Hello Tobias,
 
In a message dated 95-06-16 18:33:22 EDT, you write:
 
>        In the response from Prof. Pawley to the question about thick
>tissues, he wrote:
> ...
>>
>>Solution: use water-immersion objective with strict adherence to the
>>coverslip thickness requirements.
>...
>        This confused me because I thought that one used a water immersion
>lens IN PLACE OF a coverslip to image a cell or some thing in culture
>without mounting it or squishing it; and hence that water immersion lenses
>were designed to be used strictly without a coverslip. Can someone help me
>out here??
 
 
Microscope objective manufactures now have TWO different series of water
immersion objective lenses to solve TWO different kinds of microscopical
applications.
 
1)  PHYSIOLOGICAL  SERIES  water immersion
These are typically long working distance objectives (4X, 10X, 20X, 40X, 60X)
 with W.D.'s in the range of 1.9mm to 3.0mm that have Numerical Apertures
that are similar to air objectives.  (i.e.  40X NA 0.75  WD 1.9mm)     They
can be DIPPED into physiogical solutions directly  (NO COVERSLIP NEEDED)
 They are best suited for in-vivo work like micro-circulation microscopy,
 electrophysiological potential measurements ( patch clamp with Long Working
Distance between specimen and micro-manipulator access on up-right
microscopes) and Opthalmic corneal microscopy.
 
2) CONFOCAL and DIC SERIES  water immersion
These are specific objectives designed to have MAXIMUM numerical apertures
for work at diffraction limits that attempt to minimize the effects of
spherical aberation on thick specimens.  THEY MUST BE USED WITH COVERSLIPS.
Typical specifications are:
40X  NA 1.15   WD 0.225mm   340nm UV excitation capable ;   60X  Plan Apo NA
 1.2  WD 0.225mm  near UV (365nm)  and visible light excitation;  20X  NA
0.80  WD 0.8mm 340nm UV excitation capable  ( not yet commercialized);  These
objectives are characterized by their current high sales costs at upwards of
$8,000 each.
 
The theory is: by keeping the immersion and aquaeous specimen refractive
indicies closely homogenous ( 1.31) and nulling out the effect of the
coverglass refractive index (1.51) by use of the water immersion objective
correction collar ( it is much easier and better technique to just measure
your coverglass thickness and use only the ones that are 0.170 microns in
thickness) one can eliminate a great proportion of the spherical aberation
induced by the mismatch of refractive index caused by using an oil immersion
objective on relatively thick specimens.
 
 ALL High NA objectives are designed for use with a coverslip of  0.17mm
thickness  irregardless if they are OIL or WATER immersion.  Oil immersion
objectives will start to exhibit spherical aberation at depths just under the
coverslip (5 - 10 microns)  and moderate spherical aberation is obtained at
depths past 15- 20 microns.
 
If you would like a reprint of our journal article describing high numerical
aperture water immersion objectives please __ PERSONALLY__ send me your
mailing address.  Also read the related articles in the 2nd edition Confocal
Handbook by Pawley.
 
Regards,
 
 
Stan Schwartz
Manager, Biomedical Instruments
Nikon Inc. Instrument Group
1300 Walt Whitman Rd.
Melville, NY  11747
516-547-8529     Fax  516-547-0306
E-mail  [log in to unmask]