Quick Guide
Microscopy from Carl Zeiss
Quick Guide
LSM 5 MP, LSM 510 and
LSM 510 META
Laser Scanning Microscopes
LSM Software ZEN 2007
August 2007
We make it visible.
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............................................................................................................... 15
Storing and exporting image data ..................................................................................... 20
Switching off the system..................................................................................................... 21
Introduction
This LSM 510 / LSM 510 META / LSM 510 NLO Quick Guide describes the basic operation of the
LSM 510 / LSM 510 META / LSM 510 NLO Laser Scanning microscope with the ZEN 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, Axioskop 2 FS MOT). 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:
08/2007
!
The LSM 510 / LSM 510 META / LSM 510 NLO 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.
1
Starting the System
Switching on the LSM system
" When set to ON the REMOTE CONTROL switch
labeled System/PC provides power to the
computer. This allows use of the computer and
ZEN software offline (Fig. 1).
" 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
Fig. 1
REMOTE CONTROL switch
" 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 Enterprise UV-Ar Laser
" If the UV laser is required, switch it on via the
toggle switch (Fig. 2/1) on the power supply.
! It will be ready for operation after a few
seconds.
Fig. 2
2
Power supply of UV-Ar laser
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Starting the ZEN software
" Double click the ZEN icon on the WINDOWS desktop to start the Carl Zeiss LSM
software.
The ZEN Main Application Window and the LSM 510 Startup window appear on the
screen (Fig. 3)
Fig. 3
ZEN Main Application Window at Startup (a) and the LSM 510 Startup Window (b and c)
In the small startup window, choose either to start the system online ("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.
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3
4
Fig. 4
ZEN Main Application Window after Startup with empty image container
Fig. 5
ZEN Main Application Window after Startup with several images loaded
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Introduction to ZEN – Efficient Navigation
ZEN - Efficient Navigation - is the new software for the LSM 5 Family from Carl Zeiss. With the launch
of this software in 2007 Carl Zeiss sets new standards in application-friendly software for Laser Scanning
Microscopy.
The ZEN 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 image acquisition, image processing and
system maintenance, easily accessible via 3 Main Tabs (Fig. 5/1). All functions needed to control the
microscope and to acquire images are bundled in the Acquisition Tools (Fig. 5/3 and 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 Blocks 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.
Fig. 6
Basic and Pro Mode
A focus in the development of ZEN 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:
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5
The Pro-Basic concept ensures that tool panels are never more complex than needed. In Basic Mode,
the most commonly used tools are displayed. For each tool, the user can activate Pro Mode to display
and use additional functionality (Fig. 6).
Fig. 7
ZEN Window Layout configuration
More features of ZEN include:
"
The user can add more columns 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. 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 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 software, please refer to the User Manual that is provided with your
system.
6
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Setting up a new image document and saving your data
To create a new image document in an empty image container, click the "Single"
or the "Start"
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.
Alternatively you can create a new empty image document with the "New"
Button area or the "New" function in the File Menu.
button in the Action
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.
Note:
There is no "image database" any more like in the earlier Zeiss LSM software versions.
Fig. 8
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New image document in the Open Images Ares
7
Advanced data browsing is available through the File Browser (Ctrl-F or from the File Menu). The File
Browser can be used like the WINDOWS program file browser. Images can be opened by a 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
8
File Browser
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Turning on the lasers
" Open the Laser Tool in the left tool area (always the first in the list) and activate the lasers you need
for your experiment (Fig. 10). Remember, the argon multi-line laser has to first be put to standby for a
5 minute warm-up before it changes to on.
" Zeiss recommends operating the Argon multi-line Laser at a tube current of about 6 A (#50 %
output). This is the best compromise between laser stability/power and laser life-time. (tube-current
control can be found in Pro Mode).
$
Fig. 10
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Laser Control tool (in Pro Mode)
9
Setting up the microscope
Changing between direct observation, camera detection and laser scanning mode
The Ocular, Camera and LSM buttons switch the beam path and indicate which beam path is currently
in use for the microscope:
" Click on the Ocular button to change the microscope beam path
for direct observation via the eyepieces of the binocular tube, lasers
are blocked.
" Click on the Camera button to change the beam path of the
microscope for the port where the camera is attached for camerabased image acquisition.
" Click on the LSM button to set the beam path for the LSM 510
system.
Setting up the microscope and storing settings
Click on the Ocular button for direct observation. Then choose the Light Path tool from the Left Tool
Area. Since the Ocular button has been chosen before, the Oculars tab is pre-selected and presented as
the front tab in this tool (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. Remember the immersion
medium if the objective chosen requires it!
Fig. 11
Microscope Control window,
e.g.: Axio Imager.Z1
" 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.
10
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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 pullload button. The
down list and pressing the
current config can be deleted by pressing the
delete
Fig. 12
Microscope Control window with
Transmitted Light pop-up menu
Fig. 13
Saving Microscope configurations
button.
In Pro Mode, these configurations can be assigned
to buttons that are easier to press.
Note:
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Depending on the microscope configuration, settings must be done manually if
necessary.
11
Configuring the beam path and lasers
" Click on the LSM button.
Choosing a configuration
Simultaneous scanning of single, double and triple labeling:
! Advantage: faster image acquisition
! Disadvantage: Eventual 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 crosstalk.
! 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. 14.
$
Fig. 14
12
Light Path tool for a single track (LSM)
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Settings for track configuration in Channel Mode
" Select Channel Mode if necessary (Fig. 15).
" Click on the LSM tab (Fig. 14).
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:
Fig. 15
Imaging Setup tool for a single track
(LSM)
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 (HFT) or secondary dichroic beam splitter
(NFT) position through selection 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, META 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. 16) to visualize the activated laser lines for
excitation (vertical lines) and activated detection
channels (colored horizontal bars).
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Fig. 16
Detection Bands & Laser Lines display
13
" For storing a new configuration enter a
desired name in the first line of the
Configurations list box (Fig. 17) and click
Store.
" For loading an existing configuration select it
from the list box and click on the
button.
Load
" For deleting an existing configuration select it in
Fig. 17
Track Configurations window
the list box and click on
Delete.
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 8 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. 15,
Fig. 16 and Fig. 17).
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
Remove 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.
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.
14
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Scanning an image
Setting the parameters for scanning
" Select the Acquisition Mode tool from the Left Tool Area (Fig. 18).
" 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!
$
Fig. 18
Note:
Acquisition Mode tool
When using an Axioskop 2 FS MOT, make sure you set the correct objective manually in the
Acquisition Mode tool. This ensures correct calibration, calculation of pinhole and Z stack
optimization, etc. The Axioskop 2 FS MOT does not automatically detect the objective lens.
Adjusting scan speed
" Use the Scan Speed slider in the Acquisition Mode tool (Fig. 18) 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. 18).
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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15
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 (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. 19
16
Channels tool
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Image acquisition
Once you have set up your parameter as defined in
the above section, you can acquire a frame image
of your specimen.
" Use one of the Find, Fast, Continuous, or
Single buttons to start the scanning procedure
to acquire an image.
" Scanned images
windows.
are
shown
in
separate
" Click on the Stop button to stop the current
scan procedure if necessary.
Select Find for automatic preadjustment of detector gain and
offset.
Select Fast for continuous fast
scanning – useful for finding and
changing the focus.
Fig. 20
Image Display
Fig. 21
View Dimensions Control Block
Select
Continuous
for
continuous scanning with the
selected scan speed.
Select Single for recording a
single image.
Select Stop for stopping the
current scan procedure.
Image optimization
Choosing Range Indicator
" In the View – Dimensions View Option
Control Block, click inside the color field in the
button under the channel button
(Fig. 21).
Note: Clicking on the right hand side of the
button leads to a list of colors.
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17
The scanned image appears in a false-color
presentation (Fig. 22).
If the image is too bright, it appears red on the
screen. Red = saturation (maximum).
If the image is not bright enough, it appears blue
on the screen. Blue = zero (minimum).
Adjusting the laser intensity
Fig. 22
Image Display
" Set the Pinhole to 1 Airy Unit (Fig. 23).
" Set the Detector Gain high.
" When the image is saturated, reduce AOTF
transmission in the Laser control section of the
Channels Tool (Fig. 23) using the slider to
reduce the intensity of the laser light to the
specimen.
Adjusting gain and offset
" Increase the Amplifier Offset until all blue
pixels disappear, and then make it slightly
positive (Fig. 23).
" Reduce the Detector Gain until the red pixels
only just disappear.
Fig. 23
18
Channels tool
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Scanning a Z stack
" Open the Z Stack tool in the Left Tool Area.
" Select Mode First/Last on the top of the Z
Stack tool.
" Click on the
Action Button area.
button in the
A continuous XY-scan of the set focus position will
be performed.
" Use the focus drive of the microscope to focus
on the upper position of the specimen area
where the Z Stack is to start.
" Click on the Set First button to set the upper
position of the Z Stack.
" Then focus on the lower specimen area where
the recording of the Z Stack is to end.
" Click on the Set Last button to set this lower
position.
button to set
" Click on the
number of slices to match the optimal Z-interval
for the given stack size, objective lens, and the
pinhole diameter.
Fig. 24
Z Stack tool
" Make sure that the activation tickbox is set
.
Start button to start the
" Click on the
recording of the Z Stack.
Note:
When
is not activated, the respective tool and its set parameters are not included in
the multidimensional image acquisition. If no multidimensional tool is activated, the
button is grayed out and only single images can be scanned.
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Start
19
Storing and exporting image data
" To save your acquired or processed images, click
on the Save or Save As button in File Menu
button at the
(Fig. 25/1), click on the
bottom of the File Handling Area (Fig. 25/2)
or click the
(Fig. 25/3).
Fig. 25
button in the Main Toolbar
Save Image buttons in ZEN
" The WINDOWS Save As window appears.
" Enter a file name and choose the appropriate
image format. Note: the LSM 5 format is the
native Carl Zeiss LSM image data format and
contains all available extra information and
hardware settings of your experiment.
" Click on the SAVE button.
Fig. 26
Save as window
If you close an image which has not been saved, a
pop-up window will ask you if you want to save it.
Choosing "yes" will lead you to the WINDOWS
Save As window.
To export image display data, a single optical
section in raw data format or the contents of the
image display window including analysis and
overlays, choose Export from the File Menu. In
the Export window you can select from a number
of options and proceed to the WINDOWS Save As
window to save the exported data to disk.
Fig. 27
20
Export window
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Switching off the system
" Click on the File button in the Main Menu bar and then click on the Exit button to leave the ZEN
software.
" If any lasers are still running you should shut them off now in the pop-up window indicating the lasers
still in use.
" Shut down the computer.
" Wait until the fan of the Argon laser has switched off.
" On the REMOTE CONTROL turn off the Components switch and the System/PC switch (Fig. 1).
" Switch off the X-Cite 120 lamp or the HBO 100 mercury burner.
" Switch off the UV-Ar laser of by the toggle switch on the power supply (Fig. 2).
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