In message     Confocal Microscopy List writes:
>> From
>> the data in the pixel file I somehow always end up with much too flat
>> reconstructions. We ran a test with small latex droplets embedded in
>> plastic (these should be very round), but we got pancakes instead. It
>> appears that the scaling of the microscope is not properly calibrated.
>>
>> Does anyone know of the scaling problem and its solution?
>>
>> moi,
>> Koos Schut
>> Institute of Theoretical Physics,
>> University of Groningen, The Netherlands
>> e-mail: [log in to unmask]
>
> --The index of refraction of the mounting medium of the specimen will have an
> effect on the specimen's apparent thickness in the z-axis.  Usually this will
> result in foreshortening, as you've described.  Could this be what you're
> observing?
 
        The Depth Correction discussed above is a result of the phenomena that the
movement of the focal plane (d) does not always depend on the movement of the
 object
stage (D) alone.  A reason that d does not equal D is that the mounting medium
 often
has a different index of refraction than the immersion medium.  To a reasonable
approximation with n1 and n2 being the refractive index of the mounting media
 and
immersion media respectively: d/D = n2/n1.
        This correction can either shortening or lengthen the specimen's apparent
thickness. several examples follow:
 
Immersion Media         Mounting Media          Correction
oil                     air                     0.66
oil                     water                   0.88
oil                     fluoromount             0.98
air                     water                   1.33
air                     fluoromount             1.47
 
        A more detailed examination of this correction is available in the
 documentation
of at least one CLSM manufacturer.
 
david hanzel