Aryeh,
You have just set up "axial illumination" and generated both its benefits
and problems.
Try the following experiment: Remove the sample and an objective then
rotate the nosepiece so that you have some open working space. Fold back
the bottom quarter of a business card (white works best) to make an "L".
Rest the base of the L on the stage so that you can catch the beam emerging
from the condenser. Close the field iris then open and close the
condenser. You will notice two phenomena:
a. That the angle emerging from the condenser changes dramatically as you
open and close it (closed = axial illumination; a pencil of light with
little or no angle) and
b. that the cross over point (related to depth of field in the specimen)
is very shallow when the condenser is full open but becomes "infinitely"
deep when the condenser is closed.
This latter phenomenon is the source of your problem: very great depth of
field pulls in dust and dirt from a large range of adjacent planes, both
immediately adjacent to the sample and within the optical train (front
focal plane of the condenser, back focal plane of the objective, exit pupil
of the microscope). The rings you see around these particles result from
the very coherent beam, generated by the closed condenser, diffracting at
their edges. If the diffraction results in half wave shifts (or odd
multiples of half wave shifts), you will see a dark fringe (destructive
intereference); if it results in full wave or multiple wave shifts, you
will see bright halos.
To REALLY remove the dust and dirt can take a half a day's worth of
cleaning. Evaluate how disruptive these spots are and, if you can live
with them, just explain their origin in your write up.
For further discussions along these lines, you might want to read
"Optimizing Light Microscopy". Details are available on our website.
Hope this was helpful.
Barbara Foster,President
Microscopy/Microscopy Education
125 Paridon Street Suite 102
Springfield, MA 01118-2130
PH: 413-746-6931 FX: 413-746-9311 email: [log in to unmask]
Website: www.MME-Microscopy.com/education
America's first national consortium of microscopy specialists offering
customized on-site training in all areas of microscopy, image analysis, and
sample preparation
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At 11:01 PM 6/19/00 +0300, Aryeh M. Weiss wrote:
>I have a problem with some artifacts in my brightfield images which I cannot
>track down.
>
>When the condensor is set to a low NA, there are a lot of dark spots in
>the observed image. However, these spots do not originate in the condensor
>(verified by tilting the condensor -- they dont move).
>When the NA of the condensor matches the NA of the objective, they
>almost disappear. Problem is, in this setup, we often have to work with
>very low NA illumination (for example, with the condensor well removed its
>correct focus setting). Under these conditions, the spots are quite
prominent.
>
>Their location does not change when the objectives are changed.
>They do not appear to be in any of the image planes.
>We have cleaned all of the exposed surfaces.
>They appear both in visual observations and with two different cameras,
>so they are not detector artifacts.
>
>We can acquire them as a background image and subtract, but it is certainly
>better to get rid of them in the optics, before processing.
>
>Any ideas will be much appreciated.
>
>Thanks in advance,
>--aryeh
>Aryeh Weiss | email: [log in to unmask]
>Department of Electronics | URL: http://optics.jct.ac.il/~aryeh
>Jerusalem College of Technology | phone: 972-2-6751146
>POB 16031 | FAX: 972-2-6751275
>Jerusalem, Israel | ham radio: 4X1PB/KA1PB
>
>
|
