Zeiss 710MP confocal
Tests of stage repeatability. Imaging using 25X objective.
Cyan lines mark the edges of each 256 X 256 pixels tile collected. Each tile was scanned in 0.5 sec from top to bottom.
Blue arrow shows that at the top of the first tile, where stage has been sitting, there is no jitter.
White arrows show jitter where stage has quickly moved and imaging commenced before stage settled in place.
Green arrow shows bottom of scanend field where jittering has settled by 0.5 sec.
This is not as much of a problem when tiles are scanned slower.
Jitter is not noticeable because is is averaged out at the top of each tile due to the slow scan speed (2 sec per tile) and stops 1/8 of the way down each tile based on the calculation from the faster scan that it takes 1/4 sec to settle after imaging commences.
Zooming in shows the jitter at the very top.
But we don't know yet what the skip below is.
How repeatable is the stage over time?
The system was set to take 2 X 2 tiles every 6 seconds. The following picture is a subtraction of adjacent frames through a time stack. Where there are white lines there is a difference from one timepoint to the next. Where ther is merely speckle, the stage returned to the correct position.
The repeatability in the X axis looks dead-on but off by as much as 3 um in the Y axis.
Also shown as a single timepoint in XY at left and kymogram at right (enlarged 200%):
Conclusion:
last updated 20110204
comments, questions, suggestions: Michael.Cammer@med.nyu.edu