60x Pl Apo was giving autofluoresence from the glass
components of the objective lens following excitation with ultraviolet light
(selected by the DAPI filter).  This then masked the sample fluorescence to
the extent that no image was obtained.  The longer wavelengths (green and
blue lines) would not cause this autofluorescence.
 
In comparison, the Pl Fluotar 40x objective could cope with a larger range
of wavelengths, hence this problem was not observed with this lens.
 
IS THIS A REASONABLE ASSUMPTION?  HAVE OTHER PEOPLE EXPERIENCED SIMILAR
DIFFICULTIES?
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2.    Our application is visualisation of marcophage particles in lung
tissue.  Macrophages autofluoresce but we are trying to locate the presence
of benzopyrene within the cells.  Benzopyrene excites at 380nm and emits at
430nm. The DAPI filter seems a reasonable choice (and was recommended by
this discussion list).
 
WHAT ARE OTHER PEOPLE USING DAPI FILTERS/STAINS FOR?  ARE THERE MANY OTHER
GENERAL APPLICATIONS FOR DAPI THAT COULD HELP JUSTIFY THE PURCHASE PRICE OF
~$650 (AUSSIE DOLLARS)?
 
Many thanks for your responses,
 
Felicity Lawrence
Analytical Electron Microscopy Facility, QUT
Brisbane, Australia