Judy

     I would recommend reading the following publication:

     Aberration control in quantitative imaging of botanical specimens by
     multidimensional fluorescence microscopy

     N. S. White et al  Journal of Microscopy, Vol. 181, Pt 2, February
     1996, pp. 99-116

     This paper discusses empirical determination of axial distortion and
     attenuation, their practical calibration and subsequent computational
     correction.

     Bio-Rad MRC-1024 users should note that the axial geometric distortion
     (squashing or stretching in Z) caused by mismatched refractive indices
     (RI) is automatically corrected as long as the appropriate RI values
     for immersion medium, cover slip and mounting medium are entered into
     the 'User Setup' dialog box.


     Duncan McMillan
     Senior Product Manager
     Bio-Rad Microscopy Division
     Hemel Hempstead
     UK

     Tel (44) 1442 232552 xt 300
     email [log in to unmask]




______________________________ Reply Separator _________________________________
Subject: Refractive Index Mismatch
Author:  Confocal Microscopy List <[log in to unmask]> at
Internet
Date:    16/01/97 09:15


        Dear fellow microscopists,

        Can someone enlighten me on the potential pitfalls of RI mismatch
between coverslip and immersion oil.  I am working with someone who must
grow his cells on Aclar coverslips (RI = 1.435).  We are mounting the
specimens in Vectashield (RI = 1.4577) and are using Zeiss oil (RI =
1.515). We are trying to do confocal reconstructions of fluorescently
tagged (FL and RH) mineral aggregates in these cultures (approx. 10-20 um
thick) to visualize their substructure.  To do this we are using 100X oil
immersion lens at Zoom 4 on a Leica TCS-NT confocal.    What sorts of
aberrations could I expect in the reconstructed images??  Should I use an
oil with a lower RI?
        A related question:  Most of the fluorescent specimens I work with
have glass coverslips and are imaged with oil immersion objective lenses
(consistent RI), but are mounted in Vectashield (Lower RI) or similar
anti-photobleach medium.  What problems does this pose for confocal
imaging??

Judy Drazba, Ph.D.  ([log in to unmask])
Confocal Microscopy Facility, NC-3
The Cleveland Clinic Foundation
9500 Euclid Avenue
Cleveland, OH 44195-5001
Office (216)445-3760
FAX    (216)444-7927