Zeiss LSM 880 Operating Manual page 135

LSM 880
Find offset searches for a reflective surface (the focus reference plane) starting at the Z-level of the
imaging plane. Next, it copies the increment between the Z-levels of the imaging plane and the focus
reference into the Offset spin box. See description of Positions tool (section Multidimensional
Acquisition – Positions) for details on how to specify Offset values in multi-position experiments.
Autofocus Map mode
Autofocus Map aims to minimize the time spent on maintaining focus stability in experiments that
involve multiple cycles of data recording from arrayed samples. This autofocus mode is therefore helpful
when working with e.g. microtiter plates that are slightly tilted or feature wells with uneven bottoms.
Autofocus Map is only available in the Sample Carrier method of the Positions tool and cannot be
combined with Tile Scan.
Before starting an imaging experiment using Autofocus Map, a Focus Map has to be recorded for all
positions selected in the method Sample Carrier. If no Focus Map is present, ZEN displays the
notification
After pressing
map the reference planes in all these positions. This information is stored (but not displayed) as a Focus
Map. After the successful generation of a Focus Map, ZEN displays the notification
The fundamental requirements as well as the strategy for using Offset are identical in Autofocus Map
and Reflection (see above).
The same Offset value is applied to all positions of the sample carrier. As the Focus Map is only created
once and its information subsequently used in all acquisition cycles, Autofocus Map provides a speed
improvement when working with arrayed samples. Autofocus Map does not correct for Z-drift over time.
If such compensation is needed, the autofocus mode Reflection should be selected for arrayed samples.
Definite Focus mode
Selecting this mode activates the Definite Focus unit. Activity intervals (see below) are set like with all
other autofocus modes. See operating manual of Definite Focus for reference.
Specifying Activity intervals for focus actions
Activity intervals for focusing can be specified for
the Fluorescence, Reflection, and Definite
Focus modes.
It is advisable to minimize the number of focusing
activities in order to improve the speed of the
overall acquisition and to protect delicate samples
from laser damage.
However, optimal
determined experimentally.
As soon as one of the acquisition tools Time Series, Tile Scan, or Positions is activated, focus intervals
can be linked to these tools in Focus Devices and Strategy (Fig. 171).
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CHAPTER 1 - SYSTEM OPERATION
Left Tool Area and Hardware Control Tools
.
, ZEN uses laser reflection (see autofocus mode Refelction for details) to
conditions need to
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Fig. 171 Specifying activity intervals for
be
focus activities
ZEISS
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