Zeiss LSM 5 Pascal Confocal Laser Scanning Microscope

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					                     Operating Instructions for the
         Zeiss LSM 5 Pascal Laser Scanning Confocal Microscope

1. Starting the System
-sign into log book for mercury lamp and argon lasers
-turn on mercury arc lamp
-turn on Ar laser using power button then turn key to start
         -turn power to 11 o’clock position on Ar power supply then flip switch to run
-wait for Ar laser to glow purple from initial orange colour (if laser clicks, turn it off
-turn on power bar to turn on computer and HeNe lasers. The bottom power supply is for the
543 nm HeNe laser and the top one is for the 633nm laser
-enter username and password

2. General Operation
-initialize Pascal software. Choose Online mode to activate the complete LSM 5 Pascal
hardware and click on Start. The Offline mode allows you to process and analyze previously
acquired images, however the microscope cannot be controlled by the software
-Click on the Acquire button in the main menu
-Select the Vis mode and click on the Micro icon

2.1. Transmitted light observation
-to view the specimen in transmitted light, select a position without a reflector module in the
Reflector Turret panel and select Transmitted Light. Light intensity may be adjusted using
the slider bar or the toggle switch on the lower front of the microscope
-focus on sample through the eyepiece

2.2. Reflected light (epi-fluorescence) observation
-to view the specimen under fluorescent light, turn off the Transmitted Light, choose a filter
position in the Reflector Turret and turn the Reflected Light on
-focus on sample through the eyepiece

2.3. Configuration Control
-click on the Config button in the Acquire subordinate toolbar of the main menu
-The configuration control window offers the choice of Single Track or Multi Track. Choose the
appropriate mode for your application. In multitracking, each track is a separate unit and can
be configured independently of the other tracks with regard to channels, emission filters and
dichroic beam splitters
-click on the Store/Apply button in the Track panel
-choose the appropriate configuration and click on Apply. The stored instrument parameters
will be installed and will be displayed in the Beam Path and Channel Assignment window.
-when adding new tracks, first click on the Add Track button. Select an existing configuration
by selecting Store/Apply and choose from the list of configurations. Click on Apply.
-in multitracking, tracks can be activated or deactivated by double-clicking on the name of the
relevant tracks. A tick to the left of its name indicates that a track is active.

2.4. Scan Control
-click on LSM in the Acquire subordinate toolbar of the main menu
-click on the Scan button to open the Scan Control window
-two scanning modes are available, a one dimensional Line scanning mode and a two
dimensional Frame scanning mode. Choose the appropriate mode for your application.
-select the Mode icon

Objective Lens and Image Size Panel
-select the appropriate Objective from the list
-select the Frame Size from the default sizes. The recommended setting is 512 x 512 pixels

Speed Panel
-select the Scan Speed from the 13 preset steps using the slider. The recommended setting
for the first scan is 7. The scan time indicating the recording duration for the entire frame will
be displayed.

Pixel Depth, Scan Direction and Scan Average
-select 8 bit or 12 bit data depth (ie. 256 or 4048 gray values. The recommended setting is
12 bit)
-select the Unidirectional → or Bi-directional     scan direction. In unidirectional scanning,
the laser scans in one direction only then moves back and scans the next line. In bidirectional
scanning, the laser also scans when moving backwards. The scan time is faster in
bidirectional scanning but there may be a problem with registration
-select the desired scan average Method, mean or sum
-select the desired scan average from the available values in the Number box. The greater
the number of averages selected, the better the image quality will be. However, scanning
time will increase as will photobleaching.

Zoom, Rotation and Offset
-select the appropriate Zoom value.
-the zoom magnification control, determines the size of a pixel at the specimen. For Nyquist
sampling, the pixel should be at least 2.3 times smaller than the smallest features that you
expect to see in your specimen.
-In a Zeiss confocal system, the optimal zoom setting can be determined using the following

                                        3.92 x Na x system constant
                    Zoom factor =
                                     Number of pixels/line x Magobj x λexcitation

                        Na - numerical aperture of the objective lens
                        System Constant - 8.94 mm for an LSM 510 and LSM 5 Pascal
                        Number of pixels/line – typically set at 512 pixels
                        λexcitation - excitation wavelength

                                              system constant
                 Pixel size =
                                Number of pixels/line x Zoom factor x Magobjective

- For example, with a 63x objective (Na = 1.4), 512 pixels per scan line and a wavelength of
488 nm, the full resolving power (correct sampling) is achieved with a scanning zoom of 3.11
as a minimum and a pixel size of 88.9 nm.
-The problem in practice is that use of the zoom magnification control on typical confocal
microscopes can easily be misused in a manner that violates the Nyquist criterion. Make
sure you use the correct zoom setting.

Channel Settings
-click on the Channel button in the Scan Control window to open the Channel Settings
-set the pinhole to 1 Airy Unit for each channel 1 and 2
-adjust the % transmission for the laser line used by each channel in the Excitation of Track
window. Set the Ar laser power to <5% (2% is good) and the HeNe laser power to <30%.
-set Ampl. Gain to 1

3. Optimizing Image Quality
-deactivate all but one channel by double clicking on the appropriate track in the List of
Tracks window in the Configuration Control palette. A tick to the left of its name indicates
that a track is active.
-do all image optimization on the active channel
-click on the Find button in the Scan Control window
-in the main image window, click on Palette to open the Color Palette window

-click on the Range Indicator item and scan the specimen by clicking on Cont in the Scan
Control window. The scanned image appears in false color where red indicates bright areas
and blue indicates dark areas. On the Channel Settings panel of the Scan Control window,
set the PMT gain with the Detector Gain slider until the image has just a trace of red. To
adjust the background level, adjust the Ampl. Offset slider so that areas without picture
content show just a few speckles of blue. If necessary, amplify brightness with the Ampl.
Gain slider but only if adjusting the other settings is insufficient for image optimization.
-in the Scan Control window, click on the Stop button to stop the continuous scan
-repeat this procedure for the other channels
-in the Color Palette panel click on No Palette to deactivate the range indicator
-activate all channels and click on Single in the Scan Control panel to view the image
-when multiple channels are being scanned, click on Split xy to view all the tracks plus an
-click on Info in the main image window to review all parameters for each acquired image

4. Z Stack
-optimize image quality on the brightest section in the stack
-use only one channel to set the appropriate parameters for Z sectioning. Deactivate all but
one channel by double clicking on the appropriate track in the List of Tracks window.
-in the Scan Control panel, select the Z Settings option
-the optimum stack size is determined with the help of the Line Sel and Range functions
-click on the Line Sel button – an XY scan of the current slice is performed. The Line toolbar
will be displayed at the right hand side of the main image window.
-click on the Line arrow button → in the Line toolbar and define a straight line as the cutline
for the XZ scan
-click on the Range button. An XZ scan will be performed and displayed in the image window.
The position of the current slice is shown with a green line and the positions of the first and
last slice with two red lines. The red lines define the upper and lower limits of the Z range.
-move the green line to shift the current focus position and the red lines to shift the limits of
the stack
-once the upper and lower limits have been established, reactivate all channels and click on
the Start button to start recording of the Z stack. Ensure that the Nyquist sampling criterion
is met in the Z direction by using an appropriate z sectioning Interval.
-to view the progression of the Z stack, switch to the Gallery view. It may also be beneficial
to switch to the Range Indicator setting in the Color Palette panel.
-an alternate way to set up the optimum stack size is to select the Mark First/Last tab in the
Z settings panel.
-activate only the channel which uses the less powerful laser
-in the Scan Control window, click on the Fast XY button
-with the microscope continuously scanning the sample, use the manual focusing drive to
focus on the upper position of the specimen where the Z stack is to start. Click on Mark
-focus on the lower position of the specimen. Click on Mark Last.
-enter the appropriate value for the refractive correction in Refr. Corr.
-click on the Start button to start recording of the Z stack

4. Shutting Down the System
-shutdown the computer
-turn off power bar
-turn laser power on Ar power supply to 0
        -flip switch to standby
        -turn key to off
        -wait for fan to go off
        -turn power to off
-write down Ar laser hours in logbook
-write down Hg lamp hours
-turn power off on Hg lamp
-remove objective lens
-replace cover