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Table of contents
Starting the microscope
Remove the blue cover from the micro-scope.
Check if the system is already turned on. The LED display on the microscope tells you
is the microscope is switched on. Proceed to check the laser if already on.
Turn the microscope by turning a black switch on the left of the microscope, just below the air-table. Thus
should turn all the systems except the laser on(That needs to be switched on separatly)
Check if the computer is already on, turn the computer monitors on
Note :: Remember to turn the microscope on before running the Zeiss software in the online
mode for imaging. See switching software on the computer below.
Switch Lasers "on"
Our confocal has a single HeNe 746 nm laser. This sits on a granite table next to the microscope.
To switch on the laser, turn the key on the laser power source to your left. To actually use
the laser for scanning a sample, you would have to activate it in the Zeiss control software.
Start Zeiss Software
Double click on LSM 5 Pascal icon to start confocal operating software. The operating mode window
will appear. The control software checks for operating microscope.
If the microscope is not turned on
an error window appears. Choose offline or online mode as per use. This brings up the start menu for
the microscope.
Choose acquire mode from the tab.
Configuring the laser
Click on the acquire button in the main window and choose Laser sub window.
This brings the laser control window. To switch on the laser, choose the laser you
want turned on and click on the on tab. You can adjust the laser intensity now or later.
To set the required laser path settings, choose "Config" button from the main menu. The following
window appears.
Configuring microscope mode
To set the microscope, hit "Acquire" and choose "Micro". This should open up a control window.
Click on the objective lens icon to choose an appropriate lens. There are manual and automated
focusing wheels on the microscope. We do not have oil-immersion lenses right now. Be-aware
not to put any oil drop on the lens.
Set sample on the stage and adjust focus manually
We have a inverted configuration for the microscope. This is so that it is easy enough to set an
experiment on the microscope, and still be able to image in real time. Thus the lens sits below the
stage. For an inverted stage, you have to put the sample upside down sometimes. Use a double sided
tape to stick the sample to a glass slab. Also a cover slip on a glass slab works well too.
Be very careful not to touch the lens below the stage. Do not drop anything on the lens.
To view specimens directly down the microscope itself, pull out the lower of the two sliders
on the front face of the microscope. See the diagram on the front panel of the microscope.
For transmitted light, click "transmitted light" button at the top of the axiovert control
window. Switch the halogen light on, and vary transmitted light intensity by the scroll bar next to it.
Use the manual focus wheel to raise the objective lens until the specimen is in focus.
Configure scanning parameters
Push the bottom slider on the microscope front to right, to prepare microscope for scanning mode.
Click "Acquire", and choose "Scan" from the menu. The scan control window appears.
For a fast scan, click "Fast XY" on the right side of the control window(an image window appears).
At this point, the image need to be optimized. Choose "channels" sub window. You should see three
scroll bars. "Detector gain" sets the gain on the photo-detector. "Amplifier Offset" sets the
background, and "Amplifier gain" alters amplification factor.
Check the pin-hole factor in the channel window. The microscope can automatically set the
pin-hole size for you based on the objective lens being used.
For choosing the right settings of detector gain and laser power to be used for a sample, click on
"pallet" button on the scanning image window and choose red and blue range indicator. Now adjust the
detector gain and laser power settings such that you avoid under-saturation and over saturation of
your imaging photo-detector. Over saturation is shown by red color on the image, while under saturation
is shown by blue color. Optimize such that both these colors are minimized.
To improve image quality, you can choose the appropriate frame size. (For high quality scan choose 1024*1024,
for a low quality scan 512*512 scans are good). You can also adjust the scan speed(5 or 6 for low
scan). Magnify, move, rotate the image if needed using buttons at the bottom.
Click 'Stop' if the image has been optimized. You can click on single to get a 'single' image of the
sample. Click 'Save as' to save the image to the database. If you are using fluorescence in your
images work quickly cause you will be bleaching your sample quickly.
Set Z slice parameters
For taking images with different Z slices, the slice parameter needs to be set. This way you can
create true 3-D images of your sample. Click on 'Z-stack' button in the scan control window(this
will open Z settings panel). Choose 'Fast XY'. Choose the focus wheel to set the start of z-stack.
Click 'Mark first' in the panel. Now change the focus to go to bottom of the stack and click
'mark last'. Click 'start' to start scanning for different z stacks.
Creating 2.5D topological images
To create a 2.5D topological view of the image, click 'Topo'. This should show the view where
all the z slices are shown together, with a z scale.
You can choose a noise filter from the window panel on the right, choose either "none", "median"
or an "FFT". The intensity threshold can also be set from the panel. A useful function is to
fill holes in the 3D data by "fill holes". Various display options are also available.
Creating 3D images, storing 3D data
The Zeiss software creates a point cloud representation of the Z slices created by the
scanning of the sample. This can be used to create a rendered 3D image of the sample. Various options
for displaying the surface of the objects can be used. Click on "3D" button to open the 3D
image mode. You can choose surface type form basic, advance and full resolution. Also the 3D
image can be rotated to an view. A movie of the 3D rendered views can be saved in the
software too.
An example 3D view of the imaged somata is shown here.
To save the image in the data base, and the 3D data, click 'Save as'. Choose the location of the .mdb
file and choose the database you want to save the images in. The 3D data can be saved as a .txt file
which gives the point cloud of the 3D image.
Switching off the system
If you are the last user of the day, you need to switch the laser off and cover the microscope.
Firstly exit from the control program. Then shut down the microscope from the switch on the left side
of the microscope. In the end, switch the laser off from the key button on the left side, down below.
Do not leave the laser on if nobody is using the microscope after you. The laser has a finite life
time and replacing the laser is an expensive affair. Finally cover the microscope with the Zeiss
cover. Clean up the table after use, and keep any slides back on the rack.
Useful links for more info
Three-dimensional confocal microscopy:Volume investigation of biological specimens
Edited by John K. Stevens, Linda R. Mills, Judy E. Trogadis.
3D Laser Scanning Confocal Microscopy web page at
UBC
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