Zeiss LSM 710 User manual

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04/2009 1
Contents
Page
Contents ................................................................................................................................. 1
Introduction............................................................................................................................ 1
Starting the System ............................................................................................................... 2
Introduction to ZEN – Efficient Navigation .......................................................................... 5
Setting up the microscope................................................................................................... 10
Configuring the beam path and lasers ............................................................................... 12
Scanning an image............................................................................................................... 17
Storing and exporting image data ..................................................................................... 22
Using the ConfoCor 3 module............................................................................................. 23
Switching off the system..................................................................................................... 29
Introduction
This LSM 710 / LSM 710 NLO and ConfoCor 3 Quick Guide describes the basic operation of the
LSM 710 / LSM 710 NLO and ConfoCor 3 Laser Scanning microscopes with the ZEN 2009 software.
The purpose of this document is to guide the user to get started with the system as quick as possible in
order to obtain some first images from his samples.
This Quick Guide does NOT replace the detailed information available in the full user manual or in the
manual of the respective microscopes (Axio Imager, Axio Observer, Axio Examiner).
Also, this Quick Guide is written for a user who is familiar with the basics of Laser Scanning Microscopy.
For your safety!
Observe the following instructions:
The LSM 710 / LSM 710 NLO and ConfoCor 3 Laser Scanning Microscope, including
its original accessories and compatible accessories from other manufacturers, may
only be used for the purposes and microscopy techniques described in this manual
(intended use).
In the Operating Manual, read the chapter Safety Instructions carefully before
starting operation.
Follow the safety instructions described in the operating manual of the microscope
and X-Cite 120 lamp / HBO 100 mercury lamp.

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Starting the System
Switching on the LSM system
!Switch on the main switch (Fig. 1/1) and the
safety lock (Fig. 1/2).
!When set to ON the power remote switch
labeled System/PC provides power to the
computer. This allows use of the computer and
ZEN software offline
!To completely switch on the system, now press
the Components switch to ON. This starts the
other components and the complete system is
ready to be initialized by the ZEN software.
Switching on the X-Cite 120 or the HBO
100 mercury lamp
!Switch on the main switch of the X-Cite 120 /
HBO 100 lamp for reflected light illumination
via the power supply as described in the
respective operating manual.
Switching on the Ar-ML Laser
!If the Ar-ML laser is required, switch it on via
the toggle switch (Fig. 2/2) on the power supply
and turn the key (Fig. 2/1).
The laser is automatically kept in standby mode
for 5 minutes to warm up.
!Set the idle-run-switch (Fig. 2/3) to run. It takes
about 50s until the laser has reached the set
output power (green LED) provided the warm
up time of 5 minutes is already completed.
!Adjust the required power level with the control
knob (Fig. 2/4) (default position should be
11 o’clock).
Fig. 1 Power remote switch
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Fig. 2 Power supply of Ar-ML laser
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04/2009 3
Starting the ZEN software
!Double click the ZEN 2009 icon on the WINDOWS desktop to start the Carl Zeiss LSM
software.
The ZEN Main Application window and the LSM 710 Startup window appear on the
screen (Fig. 3)
In the small startup window, choose either to start the system (Start System hardware for acquiring new
images) or in Image Processing mode to edit already existing images. Toggle the little
symbol to
view the Boot Status display and get the additional Offline / Demo button option:
Choosing Start System initializes the whole microscope system and activates the entire software
package for new image acquisition and analysis.
The Image Processing mode ignores all hardware and activates only data handling and image
processing functionality for already acquired images.
The Offline / Demo mode reads the current hardware database but does not activate the system
hardware for use. Instead, it simulates the system hardware for training purposes.
Upon clicking the Start System button, the Image Processing button changes to a Cancel
button. Click Cancel to interrupt/stop the Startup of the system.
After Startup, the ZEN Main Application Window (Fig. 4 and Fig. 5) opens. To benefit from all of ZEN's
features, run the window in its full screen mode.
Fig. 3 ZEN Main Application window at Startup (a) and the LSM 710 Startup window (b
and c)

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Fig. 4 ZEN Main Application Window after Startup with empty image container
Fig. 5 ZEN Main Application Window after Startup with several images loaded

04/2009 5
Introduction to ZEN – Efficient Navigation
The ZEN 2009 interface is clearly structured and follows the typical workflow of the experiments
performed with confocal microscopy systems:
On the Left Tool Area (Fig. 4/D) the user finds the tools for sample observation, image acquisition,
image processing and system maintenance, easily accessible via four Main Tabs (Fig. 5/1). All functions
needed to control the microscope can be found on the Ocular Tab, to acquire images use the
Acquisition Tools (Fig. 5/3and 4). Arranged from top to bottom they follow the logic of the
experimental workflow. The area for viewing and interacting with images is centered in the middle of the
Main Application Window: the Center Screen Area. Each displayed image can be displayed and/or
analyzed with many view options available through view tabs which can be found on the left side of the
image. According to the chosen view tab, the required view controls appear in View Control Tabs below
each image. File management and data handling tools are found in the Right Tool Area (see Fig. 4 and
Fig. 5).
Color and brightness of the interface have been carefully adjusted to the typical light conditions of the
imaging laboratory, guaranteeing optimal display contrast and minimal stray light for high-sensitivity
detection experiments. The ZEN software is optimized for a 30" TFT monitor but can also be used with
dual-20" TFT setups.
A focus in the development of ZEN 2009 was to fulfill the needs of both basic users and microscopy
specialists. Both types of users will appreciate the set of intuitive tools designed to make the use of a
confocal microscope from Carl Zeiss easy and fast:
The Show all concept ensures that tool panels are never more complex than needed. With Show all de-
activated, the most commonly used tools are displayed. For each tool, the user can activate Show all
mode to display and use additional functionality (Fig. 6).
Fig. 6 Show all mode

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More features of ZEN 2009 include:
The user can add more columns for tools to the Left Tool Area or detach individual tools to
position them anywhere on the monitor. To add a column, drag a tool group by the title bar (e.g.,
Online Acquisition) to the right and a new tool column automatically opens. Alternatively use the
context menu "move toolgroup to next column". To detach a tool, click on the little icon on the
very right end of the blue tool header bar (Fig. 7).
Another unique feature in Imaging Software is the scalable ZEN interface. This Workspace Zoom
allows adjustment of the ZEN 2009 window size and fonts to the situational needs or your
personal preferences (Fig. 7).
Setting up conventional confocal software for a specific experiment can take a long time and is
often tedious to repeat. With ZEN these adjustments have to be done only once – and may be
restored with just two clicks of the mouse. For each type of experiment one can now set-up and
save the suitable Workspace Layout. These configurations can also be shared between users.
For most controls, buttons and sliders, a tool tip is available. When the mouse pointer is kept over
the button, a small pop-up window will display which function is covered by this tool/button.
These are just some of the most important features of the ZEN interface. For a more detailed description
of the functionality for the ZEN 2009 software, please refer to the User Manual that is provided with
your system.
Fig. 7 ZEN Window Layout configuration

04/2009 7
Setting up a new image document and saving your data
To create a new image document in an empty image container, click the Snap or the
Auto Exposure button. For an empty image document press the New button.
The new document is immediately presented in the Open Images Area. Remember, an unsaved 2D
image in the active image tab will be over-written by a new scan. Multi-dimensional scans or saved
images will never be over-written and a new scan will then automatically create a new image document.
Acquired data is not automatically saved to disc. Make sure you save your data appropriately and back it
up regularly. The ZEN software will ask you if you want to save your unsaved images when you try to
close the application with unsaved images still open.
There is no image database any more like in the earlier Zeiss LSM software versions.
Fig. 8 New image document in the Open Images Areas

8 04/2009
Advanced data browsing is available through the ZEN File Browser (Ctrl + F or from the File Menu).
The ZEN File Browser can be used like the WINDOWS program file browser. Images can be opened by
double-click and image acquisition parameters are displayed with the thumbnails (Fig. 9). For more
information on data browsing please refer to the detailed operating manual.
Fig. 9 File Browser

04/2009 9
Turning on the lasers
ZEN 2009 operates all lasers automatically. Whenever they are used (manually or by the Smart Setup
function) the lasers are turned on automatically. The Laser Life Extender function of the software shuts
all lasers off if ZEN is not used for more than 15 minutes.
To manually switch lasers on or off:
!Click the show manual tools tickbox and open the Laser tool. All available lasers can be operated
within this tool (Fig. 10).
"
Fig. 10 Laser Control tool

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Setting up the microscope
Changing between direct observation and laser scanning mode
The Ocular and Acquisition buttons switch between the use of the LSM and the microscope:
!Click on the Ocular button to open the controls for the
microscope beam path and for direct observation via the
eyepieces of the binocular tube, lasers are blocked.
!To set the hardware in position for using the microscope,
click Online if not yet active.
!To close the light shutters on the microscope click Offline.
!Click on the Acquisition button to move back to the
LSM system.
Setting up the microscope and storing
settings
Click on the Ocular tab for direct observation;
press the Online button for your actions to take
effect immediately. Then open the Ocular tool to
configure the components of your microscope like
filters, shutters or objectives (Fig. 11).
Selecting an objective
!Open the graphical pop-up menu by clicking on
the Objective symbol and select the objective
lens for your experiment (Fig. 11).
!The chosen objective lens will automatically
move into the beam path.
Focusing the microscope for transmitted light
!Open the graphical pop-up menu by clicking on
the Transmitted Light icon (Fig. 12).
!Click on the On button. Set the intensity of the
Halogen lamp using the slider.
!Clicking outside the pop-up control closes it.
!Place specimen on microscope stage. The cover
slip must be facing the objective lens. Re-
member the immersion medium if the objective
chosen requires it!
Fig. 11 Microscope Control window, e.g.:
Axio Imager.Z2

04/2009 11
!Use the focusing drive of the microscope to focus the object plane.
!Select specimen detail by moving the stage in X and Y using the XY stage fine motion control.
Setting the microscope for reflected light
!Click on the Reflected Light icon to open the
X-Cite 120 Controls and turn it on.
!Click on the Reflected Light shutter to open
the shutter of the X-Cite 120 lamp / HBO100.
!Click on the Reflector button and select the
desired filter set by clicking on it.
Storing the microscope settings
Microscope settings can be stored as
configurations (Fig. 13) by typing a config name in
the pull-down selector and pressing the save
button. Fast restoration of a saved config is
achieved by selecting the config from the pull-
down list and pressing the load button. The
current config can be deleted by pressing the
delete button.
These configurations can be assigned to buttons
that are easier to press.
Depending on the microscope configuration,
settings must be done manually if necessary.
Fig. 12 Microscope Control window with
Transmitted Light pop-up menu
Fig. 13 Configuration panel
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12 04/2009
Configuring the beam path and lasers
!Click the Acquisition button.
Smart Setup
The tool Smart Setup is an intuitive, user-friendly interface which can be used for almost all standard
applications. It configures all the system hardware for a chosen set of dyes.
!Click on the Smart Setup button to open the smart setup window. This window can
be accessed any time from the software to change dye combinations.
!Click on the arrow in the dye list and simply choose the dye(s) you want to use in your experiment
from the list dialogue. In this dialogue, the dyes can be also searched by typing the name in the search
field.
Fig. 14 Smart setup tool
Once finished with the input, Smart Setup suggests four alternative considerations (see below): One for
fastest imaging, one for the best signal, best compromise between both speed and best signal and
the optimal setup for later linear unmixing of the dyes.
The graphs display relative values for the expected emission signals and cross-talk. The resulting imaging
scheme (single or multitrack) is shown below the graphs.

04/2009 13
Fig. 15 Proposals panel of the Smart Setup tool
Pressing Apply, automatically sets the hardware parameters in the displayed way for the dyes chosen.
If the option Linear Unmixing is selected, the system is set in the lambda mode automatically.
Pressing the Auto Exposure button will then optimize the settings of the Gain (Master) and
offset for the given laser power and pinhole size. Further image optimisation from this point can be done
easily.
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14 04/2009
Setting up a configuration manually
Simultaneous scanning of single, double and triple labeling:
Advantage: faster image acquisition
Disadvantage: potential cross-talk between channels
Sequential scanning of double and triple labeling; line-by-line or frame-by-frame:
Advantage: Only one detector and one laser are switched on at any one time. This reduces cross-
talk.
Disadvantage: slower image acquisition
!Open the Imaging Setup and the Light Path tool in the Setup Manager Tool group to access the
hardware control window to set-up the beam path.
The open Light Path is shown in Fig. 16.
"
Fig. 16 Light Path tool for a single track (LSM)

04/2009 15
Settings for track configuration in Channel Mode
!Select Channel Mode if necessary (Fig. 17).
The Light Path tool displays the selected track
configuration which is used for the scan procedure.
!You can change the settings of this panel using the
following function elements:
Activation / deactivation of the excitation wavelengths (check box) and setting of
excitation intensities (slider). If necessary open the Laser Control tool (see above).
Selection of the main dichroic beam splitter (MBS) from the relevant list box.
Selection of an emission filter through selection from the relevant list box.
Activation / deactivation (via check box) of the selected channel (Ch 1-4, monitor diode
ChM, QUASAR detectors ChS1-8, transmission ChD) for the scanning procedure and
assigning a color to the channel.
!Select the appropriate filters and activate the
channels.
!Click the Laser icon to select the laser lines and
set the attenuation values (transmission in %) in
the displayed window.
!For the configuration of the beam path, please
refer to the application-specific configurations
depending on the used dyes and markers and
the existing instrument configuration.
!In the Imaging Setup tool the Detection Bands
& Laser Lines are displayed in a spectral panel
(Fig. 18) to visualize the activated laser lines for
excitation (vertical lines) and activated detection
channels (colored horizontal bars).
Fig. 17 Imaging Setup tool for a single
track (LSM)
Fig. 18 Detection Bands & Laser Lines
display

16 04/2009
!For storing a new configuration click and
enter a desired name in the first line of the list
box (Fig. 19), then click Ok to store the con-
figuration.
!For loading an existing configuration click
then select it from the list box.
!For deleting an existing configuration click
then select it from the list box and confirm the
deletion with Ok.
Settings for multiple track configurations in Channel Mode
Multiple track set-ups for sequential scanning can be defined as one configuration (Channel Mode
Configuration), to be stored under any name, reloaded or deleted.
The maximum of four tracks with up to eight channels can be defined simultaneously and then scanned
one after the other. Each track is a separate unit and can be configured independently from the other
tracks with regard to channels, Acousto-Optical Tunable Filters (AOTF), emission filters and dichroic beam
splitters.
The following functions are available in the List of Tracks panel in the Imaging Setup Tool (Fig. 17,
Fig. 18 and Fig. 19).
Switch track every
Line Tracks are switched during scanning line-by-line. The following settings can be changed
between tracks: Laser line, laser intensity and channels.
Frame Tracks are switched during scanning frame-by-frame. The following settings can be
changed between tracks: Laser line and intensity, all filters and beam splitters, the
channels incl. settings for gain and offset and the pinhole position and diameter.
Frame Fast The scanning procedure can be made faster. Only the laser line intensity and the
Amplifier Offset are switched, but no other hardware components. The tracks are all
matched to the current track with regard to emission filter, dichroic beam splitter,
setting of Detector Gain, pinhole position and diameter. When the Line button is
selected, the same rules apply as for Frame Fast.
Add Track button
An additional track is added to the configuration list in the Imaging Setup
Tool. The maximum of four tracks can be used. One track each with basic
configuration is added, i.e.: Ch 1 channel is activated, all laser lines are
switched off, emission filters and dichroic beam splitters are set in accordance
with the last configuration used.
Remove button The track marked in the List of Tracks panel is deleted.
A click on this arrow button will move the selected track (highlighted in light
grey) one position upwards in the list box.
A click on this arrow button will move the selected track (highlighted in light
grey) one position downwards in the list box.
Fig. 19 Track Configurations window

04/2009 17
Scanning an image
Setting the parameters for scanning
!Select the Acquisition Mode tool from the Left Tool Area (Fig. 20).
!Select the Frame Size as predefined number of pixels or enter your own values (e.g. 300 x 600) in the
Acquisition Mode tool. Click on the Optimal button for calculation of appropriate number of pixels
depending on objective N.A. and #.
The number of pixels influences the image resolution!
Adjusting scan speed
!Use the Scan Speed slider in the Acquisition Mode tool (Fig. 20) to adjust the scan speed.
A higher speed with averaging results in the best signal-to-noise ratio. Scan speed 8 usually produces
good results. Use speed 6 or 7 for superior images.
Choosing the dynamic range
!Select the dynamic range 8 or 12 Bit (per pixel) in the Bit Depth pull-down in the Acquisition Mode
tool (Fig. 20).
8 Bit will give 256 gray levels; 12 Bit will give 4096 gray levels. Publication quality images should be
acquired using 12 Bit data depth. 12 Bit is also recommended when doing quantitative measurements or
when imaging low fluorescence intensities.
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Fig. 20 Acquisition Mode tool
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18 04/2009
Setting scan averaging
Averaging improves the image by increasing the signal-to-noise ratio. Averaging scans can be carried out
line-by-line or frame-by-frame. Frame averaging helps to reduce photo-bleaching, but does not give quite
as smooth of an image.
!For averaging, select the Line or Frame mode in the Acquisition Mode tool.
!Select the number of lines or frames to average.
Adjusting pinhole size
!Select the Channels tool in the Left Tool Area.
!Set the Pinhole size to 1 AU (Airy unit) for best compromise between depth discrimination and
detection efficiency.
Pinhole adjustment changes the Optical Slice thickness. When collecting multi-channel images, adjust
the pinholes so that each channel has the same Optical Slice thickness. This is important for
colocalization studies.
"
Fig. 21 Channels tool