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.

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