To the group--
This message is posted on behalf of Johannes Helm, who forwarded it to
me after having trouble posting it from his mailserver. Johannes' note
uses my response as the starting point.
For whatever it's worth, the comparision that we did was performed using
tissue mounted in DPX, which has an index of refraction approximately
equal to that of glass.
Martin Wessendorf
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Dear Patrick--
My own very limited experience deals only with oil-immersion 60x 1.4
NA
objectives. A few years ago, when manufacturers switched from 160
mm
tube-length optics to infinity optics, our department bought a new
microscope to mount under our new confocal. I found that with the
new
60x objective I was unable to image through the full thickness of
tissue
sections that I had formerly been able to image with the comparable
60x
1.4 NA objective mounted on our older 160 mm tube-length scope.
Although the working distance for the new objective was listed as
being
0.11 mm and that for the old objective was 0.12 mm, there was a 30+
micron difference in the depth to which we could focus.
Bottom line: the manufacturers should have the numbers. However,
don't
necessarily believe them.
Good luck!
Martin Wessendorf
Good afternoon,
it might be worth to mention the fact, that "working distance" canNOT
automatically be translated into
what one might like to call "penetration depth in the specimen".
If I recall correctly what I once learnt:
"Working Distance" in case of objectives that are designed to be used
WITH coverslips is the distance
between the front lens of the objective and the upper surface of the
cover slip (lower surface in case of
inverted micrscopes) when focussing on a specimen point which is located
at an infinitely short distance
from the lower surface of the cover slip (upper surface in case of an
inverted microscope).
In case of homogenous immersion - i.e. strictly speaking the refractive
indices of immersion medium,
mounting medium and in some cases cover slip material are identical -
working distance is equal to
penetration depth.
However, if you for example have an oil immersion lens 63X/1.4 with a
working distance of 90 microns,
then these ninety microns do not translate into 90 mm in penetration
into a specimen mounted in a
medium with a refractive index of 1.3 or something like that!
Also, a water immersion lens specified for working distance d used to
view specimens embedded in
fluoromount will not necessarily allow to view d into the specimen.
Also, the specified working distance is valid only, if cover slips made
out of the correct material and with
the correct thickness are used. You cannot simply replace a 200 mm
coverslip from fused silica with a 170
mm coverslip from BK7 glass, when using e.g. a glycerine immersion
UItrafluar.
In objectives built to be used without any coverslip - such objectives
can for example be popular in
geology or mineralogy, e.g. the 100X/0.9 Ultrafluar by Zeiss, or water
immersion lenses for
electrophysiology - working distance indeed is the distance between
front lens and focal point under the
precondition that the refractive index of the specimen is identical to
that of the mounting medium (=
immersion medium in this case). On the old (160mm) Planapochromats
63X/1.4Oil by Zeiss, for example,
there is a dash "-" where you perhaps would expect a "170" or "0.17"
indicating the correct thickness of
coverslips in microns or mm. This is because the refractive index of
immersion oil at wavelengths of visible
light is approximately equal to that of cover slip glass.
That magnitude what many would like to know, the "penetration depth",
cannot be easily defined, since it
is dependent on the specimen which, unfortunately, uses to be a kind of
"biological cellsoup" with a truly
anarchic behaviour of the refractive index (I've absolutely nothing
against biological specimens, my
slightly drastic vocabulary, which I kindly ask you to excuse, is to
underline the difficulties that would
arise upon any effort to define "penetration depth").
Another point:
There do exist so called "multi immersion lenses" which are designed to
be used
with different kinds of immersion media, with and without coverslip and
so on. I suppose, but I do not
know for sure, that the mathematicians who designed these lenses,
allowed for more "compromises" and
also made use of movable lenses (you can adjust them by means of a ring
on the objective).
Also, there exist long-distance objectives, sometimes called
"LD-lenses", which allow to adjust for
different thicknesses of cover slips. Those were originally popular
among users of inverted microscopes
who wanted to focus through bottom glasses or even petri-dish bottoms
(hopefully from glass and not
from plastic, when DICcing). Some of these LD lenses need special caps
when used in order to focus
through thin cover slips. They usually have comparatively small N.A.s
and are hence not extremely
suitable for confocal microscopy.
Generally one can say, that "in a zero order approximation" lenses with
small numerical apertures will
have a "penetration depth" closer to the nominal working distance value
upon comparable index
mismatches than lenses with larger values for the N.A.
This does not answer Patrick's question, but I thought nevertheless it
might be worth to mention this.
Best regards
Johannes
--
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Paul Johannes Helm
Mail Address:
Institute of Basic Medical Sciences
Department of Anatomy
P.O. Box 1105 - Blindern
NO-0317 Oslo
Norvège
Visiting Address:
Institute of Basic Medical Sciences
Department of Anatomy
Sognsvannsveien 9
Rom 0346/0347
NO-0372 Oslo
Norvège
Voice: +47 22851159
Fax: +47 22851278
Email: [log in to unmask]
WWW: http://www.uio.no/~jhelm
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