>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?
>
>-------
>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?
 
Felicity,
 
You do not say what excitor wavelength you are using, but if it is in the
uv, the first place to look is at the design specifications for these two
types of objectives (which Leica should be able to provide).  The fluotar
objectives are specifically designed for working over a greater range in
the uv than are apochromats, but you should check their specific spectral
transmission properties.  In addition, lenses not designed specifically for
uv work may have severe chromatic aberration in the uv, which may explain
your non-detailed "ghost-like" images when using the planapo.  In
particular, what I propose is that (axial) chromatic aberration causes the
uv light to be focussed at some other plane (voxel) in the specimen than
the plane to which the detection path and pinhole are aligned for detecting
the fluorescence signal. Because you are getting only a non-focussed
illumination spot in the plane you are detecting, you get a weak,
unfocussed fluorescence signal.
 
Hope this helps,
Carol Cogswell
 
 
-------------------------------------------
  Carol J. Cogswell
  Senior Lecturer
  Physical Optics Department
  School of Physics
  University of Sydney
  NSW 2006 Australia
  Fax: +61 2 692 0923
  Tel: +61 2 351 3201
  Email: [log in to unmask]
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