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)