I didn't think a confocal could resolve z-steps of less than about 5-600
nm.  Wouldn't the 100 nm steps suggested by Leica in this post lead to
overlapping of optical sections?
 
>Hallo everybody...!
>
>This message unfortunately went out encoded, so
>I send it again in ASCII...Sorry!
>
>Some further remarks to the hot discussion about pixel shifts....
>
>The described pixel shift in Z in simultaneous scanning is clearly
>caused by the chromatic aberration of the objective lens.
>
>To match the two data sets perfectly one should use a step size in
>the order of the chromatic aberration. Take a series for each
>staining, then superimpose the two series with one series
>shifted axially by this step size. For example, the
>Leica PL APO 100/1.4 OIL has an axial chromatic aberration
>of about 100 nm between 488 and 647 nm. So, one would step
>through each series with a step size of 100 nm and then
>shift one stack of images up by 100 nm. This can be done
>easily and reproducibly using the high-resolution focussing
>stage of the Leica TCS 4D.
>
>For sequential scanning, this means acquiring FITC first then
>changing the detection filters and collecting the next series.
>Using this protocol, the observed pixel shift is only caused
>by the preparation of the specimen (immersion oil , compressing
>the cover glass, etc.)
>
>Additionally, to avoid specimen induced shift, one can change
>filters after acquiring one frame for one stain, change filters,
>acquire second frame for second stain, and then step to the next
>level.
>
>This feature can be extended also for a triple labelled specimens.
>
>For further information, please contact us at
>Leica Lasertechnik GmbH
>Martin Hoppe, Ph.D. or Werner Knebel, Ph.D.
>Leica Lasertechnik GmbH,
>Im Neuenheimer Feld 518, D 69120 Heidelberg, Germany
>Phone+49-6221-41480
>Fax +49-6221-414833
>Email: [log in to unmask]
>or the general Leica support adress: [log in to unmask]
 
Thomas E. Phillips, Ph.D.
Associate Professor of Biological Sciences
Director, Molecular Cytology Core Facility
3 Tucker Hall
University of Missouri
Columbia, MO 65211
(314)-882-4712 (voice)
(314)-882-0123 (fax)