Standard Operating Procedure
Carl Zeiss Inverted TIRF Microscope
Tool Manager: Donald Lee
TM e-mail: [email protected] TM Phone #: 510-512-2807
Daniel Group Contact: [email protected]
You must be trained to use this equipment and be familiar with the SOP before using it
unsupervised. Incorrect usage may lead to injuries and damages!
Safety Warning:
 You must complete all safety training required for this lab. Training listed is on the door.
 3B Lasers – eye damage possible. Laser safety goggles are located at the lab entrance.
 Moving stage – careful of injuries.
Equipment Damage Warning:
 Only wipe objectives with lens paper. Do not apply pressure on the lens.
 Keep loading solutions only on the staging area (right side of black stage).
 Keep large solutions away to prevent catastrophic spillage damage.
 Do not over-focus objective to the point that it pops out the sample holder.
 Do not bend fiber optic cables (silver-colored wiring on right side).
Equipment Sharing Policy:
 Many people’s projects depend on a properly working scope - so please be careful!
 Please reserve the scope 12 hours in advance through our Google Calendar. Contact the
Tool Manager for access to the calendar.
 If you make/cancel an appointment before 12 hours until usage, please notify the Daniel
Group so that others can change their plans.
 Document and report any malfunction to the Tool Manager.
 For immediate assistant, ask anyone in the Daniel Group for help (Olin Hall 265).
Startup procedure:
1. The microscope should already be ON. If not,
a. Turn on Microscope Power Supply box.
b. Turn on the microscope (circle silver button on left side towards back).
2. Turn on Laser Generator box (switch is next to power cord).
3. Turn on Stage Power Supply box. This is for the automated stage.
4. On the Laser Control Box,
a. Turn the laser safety key interlock to ON position.
b. Turn on the lasers you want to use.
5. Turn on Camera Power Supply. Wait at least 20 min for the camera to warm up.
6. Turn on Input/Output Controller. This controls camera-software timing.
7. Turn on Definite Focus Controller. This allows you to keep focus while moving stage.
8. Turn on TIRF Controller. This controls the laser power.
9. (Optional) Turn on EXPO mercury lamp. This provides white light.
Sample loading procedure:
1. On Microscope Control Box, do the following:
a. Home  Load position  Set work position. This lowers the objective lens.
b. Control  Reflector  74 HE GF/mRF
Control  Objectives  100x Oil.
2. On the 100x objective, drop immersion oil (Immersol 518F) onto lens without letting the
dropper touch the lens.
3. Load your sample onto the stage. Note: Wipe the glass slide before loading. Make sure
the sample holder is securely fitted inside the stage. Sometimes the holder pops out.
4. Use the coarse focus adjuster knob to bring the oil on the lens in contact with sample.
Stop immediately when oil spreads. Then use finer adjuster knob to spread the oil evenly.
5. Use joystick to move lens to the correct place. Note: If the stage moves too fast/slow,
push the joystick button to change the speed.
6. Place the laser safety cover on top of the stage.
Data acquisition procedure:
1. Start the AxioVision software on the computer.
2. Go to Microscope  Microscope
a. Under Reflected tab, select “74HE GFP/mRFP”.
b. Under Laser tab, set laser power based on how bright your samples are.
c. Set angle using this button
and set it 70 degrees.
WARNING: Do not use
, otherwise laser will point towards you and could
hurt your eyes!
d. Turn on laser and make sure the lasers are visible on the stage.
WARNING: Do not look at lasers closely when checking! It should be visible
through the laser safety cover.
3. Go to Hardware
a. Change camera exposure time. (50 - 100 ms is okay.)
b. Enable EM-CCD. This enhances signal.
Note: If camera is oversaturated, it will turn off and make an alarm noise. Uncheck
and Check the EM-CCD checkbox to turn off the alarm noise.
4. Go to Live View.
a. Select the image contrast option. Min/Max or Best Fit.
5. Find focus using Live View.
WARNING: Do not over focus! Can cause damages to lens. Visually check the lens
frequently when focusing for the first time.
6. Go back to Hardware. Go to Definite Focus tab and set it to ON. This keeps the sample in
focus. Confirm that autofocus is working through Live View.
7. When ready, exit Live View.
8. To record a picture, click Snap Shot.
9. To record a movie, go to Fast Acquisition.
a. Go to T tab and set time interval in between images and time duration.
b. Go to C tab Edit Hardware Setting. Change your laser setting for before and
after each image.
c. Go back to Experiment tab. Change file name and save settings if you want.
d. When ready, click Start. This opens up the acquisition box.
e. Click start again when ready. Check to make sure images are being acquired..
f. When done, click on “Start Cutter”.
g. Then click on “Convert All”.
10. Save your video file as .zvi file. You can view files using ImageJ software.
Changing sample procedure:
1. Make sure to disable Definite Focus in AxioVision, otherwise objective will not move
down.
2. On the Microscope Control Box, go to Home  Load position.
3. Take off the laser safety cover.
4. Change sample. Make sure there is some oil on lens. Add more if needed.
5. On the Microscope Control Box, push
.This raises the objective back to the last
known position. Make sure oil spreads.
6. Put back the laser safety cover.
Shutdown procedure:
1.
2.
3.
4.
Disable Definite Focus in AxioVision.
Go to the microscope control box  Load Position  Set Position.
Unload sample.
Wipe off oil from lens by using lens paper and gently brushing over it. It is okay to leave
some oil behind.
WARNING: Do not directly apply pressure over lens, as it may get scratched.
WARNING: Do not wipe lens with solvent as it may dissolve the lens glue.
5. Turn off all equipments EXCEPT the Microscope Power Supply and the microscope.
6. Clean and disinfect microscope staging area.
WARNING: Must not leave any salt solution behind or corrosion may occur.
7. Clean surrounding area and leave no samples behind. This area will be cleaned out if no
one is there. To preserve samples, cover it, label it, write your contact info, and put it
away from the scope to prevent accidental bumping.
Accidental Spill on Microscope:
 If small volume spill occurs on stage:
1. Wipe dry with paper towel.
2. Use 70% ethanol to disinfect and also dilute out salt in spilled solution. Dry again.
3. If spill reaches hard-to-reach areas, let the tool manager know.
 If small volume spill occurs over objective lens:
1. Use lens paper only to soak up liquid. Do not press on lens, just brush over it.
2. Dab 100% ethanol onto lens paper, and then gently wipe over spilled area.
Important that no salt solution dries on objectives.
 Large volume spill (10 mL +) over microscope components:
1. Turn off microscope to prevent short-circuiting problems.
2. Get paper tower to soak up most of the spill and prevent leakage into electronics.
3. Contact tool manager immediately to get the leak cleaned up.
4. Explain how the spill happened so that more spill guards can be added if needed.
5. Buy TM coffee & cookie to eat outside lab. The TM will be able to work better to
fix the equipment quicker.
6. Be careful next time! Accidents are expected. Complete negligence is not okay!
Troubleshooting / Common Problems
 Cannot move objective up or down
1. Restart microscope by turning it on and off. Also restart AxioVision.
 Lasers will not turn on
1. Check that both the TIRF and Safety LED are lit on the right side of the
microscope, where the silver fiber optics comes in.
 If not lit, then make sure the magnets on the stage cover are touching the
stage corners properly.
2. Check that you are using the right reflector to allow the laser through.
3. Check to see if laser safety interlock key and laser switches are on.
4. In AxioVision, go to Hardware  Microscope. Make sure the lasers setting
modulation is not 100%. This means to cut laser power by 100%, hence, no laser
comes out.
 AxioVision froze
1. This is caused by a software glitch. Sometimes caused by having Live View on
while recording data with Fast Acquisition.
2. Save files you want to keep.
3. Restart the software.
 Can’t move the objective to the “Load Position”
1. Make sure Definite Focus is disabled in AxioVision. The autofocus feature
overrides the Load Position feature.
 Can’t find focus
1. Make sure you are using the right laser and reflector for your fluorescent sample.
2. If using microfluidic channels, make sure you loaded the right sample with
fluorescent molecules and that you are in the right channel.
3. See if the the laser is totally internally reflecting (should not shine laser above your
sample).
4. Use a higher laser power to detect any fluorescence signal.
5. Check the camera setting. EM-CCD should be enabled and increase camera
exposure time (100 ms usually works).
6. If all above fails, ask for help.
 There is a huge delay in recording images.
1. Turn on the Input/Output Controller box. Restart software.
 Uneven focus (one side is focused, the other is not)
1. Check to make sure the sample is securely and flatly placed in the holder.
2. Check to make sure the stage holder is securely fitted into the stage. This holder
can pop out some times.
a. If it is popped out, lower the objective so that the oil doesn’t contact it.
b. Move the stage holder in place.
c. Bring up the objective back up and find focus again.
Updated Nov 20, 2014