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]