So what is the refractive index of the bead?  Wouldn't a mismatch there
also cause distortion in the z axis?

--Jeff
[log in to unmask]

>----------
>From:  Benedikt Kost[SMTP:[log in to unmask]]
>Sent:  Monday, January 20, 1997 11:46 AM
>To:    Reece.Jeffrey (EX); [log in to unmask]
>Subject:       football
>
>We have a Zeiss LSM410 equipped with a 100x/1.3 oil immersion lens. When we
>take x/y sections (20 sections, 0.5 micrometer apart) through a spherical
>fluorescent bead (3.9 micrometer diameter, Multi Speck, Molecular Porbes,
>M-7900) and apply built-in Zeiss 3D reconstruction functions, we obtain
>something that looks like a football! The long axis of the football is
>oriented along the z-axis and is about 1.5 x longer than the actual
>diameter of the bead (z and x extension were both measured on an orthogonal
>section). According to Molecular Porbes, the beads are embedded in a medium
>with a refractive index matching the one of oil and sealed under a 1.5
>coverslip. We therefore took the x/y sections using "1" as a refractive
>index correction factor.
>
>Our Zeiss representative tells us that what we see is "normal" and can't be
>improved by adjusting the microscope. She suggest us to enter the z/x ratio
>we determined (1.5 x, see above) as  refractive index correction factor
>next time we take a stack of sections through a sample.
>
>To us, the fluorescent beads appear to be a good model for a cell with a
>fluorescent cytoplasm. Do we have to live with the fact, that images taken
>under seemlingly optimal conditions are considerably distorted?
>
>Thanks for comments and suggestions!
>
>Benedikt Kost
>
>Box 162
>Lab of Plant Molecular Biology
>The Rockefeller University
>1230 York Avenue
>New York, NY 10021-6399
>USA
>