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