LSM PASCAL 5 Instructions
Modified from Geoffrey Lambright's Protocols
Axiovert 200 Microscope
Instructions for operating PASCAL 5 Confocal Microscope
If you have problems with the instructions DO NOT continue, please find someone to help you. All
items are labeled with white tape. Call Hui at (415) 476-1081 if you have any question.
A. Start Up
1) Turn on air conditioner on the wall behind the microscope!
2) Turn on the mercury lamp control (white box underneath the table)
Note: Once the mercury lamp is on, it has to be on for at least 20 minutes. Furthermore, if it
has been shut down, you have to wait for at least 20 minutes to turn it back on.
3) He/Ne lasers should be on all the time.
4) The procedure to turn on the Argon Laser (Paying attention to all the steps)
A. Be sure that the Argon laser power switches 1, 2 (in the above graph, Left) is
at 0 and 4 (in the above graph, Left) is at “Stand By” position.
B. Turn on the Argon laser power via toggle switch 2 (in the above graph, Left)
and turn the switch 1 (in the above graph, Left) to position “I”.
C. then WAIT 5 minutes.
D. Toggle the switch 4 (in the above graph, Left) to the position “Run”
E. Turn on Voltage-meter and set to the 2 Volt scale.
F. The initial read-out will be around 300 mV and adjust the voltage from 0.3 V to
0.63 V via the switch 3 (a small black knob, before the indicator turns “Red”)
5) Turn on the microscope. (the power strip is tied to the left leg of the microscope table)
6) Turn on the computer. (Should be on all the time and just log in with your account and
B. SHUT DOWN
1) Close and exit Zeiss PASCAL software.
2) Turn off the microscope via the power strip at the left of the microscope.
3) Turn off the mercury lamp power control underneath the table.
4) Turn off the Argon Laser
A. Turn on Voltage-meter and lower the voltage from 0.63 V to around 0.3 V via
switch 3 (the small black knob).
B. Turn off Voltage-meter.
C. Turn switch 4 to “Stand By” position and then wait for a couple of seconds.
D. Turn switch 1 to “0” position.
E Wait for electric fan to stop (WAIT until electronic fan stops!!!
approximately 5 minutes)
F Turn switch 2 to “0” position.
10) Please write the sign-up book!!!
C. Acquiring Confocal Images on LSM PASCAL 5
1. Follow the Startup and Shutdown procedure.
2. Once the microscope system is fully turned on, you will need to start Pascal software
by double-clicking the "PASCAL" icon on the desktop.
3. At the beginning, it has the screen shown as below.
4. Select “Online Mode” and click the “Start” button to load the "PASCAL" software.
Note: “Offline mode” is for diagnostic and file management only.
5. Once the "PASCAL" program starts, a main menu will pop up, shown as below
6. You first choose the submenu, "Acquire" (the diagram above) and then open three
submenus under "Acquire" by clicking three buttons, “ Micro”, “Config” and “Scan”.(
shown from the left to the right as below)
7. Click the "Objective" button in the “Microscope Control” window and select a proper
objective for your application through a pull-down menu.
8. For the visual observation, you click the “VIS” button on the main menu (Circled in red
9. Click on the “Transmitted Light” button in the "Microscope Control" window and
adjust the light intensity through sliding the slider in the window below (usually, around
10. Set up your sample on the microscope and adjust for Kollher illumination.
11. If you want to observe fluorescence images through eyepieces, click on the “Reflected
Light” button in the "Microscope Control" window and then select the proper filter in the
pull-down menu for your staining.
12. In the configuration control window, you will have two main choices, Single track or
Multi track. After you select "Single track" or "Multi-track" mode, you click the
“Config” button, which is circled in the "Configuration Control" window below.
12. Once you click “config”, a new window will be popped up. You can use the pull-
down menu to select the existing setting (top) or create your own setting by typing in a
new name (bottom).
I. How to set up confocal imaging configuration:
1. You need to adjust the parameters in the "Configuration Control" window for your
This is great website to show you how to optimize your filter setting.
II. Adjustments on the "Configuration Control" window:
A. HFT is for the laser line selection through main dichroic mirror (Beam Splitter) and
you choose all the laser lines, which you will use in your application. Under "Single Track
Mode", you click the button "Excitation" and choose the initial amount of laser power
you want to use and need to be adjusted later on during the imaging process. (Note:
Typical laser powers are as follows: 488 nm (output power, 25 mW) = 5-10%; 543 nm
(output power, 1 mW) = 50-100%; 633 nm (output power, 5 mW) = 50%)
B. NFT is the secondary Dichroic filter and usually is set outside the emission range of
Ch2. After NFT before each channel, there is the emission filters, you choose as the
C. Since PMT only uses Grey scale, right now we need to assign pseudo-color to each
individual channel based on the fluorephore by clicking the channel button and choosing a
color from the color pallete. (Using ChD for transmitted light image)
Finally, if you are setting up your own setting, name this setting as this way,
III. Adjust the parameters in the "Scan Control' window for acquiring high quality
of confocal image:
Until this moment, you should have found and focused your sample through the
eyepieces and set up the filter configuration in the "Configuration Control" window.
Next, we will start to acquire confocal image. The "Scan Control" window includes
"Mode", which allows you to change the resolution, the scan speed, color bit depth,
zoom and; "Channel", which allows you to alter the pinhole size, detector gain and
PMT’s gain and offset, the last one is about Scan mode, including "Line", "Frame", and
"Z stack". You can choose any of these scan mode.
A. You first use "Find" (only work at Frame Size 512x512) to find the initial parameters
for your sample in gain etc. From here, you can adjust for the following three parameters:
B. In the submenu, "Channel", set the Pin-hole size at 1 airy unit and in the submenu,
“Mode”, set “Frame Size” to 1024 x 1024 for 2D image and 512x512 for 3D image
acquisition, Data Depth to 12 bit, Scan Direction to “ ”.
C. Change the laser power through laser control in the "Configuration Control" window or
in the submenu, “Channel” in the “Scan Control” window. Using “cont” mode to adjust
your best image quality, changing “gain” setting for avoiding over-saturation of your
signal and changing “ Amplifer offset” for acquiring the best background under “pallette
Note: Don’t use “Amplifer Gain”!
D. Decrease scan speed (the smaller of scan speed is, the longer time each individual scan
will take and the better quality your image will have).
Note: Aware of the bleaching! Normally, “Scan Speed” was set at 7-8, (3-4 sec for each
frame scan); If image quality is still not good, you can do “Average” up to 4 times or
increase the Laser power.
E. Increase how many times of scans each image will average, the high of scan number is,
the better quality your image will be and the long time the acquisition of your image will
Note: The basic rule is that you should keep the ratio of signal to noise at the level of at
least 2-3 folds. (Object light intensity/Background intensity, judging through the
F. Finally, if your signal to noise ratio is still not good enough, you can increase the size
of pin-hole a bit but keep in mind that this will result in the loss of “confocal-ness”
through pin-hole size control in the submenu, “Channel” in the “Scan Control” window.
If you do satisfy with your initial image, you need further adjust detector (PMT) gain,
Amplifier gain and offset based on the histogram of your image. (Only need minor
adjustment and with more experiences, you will get better at it)
G. If you are doing multiple channels in “Single Track” or “Multi Track”, adjust each
channel as above.
H. Using "Fast XY" to find the best focused plane and click on "Single" to acquire a
D. 3D imaging
1. To acquire 3D images, first you must find your sample as you would to take a normal
2D image. This means, you need to set in every channel you want to use; you will need to
optimize each channel for the particular sample you are looking at, then you may begin to
take a z-stack.
2. So after getting your imaging parameters set, select the z-Stack button in the “Scan
Control” window. This will bring up all new options for acquisition of a z-stack. Note
that once the window is open, you will see the Z Settings area which has 3 tabs of its
own, Z Sectioning, Mark First/Last and Hyperline Z Sectioning. You will need to select
Mark First/Last in order to continue forming your image. From here, you will want to put
in the distance between individual images the laser will scan. The smaller the distance the
better the image; however, the smaller the distance the more images that will be taken. If
you take a lot of images, this will take a very long time to scan (upwards of 20 minutes
per stack), your files will be enormous (up to hundreds of MB) and there is a chance of
bleaching before you make it through the whole sample, rendering your image useless. So
you will need to first find where the beginning of your sample and the end of your sample
are before deciding the interval. To do this, you will need to move the focal plane in a
respective direction and hit “Mark First”. Then you may go to Move to, “First.” This
will show you where the focal plane lies in your sample. Once you have reached a point
where the focal plane is near the end of your sample you can remark it as first and leave
it. Now do the same thing for the other direction. But you will be marking this direction
as “Last.” Once you have both “First” and “Last” marked, not the total size of you
In the image above, the sample region is -50 microns to 45 microns, making this sample
.95 microns thick. At an interval of .05, you will have 19 images to put together for the 1
stack. For average scans, you will want between 12 and 20 images comprising your whole
stack. After your first and last are marked and you have your intervals set, select “Start”
from the side menu to being your scan.
3. Once you are finished scanning, you will want to make this Image 3 Dimensional. To
do this, you will need to go to the top Menu and select the tab for “3D View”. This will
bring up a subset of buttons and options, you will need to select Projection at that time.
Once you have selected Projection, this is the new window that will appear. Here you
will begin to build your 3D stack. First thing to note is that you have a choice of view for
this projection, the x-axis, y-axis, and z-axis. Use the best one that shows the data you
need to view. Next thing to do is select the “Preview” box at the bottom. This will allow
you to see what you are working with as a projection. The number of projections you
want to use to make your image affects the clarity of the whole thing when you are done,
but also the size. If you want to make a whole 3D stack flat, then select 1 Projection.
This can be useful if you want to view a whole nerve that runs through a section of tissue,
that isn’t in cross section format. Once you select the number of projections you want,
you will want to move the Circle around to tell the computer how much to rotate your
image when animated. Half of the circle, as shown above will have your image rotate
180 degrees, then go back. If you complete the circle, the image will constantly rotate in
1 direction. When you are done preparing your 3D stack, hit the “Apply” button to have
the algorithm compile your image. Once the image is compiled, you may use the Object
window to animate and view your new 3D image. Save this image .
E. Using Batch Export database
To export these images, go to the Macro button on the top menu and select, “Batch
Export”. A window will appear asking you to locate the database of images you wish to
export. Find your files and select them. Highlight all the images you want to export and
make sure you note the following. The image file type (default is LSM-4 TIFF), which
you want to be either .JPG or .TIFF. Also note if you are exporting a single image or a
series (3D Stack). Finally, note the channels being exported. While imaging, you had
the red and green laser lines in certain channels while you made your image. Ensure that
the right channel for the right color is being exported.
A. Open “Macro” menu, choose “Batch Export” and open your image database.
B. The Batch export window will be like the figure above and highlight the files which
you want to export. Without choosing “monochrome”, you will export merged image
only. If you want to export each individual channel , you choose “monochrome” and each
channel will be exported separately as shown below.
C. Finally, click export file name for a new name and “Start Batch Export”