• Before use read basic_use_of_2p_system paying particular attention to the safety instructions.
• The main shutter of the laser must always be closed (either by the automatic laser interlock but the door of the hood or manually during e.g. alignment procedures) before manipulating anything and opening the hood. Laser light is incredible dangerous for the eyes
• Also, take special care of the laser, the PMTs and the objective. They are very expensive objects that can easily be damaged – in the case of the PMTs, very easily by excess light.
• Even though this manual aims to cover all the information needed to run the 2P, one should always ask for training before trying to image alone

There are two ways to check and close the main shutter of the laser.

1. preferred method Use the Chameleon Vision interface on the Windows computer (GLAT transit software icon link on Desktop if not open). In the system control tab of Chameleon Vision, check that the shutter is closed. If the shutter is open (and the laser is on or on stand-by), you see a yellow triangle. If that is the case, close the shutter. - since moving to 3N390 the software interface can't detect the laser, only the spectrophotometer, main shutter is controlled by button on laser controller
2. On the laser controller, there is a button 'Shutter open' and a LED that turns on when the shutter is open. Press the button to close the main shutter.

1. Turn on the Windows computer on (login: Jefferis Lab with labpassword).
2. Open Chameleon Vision (GLAT transit icon on Desktop). - currently only reads spectrophotometer data, not essential
3. Switch the laser from 'stand-by' to 'on'. (The main shutter should be still closed)
4. Turn on the imaging hardware on the rack with the PC screen:
5. Turn on the MP-285 micromanipulator controller (and press the green button on the right - this used be necessary, not anymore).
6. Turn on the MDR-6 galvo scanner controller
7. Check that the two channels on the PS-2 are set to 'off'.
8. Turn on the PS-2 PMT amplifier
9. Turn on the Pockels cell (under the table, Conoptics box)
10. Turn on the epi light pathway:
    1. Turn on the LED controller using the Thorlabs cube knob on the table (it says Blue light).
    2. Open the shutter on the Olympus filter wheel.
    3. Switch both servomotor controller to wide-field (WF). (next to keyboard of the PC)
11. Put your sample under the objective with minimum amount of solution needed to avoid spills on the equipment.
12. Check that the sample is lower than the objective.
13. Turn off the main light in the room.
14. Turn on the sign 'Laser in use'.
15. Locate your sample:
    1. Lower the objective to about 2mm above the sample by eye.
    2. Ensure the objective is as well centered on the sample as possible.
    3. Add a drop of saline solution or water.
    4. Open MicroManager on the Mac computer, and load the config file of the Grasshopper camera (should be default)
    5. Click on Live.
    6. Use the XYZ controller to move the objective and locate your sample. The controller has a fast and slow mode. Press the red button '' to switch to slow mode; an LED will light up in slow mode.
    7. Focus in the middle of your sample.
16. Turn off epi light pathway:
    1. Turn off LED controller using knob.
    2. Close shutter on filter wheel.
    3. Remove any objects that would block the hood from closing and CLOSE THE HOOD.
    4. Switch both servomotor controller to '2P'.
17. On the Windows computer, open Matlab, type and run scanimage.
18. Select MachineDataFile_Pockels.m which will run a Pockels Cell calibration and open a GUI.
19. Open the laser light pathway:
    1. Open the main shutter on the laser controller / in Chameleon GUI
    2. Switch the two channels of the PS-2 to on. Indicated values should be between 0.400 and 0.450.
20. To view your sample, click on 'FOCUS'.
21. check the power meter standing on the laser, this indicates the laser intensity permitted to go through by the Pockels (that reaches your sample)
22. Move the objective 80 microns up to locate and focus on your sample. The focus of the laser light is slightly higher than the focus of the epi light. However, be VERY careful when lowering the objective to avoid touching the sample or anything with the objective, always operate in slow mode while using the 2P. Be aware of the anatomy of your sample, e.g. if you reach the esophagus of the fly you shouldn't go too much further down.
23. When you are happy with the imaging plane, PMT gain, and laser power, click Abort to terminate the Focus acquisition on ScanImage.
24. In ScanImage select a folder to save files to, otherwise it will abort any effort to do this
25. To save images, click on 'GRAB'. Enter a name for the images (e.g. YYYYMMDD_something_).
26. For external triggering tick the box External Trigger and send trigger pulse from Igor (Mac) to start Grab or Loop acquisition
27. Igor can send a trigger by running e.g. Preview (and selecting an ODD file, depending on your settings)
28. you can select 'LOOP' instead of 'GRAB' to image multiple sweeps; these can be all triggered externally by running a multi-sweep protocol in Igor

1. Close the main shutter of the laser.
2. Switch off both channels on the PS-2.
3. Switch light path from 2P to wide field with servo-motor controller
4. Lift the hood until the click.
5. Turn off 'Laser in use' sign.
6. Turn on main light in room.
7. Move objective away from sample using the x-y-z controller.
8. Remove objective with lever - currently not used at 2P1 to have more space at the side of the camera
9. Remove saline from sample to avoid spills when removing the sample.
10. Remove sample.
11. Clean objective :
    1. Rinse it abundantly with water from all sides while holding a beaker underneath the objective to collect the water.
    2. Wipe the objective with a sheet of Thorlabs objective paper. No scraping, only a single wipe in one direction.
    3. Repeat the rinse and wipe with a clean objective paper.
12. Close matlab.
13. Switch off PS-2.
14. Switch off MDR-6.
15. Switch off MP-285.
16. Switch off pockel-cell.
17. Switch laser to 'stand-by'.
18. Copy data from computer to hard-disk and delete it from computer.

• Main: not needed
• Prompt: not needed
• System Control: shows the power spectrum of the laser
  • controls main shutter of the laser
  • adjust the wavelength of the laser
  • adjust power of laser (should be ~2100mW)
  • can lock the front panel of the laser
• Tunes: enables jumping between wavelengths, normally not needed.

• Focus: Acquires images but does not save them.
• Grab: Acquires and save a set of images.
• Loop: Repeats 'Grab' a given number of times.
• Channel 1 is 605/75nm - for any red fluorophore, JF549, mCherry, tdTomato, RFP
• Channel 2 is 525/70nm - can be used for GCaMP, mVenus, GFP

• The chiller solution needs to be replaced every 3 months (→ Erika).
• In order to keep track of the laser performance over time a “Data Run” should be performed every time the coolant is exchanged (Erika). As sweap parameters use 10nm and 10sec.
• The crystals of the desiccator need to be replaced when they turn from blue to purple.

1. Check whether the x-y-z controller is in slow or fast mode.
2. Check that the MP-285 indicates x-y-z positions. If it does not, press the green button again. If that does not help, restart ScanImage++ or matlab and press the green button on the MP-285.

1. Check that the epi pathway is fully open:
   1. LED controller switched on.
   2. Shutter on filter wheel is open.
   3. Both servomotor controllers are switched to 'WF'.
2. Check that your sample is still there.
3. Change the focal plane by moving objective. Be careful when going down.

1. Check that your laser light pathway is open:
   1. Main shutter is open.
   2. imaging shutter is open (clicking sound when you press FOCUS)
   3. Both channels on the PS-2 are switched on.
   4. Both mirrors are set to 2P on the servo-motor controller
   5. Laser is on (not in 'stand-by').
2. Check that your objective actually moves.
3. Check that you have sufficient laser power (power meter is placed after Pockels)
4. The laser might be misaligned, alignment requires training
5. Change the focal plane. Move downwards and upwards but be careful with the objective!
6. Switch back to epi light and check that your sample is still in place:
   1. Close laser main shutter.
   2. Turn both channels on PS-2 off.
   3. Open hood and return to epi light pathway.
   4. Check the sample is in place and in focus.

• Laser (Chameleon GDP 1185894.7308): head, pump laser and piezo mirrors
• Laser controller Coherent MRU X2: controls the wavelength [680-1080nm], power, and main shutter.
• Desiccation unit MRU X2 containing crystals. A green LED indicates functioning. The crystals should be blue. They will gradually turn purple and then need to be replaced.
• Chiller containing cooling fluid for laser. It should work at all times (visible movement or bubbling of liquid), and the temperature should be around 20°C (not approaching 30°C). Do not block the ventilation.
• Tubing from chiller and laser: do not step on it or place something on it, it might prevent correct circulation of the cooling fluid.
• Beam splitter to devide the laser beam into two 50% beams, one for each 2-photon microscopes.
• Polarizer and half-wave plate with beam dump.
• Thermal power meter receive part of beam (?%)
• Galileon telescope to enlarge the beam
• Pockels cell (ConOptics, model: 302): electronic control of a crystal, which determine how much laser light goes to a beam dump and to the sample.
• MP-285: controls the power of the motors of the objective in xyz.
• MDR-6: controls the power of the galvo-mirrors
• PS-2: controls the power of the PMTs
• dichroic mirrors ( IR light goes to sample but cannot return, light emitted from sample goes to PMT)
• High-load objective scanner P-726.1CD https://www.physikinstrumente.com/en/products/linear-stages-and-actuators/piezo-stages/p-726-pifoc-high-load-objective-scanner-200380/ controlled by PI-E665 Piezo amplifier
• Objective: 20X, water immersion, WD ~2-3mm?,
• 2 photo multiplier tubes (Hamamatsu: H10770PA-40 & ?)
• Optic cables: do not bend them, it can break the optic fibers!