Instructions for the Confocal Microscope
Susan J. Kohler
Nov. 18, 1999
Revised Jan. 26, 2000
It is important that you sign the log book so that we may keep track of the use of the microscope.
You must turn on the various components of the confocal system in the proper order. Follow the numbers on the devices. Numbers in parentheses are optional, depending on which lasers you plan to use. If you do not need a particular laser, don’t turn it on. The proper order is: mercury lamp power supply, microscope, computer monitors, scan electronics, HeNe laser (turn key), computer. Wait for the computer to start, and log on as tcs_user with the appropriate password (neuron). Then turn on the Argon and Krypton lasers. To do this, make sure the knobs are fully counterclockwise, then turn the electronics on with the big red button. Once you hear the fan, you may turn on the laser itself with the ignition key (turn fully clockwise, then release to the ON position). Look to see that the little yellow light comes on. Then turn the knob to the "9 o’clock" position to adjust the laser power.
Double click on the TCS_NT icon to open the confocal imaging program. When the program has finished loading and the hour glass cursor disappears, click on the Acquire button. A window will pop up and ask you for a filter choice. There is a list of fluorescent dyes or dye combinations to choose from. Choose the dye appropriate for your speciman. FITC is fluorescein and RITC is rhodamine. The chlorophyll entry works well for chlorophyll autofluorescence. Custom filters may be made; see Sue Kohler for help on this. Once the acquisition window is set up, go back and observe your sample directly through the microscope.
Place your sample on the microscope stage and secure it with the two clips. Remember that this is an inverting microscope, so the cover slip must go down. Be sure to use #1.5 cover slips for best results.
Use the lower button on the right to lower the nosepiece, then use the buttons on the left of the microscope to choose the 10X objective, or higher objective of your choice. The higher objectives require the use of oil.
Use the upper button on the right to raise the nosepiece until the objective is close to your sample.
Adjust the beam stops and filters for visual viewing. Pull OUT the stop on the left, push IN the stop on the right, and set the filter wheel on the left to the appropriate filter.
The focus knob has electronically-controlled coarse/fine adjustments. Use the STEP button on the front of the microscope (extreme right) to adjust the step size. The current step size is displayed, and ranges from S0 (superfine) to S3 (coarse). Set the control appropriately, and focus on your sample.
Once the focus is set, use the button on the front of the scope to record this as the zero position. Do this by depressing the up arrow key until the message Set ? appears. Then press the same button again until 0 uM appears.
Change to a higher objective if desired. If you use any objective higher than 10X, oil is needed. Use ONLY the oil provided. The proper technique for applying the oil will be demonstrated. Verify that your sample is still in focus.
Push IN the beam stop on the left, pull OUT the stop on the right, and set the filter wheel on the left to SCAN. The upper wheel should normally be on BF (bright field) and the ICT/P bar should be in the out position. Make sure that the microscope head is in its upright position and not tilted back.
With the acquisition open, follow the flow of buttons from left to right.
Open the filter window. This shows you the laser excitation lines, the number of active detection channels, and the detection bandwidths. These are all adjustable. If you change them, make sure that the dichroic mirror which is chosen is consistent with your adjustments.
Set the lens. It is important for you to tell the software which lens has been chosen on the microscope.
Check the pinhole size. You can view the value of the pinhole size by looking at the parameter list next to the image display. The proper default value is 1.00. Sometimes this changes due to a software bug. It can be reset to 1.00 by pressing the F9 key on the keyboard.
You may change the Mode & Format if you desire. The defaults are XY mode and 512x512 image size. Usually the defaults will suffice.
Set the signal range. Click on the big scan button to start scanning. Depending on the way you have set up the filters, you may be observing one, two, or three channels. Use the big PMT knobs to adjust the photomultiplier gains to see images. You must also use the Offset knob to adjust the dark level. These adjustments will be demonstrated. Stop the scanning by clicking on the Scan button. Note that the image is rotated 90 degrees clockwise from the direct view through the microscope, and the field of view is somewhat reduced. If you need to reposition your speciman by moving the microscope stage, turning the upper (big) knob counterclockwise moves the image up and turning the lower (small) knob counterclockwise moves the image to the right.
(If you want a single scan of the current focal plane, skip the Z positioning step which follows, and continue with Set Scan Parameters, setting the # Sections to 1.)
Set the Z Position. While still scanning, use the big Z POS knob to move up in your sample to the starting position for your z scan. Then press the Begin button on the left screen to register the starting position. The button looks grey before a value has been set and white (depressed) after it is set. Then use the Z POS knob to move down in the sample to the final position. Depress the End button to set this position. Now click on the Scan button to stop scanning. If you "run out of room" with the Z POS knob and the distance you need to cover is less than 176 uM, see the notes about Strategy for a Complete Z Scan which are posted on the wall. If you need to cover more than 176 uM, see the notes for a Z Wide scan.
Set Scan Parameters. Click on the S Accum button to set the number of times to scan each slice. Click on the # Sections button and set the number of sections you wish to acquire. If you choose User Defined, you can set the number of sections to fit your needs. You may have to compromise either the number of sections or section thickness to accommodate your Z depth.
Check the pinhole size again. You may need to reset it to 1.0 again. You can also reduce it to get better resolution at the expense of light intensity or increase it to get better light intensity at the expense of resolution.
Scan the Series. Click on Series to perform the scan. As a rough guide, 1000 passes at medium speed (the default) and 512x512 size take approximately ˝ hour. That would be something like 62 sections with 16 accumulations of each section.
ZOOMING: If you wish to zoom in on a region you may either switch to a higher objective or use the Zoom function. An easy way to zoom is to use the graphic method. Click on the button to the far right of the acquisition controls. A box will appear on the image display. You can drag this box by moving the cursor near the border of the box until it changes to two double-headed arrows in a cross. When you see this, press and hold the left mouse button and drag the box. Similarly you can resize the box when a single doubleheaded cursor appears. The zoom will be effective when you start scanning again. You can use a single image or a projection image to set up the graphic zoom.
Use the buttons on the monitor on the right to control how the images are displayed. You have choices about the number of channels to display (Single vs. Tiled), as well as whether to display a single image or multiple images (Gallery).
For maximum viewing options, choose View, Pseudo-3D, or 3D from the main button bar. From the View page, you may combine all of the sections into a single image with the Proj. button. You may also view your sections in movie mode. The Pseudo-3D page allows you to select XY, XZ, and YZ cuts through your data.
To print the image(s) on the display screen, hit the Print button at the bottom of the buttons which control the display. The image display will pop to full screen and another print button will appear in the upper left. Press it. Then click on Properties on the next screen and set the paper size to letter. You can choose either the default printer for black and white, or the Fujii printer for color glossies. Click Okay. A window will pop up saying that the job is in progress. Do NOT press the button on this screen, since it is used to cancel the job!
The saving options are a little bit tricky! You should first use the mouse to click on the images you wish to save to make sure that they are in the active window. The Save As option (under the main File menu) will save the entire data file. The Save Selection option saves only the highlighted image(s). You should also pay attention to the formats which you can use for saving. Saving data as a ScannerFile saves images in a multi-dimensional TIF file which is readable by the Leica confocal software but not by other software. Saving in TCS Export format creates a whole series of TIF files which are readable by other software but not by Leica confocal software.
Make sure your data are saved.
Click on the button Main to get back to the main window. Then choose Exit Program.
Use the CD Creator program to make a CD of your data.
Turn off the argon and krypton lasers by turning the knobs fully counterclockwise, wait a few seconds, the turn the keys to the off position. Do NOT turn off the big red power buttons yet. The fans must run for 15 MINUTES longer.
Shut down the computer by holding the cursor over the Start button and then selecting the Shutdown option. The computer takes a long time to shut itself down, and you must finally push the power button to turn it off when it tells you that it is ready.
Shut down the HeNe laser, then the scan electronics, then the monitors.
Clean the excess oil off of the microscope objectives (Just wipe off the excess oil with a piece of lens paper. This will be demonstrated.)
Move the 10X objective into the viewing path, lower the nosepiece, and turn off the power.
Turn off the mercury lamp power supply.
Cover the microscope.
After 15 minutes, turn off the power for the lasers to stop the fans.
Sign out of the log book.
You may access the computer for the confocal microscope remotely by looking in Network Neighborhood. Choose Leica_TCS_NT. After a minute, it will ask you to log on. The user is TCS_user, and you must supply the password. Look in the Data folder, and then your subfolder. This only works if the Leica computer is turned on! You can also drop your data in the 24 Hour Drop.
You can focus the microscope and move the microscope stage to reposition your speciman while doing confocal scans.
You can use the F9 keyboard key to reset the pinhole to the default value of 1.00.
You can see the effects of averaging by doing a series scan when only one of the start or end positions have been defined. The system will take just one section with the defined number of accumulations.