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Elaine Kunze wrote:
>
> Has anyone ever used confocal microscopy to measure the volume of pits
> in a metal surface?  I have tried reflective measurements, but can't
> tell where the pit begins or ends.  I know its a long shot but I
> figure if anyone knows, it will be this group.
>
> ****************************************************************************
>         Elaine Kunze
>         Flow Cytometry.....Image Analysis...
>         Life Sciences Consortium
>         http://www.lsc.psu.edu/stf/cqca/home.html
>         8B Althouse Laboratory  (814-863-2762)
>         Penn State University
>         University Park, PA 16802

Elaine,

The main problem with this sort of reflective imaging is that you will
obviously not be able to image surfaces where the reflective light
approaches the lens at angles that fall outside the acceptance angle
(defined by the NA). Another related problem, particularly if youy wish
to do roughness measurements as well as volume, is that you get massive
intensity variations between the different reflecting surfaces as they
vary between horizontal (maximum reflection) to vertical (no
reflection). Hence you get extremely "noisy" images, and volumes
measurements are "tricky".  Some of this can be corrected by using high
NA lenses and imaging processing, but I have found it far easier to work
with fluorescence rather than reflection. Fo largish objects, you may be
able to make a fluorescent caste of the object, but for fine detail (eg.
imaged through a >40x oil lens) you can flood the specimen with
fluorescent oil and image the oil. In both cases you end up making a
negative/inverse image of the object which can be measured directly, or
can be inverted so a pit looks like a pit. I've used oil saturated with
eosin (or oil red) to image metal surfaces with deep pits or scratches.

Hope this helps,

Ian.



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Dr Ian S. Harper, Director
Monash Micro Imaging
The Microscopy & Imaging Research Facility
Building 13C, Monash University,
Clayton, VIC 3800, Australia

Email: [log in to unmask]
Tel: +61 3 9905 5635;  Fax: +61 3 9905 2766
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