Zeiss 880 Installation guide

Zeiss 880
Training Notes
Zen 2.3

Turn on
Main Power
Switch
Do you need the Argon
Laser
458, 488, or 514 nm lines?
Yes
Turn on the Systems PC
Switch
Turn on the HXP 120V Lamp
Turn on the Components
Switch.
Turn on the PC and log into
your account.
Start Zeiss Zen via Elan
Tracking Software.
No
Create
scanning
configuration
880 Start-Up Sequence
Set up sample on microscope.
Go to the Acquisition Tab and
initiate the warm up
procedure for the laser.
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Logging into the 780
•Log into the computer with your UCHC or
CAM credentials; be sure you are logging
into the proper domain.
•Double click on the Zen icon, the one
labeled with “AUT-Zeiss LSM 880” or
similar text.
The idea elan tracking and billing software will
initialize after clicking on the Zen icon.
•Enter your UCHC email. Once it has been
recognized, it will bypass the password
requirement and proceed to the session
confirmation window.

Starting Zen with idea elan
•Select your scheduled appointment
and/or click the login button as directed
by the software.
•Confirm your appointment and the Zen
software will be initialized.

Starting Zen 2.3
Select Start System from the Zen 2012 login
window. This will start up the hardware and
software.
Image Processing is for using the system in an
offline mode and this will ignore all the
hardware components.

Introducing the Zen Workspace
The Zen workspace is designed in a
tabular fashion allowing you to
step you through initially looking
at your specimen via the Oculars
tab, in the control panel, to
acquiring images via the Acquire
tab. The remaining tabs add off-
line Processing and
Maintenance options.
Panels can be moved around
to customize the workspace.

Introducing the Zen workspace
Your imaging configuration and your
work space configuration can be
stored separately.
In addition, your imaging configuration
and acquisition parameters can easily be
retrieved from the Reuse function when
you have a previously saved image loaded
in the workspace.

Getting Light to the Oculars
Locate Tab
1. Click on the objective icon to access the list
of possible objectives. Be sure to note the
immersion type; air, oil or water. Please do
not change from oil to air/dry or from
water to oil and oil to water without
cleaning your specimen first.
2. Select your light source from:
Transmitted ON
Transmitted OFF
DAPI
FITC
Rhodamine
3. Be sure to turn the Reflected Light “Off”
when you are finished looking at your
specimen.
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Getting all the tools
Acquisition Tab
In order to view all the options available via the software,
be sure to go to:
1. View>Show All (global)
2. Select >Show all Tools.
Make sure to do this each time you use the software
otherwise the required features, like the lasers, will not
be accessible to you.
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Open/close the
submenu with the arrow.
Click the arrow to detach/attach panel.

Acquisition Tab
1. Experiment Manager
The manager allows you to save your scan configuration
settings for your experiment and reload the settings for
a quick start. This method does not record the
acquisition parameters.
2. Scan Control Tool Bar
•AF (Find Focus) – computer determines focus based
on contrast settings.
•Set Exposure – the computer automatically
calculates the gain and black level settings.
•Live – initiates a fast scan regardless of the scan
speed and continuously scans until Stop is selected.
•Continuous – continuously scans according to the
selected scan speed until Stop is selected.
•Snap – performs a single scan according to the
selected scan speed.
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Using Smart Setup
Smart Setup
The best and easiest way to get started is to use
the Smart Setup tool.
1. Click on the down arrow to open the dye
selection menu.
2. Select your dye from the drop down menu.
Repeat this procedure to select additional
dyes.
Depending on your imaging needs, the Smart
Setup will offer you up to four different
configurations for imaging your sample:
•Fastest
•Best Signal
•Best Compromise
•Linear Unmixing.
Whatever configurations you are presented with,
you can always manually modify the configuration
in the Light Path panel.
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Smart Setup - Fastest
Acquires the image with the simultaneous
excitation and detection of one or
multiple probes. This option produces a
single image, with a single scan however
it may result in bleed through between
the different channels.

Smart Setup – Best Signal
Acquires the image with the sequential
excitation and detection of the different
probes. Each probe is scanned separately
and the final image is the result of an
overlay of the individual channels. This
option avoids bleed through between the
different channels however multiple
scans are require to produce the image
which can lead to bleaching or other laser
induced damage.

Smart Setup – Smartest (Line)
Smartest acquires multiple channels at the
same time while quickly toggling laser
lines to acquire a second track.
There are limitations to this option, especially
if you are acquiring 4 different probes.
a) You must you use the same
dichroics between the two tracks.
b) You can’t send two probes, of
different intensities to the ChS
detector.

Smart Setup – Linear Unmixing
This option creates a confirmation for
computationally subtracting bleed through
between channels. This can be performed
after you acquire individual, pure spectral
profiles of the probes you are using.

Using the Advanced Scanning Modules
Advanced scanning modules are available as a
single option or they can be used in combination
with multiple modules. The modules are initiated
via the Start Experiment button.
1. Time Series - collect a series of images based
on a specific time interval and duration.
2. Z-Stack – collect a series of optical sections
that can be reconstructed as a 3D image.
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Using the Advanced Scanning Modules
(cont.)
Bleaching – module for
FRAP (fluorescent
recovery after
photobleaching.)
Regions – drawing tools used
to customize and restrict the
scan area.
Tile Scan – create larger
images by tiling/stitching
smaller images together.
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Using the lasers
The lasers that are available for use are:
Diode 405 nm
Argon 458,488,514 nm
DPSS (Diode-pumped solid state) 561 nm
HeNe (Helium-Neon) 633 nm
The Argon, has a start-up and shut-down procedure. This can
be monitored by opening the Laser Properties panel.
The red highlighted color indicates the laser is not available
for use. ( Please note this color will go away, it will not turn
green, when the laser is warmed up.)

Looking at the Light Path
1. Tracks
•Create and/or delete tracks which contain the
scanning configuration for each probe or
combination of probes that are scanned. The
configuration consists of the laser line, excitation
dichroic, detector and emission range that is
captured. If you use the Smart Setup application,
your tracks will automatically be configured for
you.
2. Switch track every
•Line – tracks are switched line by line. Laser line,
laser intensity and channels can be changed
between tracks.
•Frame – tracks are switched frame by frame.
Laser line and intensity, filters, beam splitters,
gain and offset, and pinhole setting can all be
changed between tracks.
•Frame Fast – scanning procedure can be made
faster. Only the laser line intensity and Amplifier
offset can be changed between tracks.
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Imaging Setup and Light Path
1. Track – defines the fluorophore(s) that is(are)
being detected. A maximum of four tracks can
be created which specify the dye, laser
excitation line and the emission detection
range.
2. Visual representation of excitation line(s) and
the emission profile(s) which you can set by
adjusting the slider bars or alternatively you can
enter the range at the drop down arrow.
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