Zeiss LSM 880 Operating Manual page 134

ZEISS
Off mode
If Off is selected, autofocus is inactive and will not be used.
If a multi-track acquisition includes a track that employs a camera for data capture, the autofocus mode
is automatically set to Off in Focus Devices and Strategy.
Fluorescence mode
Fluorescence uses the current track configuration for detecting fluorescent signals (as specified in the
Channels tool) for identifying the focus reference plane.
In LSM-based multi-channel acquisitions (channels in a single or multiple tracks), the identification of the
focus reference plane is based upon the intensity maximum of all channels.
Choosing the Fluorescence modeis useful e.g. when imaging monolayers of cells with stained nuclei.
Here, the fluorescent signals of their nuclei provides an ideal reference plane for imaging the cells.
The Fluorescence mode is not an ideal choice for thick samples that do not feature a clearly detectable
intensity maximum.
Reflection mode
In contrast to the Fluorescence mode, the LSM-based Reflection autofocus mode uses incident laser
light reflected at surfaces (e.g. glass surfaces of cover slips) for identifying reference planes.
Measuring the Z-position of glass surfaces through laser reflection is only possible in the presence of a
refractive index mismatch. Readily detectable laser reflection occurs at the interfaces of either glass and
air or glass and water-like media (e.g. cell culture media in glass-bottom dishes). The Reflection mode is
ideal for maintaining focus stability in long-term observations of adherent live cells kept in glass-bottom
dishes. Reflection cannot be used if no laser reflection is expected to occur at any of the glass surfaces
(e.g. when using immersion oil and mounting media with a highly similar refractive index as glass).
Among all laser lines activated in the imaging tracks (see Channels tool, section Acquisition Parameter
– Channel), the Reflection mode uses the one with the longest wavelength for focusing. ZEN
automatically sets the output power of this laser line and also configures the Imaging Setup for
detecting laser reflection.
Fig. 170 The offset value is either specified
manually or determined
automatically.
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CHAPTER 1 - SYSTEM OPERATION
Left Tool Area and Hardware Control Tools
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The distance between the Z-level of image
recording and the Z-level of the reference plane
(e.g. surface of a cover slip) is specified by the
parameter Offset.
Offset is available
Autofocus Map autofocus modes (see below).
The Offset value can either be entered manually
or measured automatically using Find offset
(Fig. 170).
The system requires automatic measurements of
Offset to be focused on the desired imaging plane
(e.g. by visual inspection)
.
LSM 880
in the Reflection
before pressing
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and