On Tue, 20 Feb 1996 06:44:26 CST, Ian Gibbins wrote:
 
>Hello Confocalists!
>
>Over the last few months there has been illuminating discussion of the
>benefits of coverslip-free water / saline immersion lenses... Several
>companies now produce fabulously expensive immersion lenses that are
>supposed to have highly corrected symmetrical point spread functions. I
>understand the theoretical value of such a lens, especially for
>deconvolving images, but I am wondering how much improvement they provide
>in the real world of living tissues and optically mismatched balanced salt
>solutions... Has anyone tried and compared such lenses with more
>conventional long working distance immersion lenses? Are they worth paying
>3x - 5x more for?
>
>Thanks!
>IAN
>Professor Ian Gibbins
>Department of Anatomy and Histology
>Flinders University of South Australia
>Phone:  +61-8-2045271
>FAX:    +61-8-2770085
>e-mail:  [log in to unmask]
 
Yes, I've done comparisons of distilled water immersion, saline-immersible,
oil immersion, glycerine immersion, and dry lenses. My test preparation was
whole, intact, living, rat EDL muscle (a leg muscle approx 5mm thick). My
experimental aim was to measure intracellular pH as deep into the muscle as
possible using confocal and SNARF-1. And the answer to your question really
depends on what data you wish to collect and from what type of specimen.
The "fabulously expensive" distilled water immersion lenses you speak of
are also fabulous lenses if you have a living or aqueous mounted specimen.
They have correction collars for the thickness of the coverslip and this
must be set accurately to get the performance out of the lens. Each
individual coverslip thickness must be measured before use. I don't belive
you'll get better quality images with other types of lenses. However, if
your aim is not to get the best resolution images from, for example, single
living isolated cells on a coverslip.
 
But if you wish to get a physiolgical measurement of say Ca+ or H+, and
your priority is to get these measurements from deep within a thick
specimen. And if you are prepared to sacrifice the quality of images
inorder to optically section deep into the tissue. Then I would recommend a
saline-immersible lens with a lower NA, eg. 40X NA 0.7 WD 3mm. I found
these to perform best at deep optical sectioning of living tissue. But I
wouldn't want to do precise 3D reconstruction with the images. The lower NA
saline immersible lenses are a lot cheaper too. I think the reason why the
lower NA lens, while producing poorer quality images can optically section
deeper into the tissue, is that the lower NA lens may not be so sensitive
to slight refractive index mis-match from going from saline to living
tissue. My hypothesis is that the lower NA lens would have thicker optical
sections than the higher NA lenses and perhaps this is the reason that the
lower NA lenses appear more forgiving in thick specimens. If my guess is
correct then using a high NA distilled water immersion lens, and using the
confocal with the pinhole wide open (ie. sacrifice thin optical sections),
then perhaps we would expect deeper optical sectioning. I have not tried
this yet. I would welcome comments from the group re. the above statements.
 
I've also found that glycerine immersion lenses may be preferable if you
have a thick fixed specimen, mounted in a glycerine-gelatin mountant. Oil
lenses best if your specimen is mounted in say DPX (DEPEX). Dry lenses are
not crash hot in deep optical sectioning, but if you really only want
images from the surface of a specimen, that is right next to the
coverglass, the results can be quite acceptable.
 
I hope that there is a move soon for lens manufacturers to bring out higher
NA saline-immersible lenses. These should still be cheaper than the
distilled water immersion lenses as there is no need for coverslip
thickness correction optics.
Stephen H. Cody,                                        __    /
Biomedical Confocal Microscopy Research Centre        _/  \__/ \
Department of Pharmacology,                          /          \
University of Western Australia,                    /            \
Nedlands WA 6009,                                   \*  ____     /
Australia.                                           \_/    \_ _/
         email:  [log in to unmask]              __
         Phone:  61 09 346 4569   Fax: 61 09 346 3469         \/