Zeiss LSM 510 Inverted User manual

LSM 510 and LSM 510 META
Laser Scanning Microscopes
Brief Operating Manual
Release 3.5
July 2005

2 07/05
Contents
Page
Starting the System................................................................................................................3
Setting the microscope...........................................................................................................6
Configuring the beam path and lasers..................................................................................8
Scanning an image ...............................................................................................................11
Storing an image ..................................................................................................................15
Switching off the system.....................................................................................................15
Introduction
For your safety!
Observe the following instructions:
− The LSM 510 and LSM 510 META 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
HBO 100 mercury lamp.

07/05 3
Starting the System
Switching on the LSM system
• When set to ON the REMOTE CONTROL switch
labeled System/PC provides power to the
microscope and the computer. This allows to
use the microscope and the computer without
running the LSM Software (Fig. 1).
• To switch on the system completely put the
Components switch also to ON. Now the
complete system is ready to be initialized with
the LSM Software.
Switching on the HBO 100 mercury lamp
• Switch on the HBO 100 mercury lamp via the
switch of the power supply, see operating
manual of the mercury lamp or microscope.
Switching on the Enterprise UV-Ar Laser
• If the UV laser is required, switch it on via the
toggle switch (Fig. 2/1) of the power supply.
− It will be ready for operation after a few
seconds.
Fig. 1 REMOTE CONTROL switch
Fig. 2 Power supply of UV-Ar laser

4 07/05
Starting the LSM 5 software program
LSM 510
• Double click the LSM 510 icon on the desktop of WINDOWS to start the LSM 5 software
program.
The LSM 510 Switchboard window appears on the screen (Fig. 3).
• Click on the Scan New Images button in the LSM 510 Switchboard window.
Clicking on this button activates the complete LSM hardware (on-line mode).
• Click on the Start Expert Mode button in the LSM 510 Switchboard window.
The LSM 510 - Expert Mode Main menu appears on the screen.
Use of this mode requires to be thoroughly familiar with the exact microscope procedures
and interrelations.
Main menu toolbar
Subordinate toolbar
Main menu (pull-down)
Fig. 4 Main menu for Expert Mode
Fig. 3 LSM 510 Switchboard menu

07/05 5
Creating a database for acquired images
• Click on the File button in the Main menu toolbar.
The File subordinate toolbar appears in the Main menu.
Fig. 5 Main menu with File subordinate toolbar
• Click on the New button in the File subordinate toolbar.
The Create New Database window appears.
• Select drive C: or D: from pull down menu. • Create a new directory if needed.
Fig. 6 Create New Database window Fig. 7 Create New Database window
Turning on the lasers
• Click on the Acquire button in the Main menu to open the Acquire subordinate
toolbar.
Fig. 8 Acquire subordinate toolbar

6 07/05
• Click on the Laser button to open the Laser Control window.
• Select the appropriate Laser Unit by clicking on
the name of it.
• Click on the Standby button to switch required
laser(s) to Standby.
• When status is Ready click on On button.
• Set Output [%] so that the Tube Current is
about 4 A (50% of the output power).
Setting the microscope
Changing between direct observation or laser scanning
The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set in the
binocular tube of the microscope:
• Click on the VIS button to set the microscope for direct observation via the eyepieces
of the binocular tube, lasers are off.
• Click on the TV button to set the microscope camera observation (if connected) via
camera adapter of the binocular tube.
• Click on the LSM button to set the microscope screen observation via laser excitation
using the LSM 510 and software evaluation.
Setting the microscope and storing the settings
• Click on the VIS button for direct observation.
• Click on the Micro button in the Acquire subordinate toolbar to open the
Microscope Control window of the used microscope.
The Microscope Control window appears (Fig. 10).
Fig. 9 Laser Control window

07/05 7
Selecting an objective
• Open the graphical pop-up menu by clicking on
the Objective button (Fig. 10).
• Click on the objective you want to select. The
selected objective will automatically move into
the beam path.
Focussing the microscope for transmitted light
• Open the graphical pop-up menu by clicking on
the Transmitted Light button (Fig. 11).
• Click on the On button. Set the intensity of the
Halogen illuminator using the slider.
• Click on Close to close the pop-up menu.
• Place specimen on microscope stage. The cover
slip must be facing up.
• Use the focusing drive of the microscope to
focus the required 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 button to open
the shutter of the HBO 100 mercury lamp.
• Click on the Reflector button and select the
desired filter set by clicking on it.
Storing the microscope settings
Microscope settings can be stored and up to 8
buttons assigned for fast retrieval and adjustment
using the Microscope Settings panel.
The Store button permits existing microscope
configurations to be stored under any name.
The Apply button permits existing stored
microscope configurations to be loaded.
The Delete button permits existing microscope
configurations to be deleted.
The Assign button permits the assignment of a
microscope configuration to a button.
Note: Depending on the microscope
configuration, settings must be done manually if
necessary.
Fig. 10 Microscope Control window,
e.g.: Axiovert 200 M
Fig. 11 Microscope Control window with
Transmitted Light pop-up menu

8 07/05
Configuring the beam path and lasers
• Click on the LSM button in the Acquire
subordinate toolbar for laser scanning.
Choosing the configuration
Single Track
− Use for single, double and triple labeling;
simultaneous scanning only
− Advantage: faster image acquisition
− Disadvantage: cross talk between channels
Multi Track
− Use for double and triple labeling; sequential
scanning, line by line or frame by frame
− Advantage: when one track is active, only
one detector and one laser is switched on.
This reduces cross talk.
− Disadvantage: slower image acquisition
• Click on the Config button in the
Acquire subordinate toolbar to
open the Configuration Control
window.
The Configuration Control window
appears (Fig. 12).
Setting for single track configuration in Channel Mode
• Select Channel Mode if necessary (Fig. 12).
• Click on the Single Track button in the Configuration Control window (Fig. 12).
• Click on the Descanned button if necessary.
The Beam Path and Channel Assignment panel of the Configuration Control window 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). Open the Laser Control window via the Laser button.
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 by
assigning an existing color icon or defining a new one.
Fig. 12 Configuration Control window for
Single Track

07/05 9
• Select the appropriate filters and activate the
channels.
• Click the Excitation button 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.
• Clicking on the Spectr button
opens the Detection Spectra &
Laser Lines … window (Fig. 13) to
display the activated laser lines for
excitation (colored vertical lines) and
channels (colored horizontal bars).
• Clicking on the Config button
opens the Track Configurations
window (Fig. 14) to load, store or
delete track configurations.
• For storing a new track configuration enter a
desired name in the first line of the
Configurations list box and click an Store.
• For loading an existing configuration select it in
the list box and click on Apply.
• For deleting an existing configuration select it in
the list box and click on Delete.
Setting for multi track configuration in
Channel Mode
The Multi Track function permit several tracks to
be defined as one configuration (Channel Mode
Configuration) for the scan procedure, 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 of the other
tracks with regard to channels, Acousto-Optical
Tunable Filters (AOTF), emission filters and dichroic
beam splitters.
• Select Channel Mode if necessary (Fig. 15).
Fig. 13 Detection Spectra & Laser Lines …
window
Fig. 14 Track Configurations window
Fig. 15 Configuration Control window for
Multi Track

10 07/05
• Click on the Multi Track button in the Configuration Control window (Fig. 15).
• Click on the Descanned button if necessary.
The following functions are available in the List of Tracks panel (Fig. 15).
Modes
Switch tracks after each
Line button Tracks are switched during scanning line by line. The following settings
can be changed between tracks: Laser line and intensity and channels
incl. settings for gain and offset.
Switch tracks after each
Frame button 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 button The scanning procedure can be made faster. Only and the laser line and
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 Line button is selected, the same
rules apply as for Frame Fast.
Settings
Add Track button An additional track is added to the configuration list. The maximum of
four tracks can used. One track each with basic configuration is added,
i.e.: one Ch 1 channel is activated, all laser lines are switched off,
emission filters and dichroic beam splitters are set in accordance with the
configuration last used.
Remove button The single track previously marked in the List of Tracks panel in the
Name column is deleted.
Store/Apply Single Track
button Opens the Track Configurations window. A selected track defined in a
Channel Mode Configuration can also be stored as a single track for
single tracking applications. Also, it's possible to load a single track in a
multi tracking configuration.
A click on this arrow button will move the selected track (highlighted in
blue) one position upwards in the list box.
A click on this arrow button will move the selected track (highlighted in
blue) one position downwards in the list box.
• Configure each desired track for Multi Track function as described for Single Track.
• For storing/applying or deleting a Channel Mode Configuration use the Config button.

07/05 11
Scanning an image
• Click on the Scan button in the Acquire subordinate toolbar to open the Scan
Control window.
The Scan Control window appears (Fig. 16).
Setting the parameters for scanning
• Select Mode in the Scan Control window.
• Select the Frame Size as predefined number of
pixels or enter your own values (e.g. 300 x 600)
in the Objective Lens, Image Size & Line
Step Factor panel. Click on the Optimal
button for calculation of appropriate number of
pixels depending on N.A. and λ.
The number of pixels influences the image
resolution!
Note: When using an Axioskop 2 FS MOT, indicate
the objective that is in use in the Scan Control
window. This ensures correct calculation of
pinhole, Z stack optimization etc.
Adjusting the scan speed
• Use the slider in the Speed panel (Fig. 16) 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. Us speed 6 or 7 for superior images.
Choosing the dynamic range
• Select the dynamic range 8 or 12 Bit (per pixel) in the Pixel Depth, Scan Direction & Scan Average
panel (Fig. 16).
8 Bit will give 256 gray levels, 12 Bit will give 4096 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.
Setting the scan averaging
Averaging improves the image by increasing the signal : noise ratio. It can be achieved line by line or
frame by frame. Frame averaging helps reduce photobleaching, but does not give quite such a smooth
image.
• Select the Line or Frame mode for averaging.
• Select the desired scan average method Mean or Sum in the Method selection box.
If you are using the Mean method, the image information is generated by adding up all scans pixel by
pixel and then calculating the mean value.
Fig. 16 Scan Control window, Mode settings

12 07/05
In the Sum method, the pixel values of all scans are only added up, without a mean value being
calculated.
• Select the Number for averaging.
Continuous averaging is possible in the Frame mode. In this case a Finish button for ending continuous
averaging is displayed instead of the Cont. button.
Adjusting the pinhole
• Select Channels in the Scan Control window.
• Set the Pinhole size to 1(Airy unit) for best
compromise between depth discrimination and
efficiency.
Pinhole adjustment changes the Optical Slice.
When collecting multi channel images, adjust the
pinholes so that each channel has the same
Optical Size. This is important for colocalization
studies.
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 XY, Single or Cont.
buttons to start the scanning procedure and
acquire an image.
Scanned images are shown in separate windows.
• Click on the Stop button to stop the current
scan procedure if necessary.
Select Find for automatically pre-
adjustment of detector sensitivity.
Select Fast for continuous fast
scanning – useful for finding and
changing the focus.
Select Single for recording a single
image.
Select Cont. for continuous scanning
with the selected scan speed.
Select Stop for stopping the current
scan procedure.
Fig. 17 Scan Control window, Channel settings
Fig. 18 Image window

07/05 13
Image optimization
Choosing a lookup table
• Select Palette in the Image window of the
scanned image (Fig. 19).
The Color Palette window appears.
• In the Color Palette List panel, click on the
Range Indicator item (Fig. 20).
The scanned image appears in a false-color
presentation (Fig. 19).
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
• Set the Pinhole to 1Airy Unit (Fig. 21).
• Set the Detector Gain high.
• When the image is saturated, reduce AOTF
transmission in the Excitation panel (Fig. 21)
using the slider to reduce the intensity of the
laser light at the specimen.
Adjusting gain and offset
• Increase the Amplifier Offset until all blue
pixels disappear, and then make it slightly
positive (Fig. 21).
• Reduce the Detector Gain until the red pixels
only just disappear.
Fig. 19 Image window
Fig. 20 Color Palette window
Fig. 21 Scan Control window, Channel settings

14 07/05
Scanning a Z stack
• Select Z Stack in the Scan Control window.
• Select Frame if necessary.
The Z Settings panel appears.
• Select Mark First/Last on the Z Settings
panel.
• Click on the XY cont button.
A continuous XY-scan of the set focus position will
be performed.
• Use the focusing drive of the microscope to
focus on the upper position of the specimen
area where the Z Stack is to start.
• Click on the Mark 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 Mark Last button to set this lower
position.
• Click on X:Y:Z=1:1:1 button to set the Z-
interval in such a way that the voxel has
identical dimensions in the X-, Y- and Z-
directions (cube).
• Click on the Start button to start the recording
of the Z Stack.
Fig. 22 Scan Control window, Z Stack settings

07/05 15
Storing an image
• Click on the Save or Save As button in the Image window or in the File subordinate toolbar of the
Main menu.
The Save Image and Parameter As window appears.
Fig. 23 Save Image and Parameter As window
• Enter file name, description and notes in the appropriate text boxes.
• Click on the OK button.
Switching off the system
• Click on the File button in the Main menu and then click on the Exit button to leave LSM 5 software
program (Fig. 5).
• 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 fan of Argon laser has switched off.
• On the REMOT CONTROL switch turn off the Components switch and the System/PC switch (Fig. 1).
• Switch off the HBO 100 mercury lamp.
• Switch the UV-Ar laser of via the toggle switch of the power supply (Fig. 2).