This SOP is published only for a social media discussion. The author does not take any responsibility for the utilization of this procedure. The system discussed here is a customized two-photon microscope, based on a Coherent Chameleon Vision 2 and Leica SP5. The optical path is fully enclosed and the SOP is written for maintenance.
Basic rules
1. Align laser with a lid room (smaller iris, smaller damage to the eye)
2. Never align eyes with the height/direction of the laser beam
3. Use the most appropriate personal protective equipment such as goggles and a white lab coat
4. Perform laser alignments with the least number of people present in the room. Ideally, alignment is a 1 person job and a colleague is aware you are performing this task, within core hours
5. Use devices such cards, cameras and viewers to visualize the laser beam
6. Take short breaks every ~45 minutes of work. Do not continue alignment if too tired. Alignment of optics can be a stressful and lengthy procedure; try to identify the right moment to take a long break to relax
7. Alignment is carried out only by authorized users
PPE for Ti:Sapphire laser
– VC5 IR card viewer from Thorlabs. WARNING: card viewers reflect part of the laser beam. Therefore, they must be used with caution, strictly using protective goggles, directing reflection away from the eyes
– Hand-held IR viewer from Newport. WARNING: hand-held IR viewers limit dexterity and must be used always with protective goggles.
– LG9 Amber lenses from Thorlabs. OD5+ on the 720-1090nm range; OD7+ on the 750-1064nm range. WARNING: goggles never fully protect from direct high power laser beam.
List of authorized users
Alessandro Esposito (MRC Cancer Cell Unit)
Coherent’s field engineer can align the laser under their own responsibility. Coherent’s field engineer can align the beam path until after the Pulse-Picker. The rest of the optical path must enclosed at any time or isolated with a beam stop.
Leica’s field engineer can align the complete beam path under their own responsibility with the exception of the Pulse-Picker. Alterations of the beam path have been discussed with Leica representatives.
Standard Operating Procedure
Room preparation
1. Show warning at the door
2. Lock the door
3. Switch on the system as needed (shutters ON)
4. Wear PPE as appropriate
5. Open the beam path as needed (keep lens tube arriving to the scan-head until the last moment)
6. If a large section of the beam path is opened, always block the laser beam with the beam stop after the optical element that is aligned in order to avoid the laser beam being reflected in dangerous directions (eye, skin, fire hazard) when misaligned
Beam alignment
7. Always activate laser shutter when the beam is not undergoing alignment
8. Apply #6 every time a section of the laser beam is aligned
9. Start laser alignment, proceed with pairs of mirrors from the position closer to the laser up to the scan-head, trying to operate the laser beam within a central part of the mirrors
10. Always ascertain that all optomechanics is stably connected to the optical table and that no optical device can fall, tilt, flip…
11. Re-aligned section should be covered (at least temporarily) while progressing towards the scan-head
12. When arrived at the EOM, remove the device (Leica’s shutter and half-plate may be removed as well). WARNING: the entrance window of the EOM is located within a brass cavity. Upwards reflections of the laser are possible.
13. Using irises, make sure the laser beam is parallel to the table
14. Reposition the EOM, coarsely aligned to the laser beam. WARNING: after the EOM there is a periscope. Use a beam-stop before the periscope, beam reflection towards undesired direction is otherwise possible.
15. With a power meter, measure power of the laser before the EOM. Relocate the power meter after the EOM and iteratively maximize power through the power meter with the EOM in “high” state.
16. Coarsely align the periscope if necessary, then reintroduce Leica’s shutter and half-plate if previously removed. BE SURE the periscope is locked to the optical table in a stable manner.
17. Remove lens tube and MFP cover.
18. Install Leica’s alignment tool on the scan-head
19. Iteratively align the front iris of the alignment tool and the back aperture of the alignment tool.
20. WARNING. During the iterative alignment of scan-head, PPE is usually hindering an already lengthy procedure. Avoid removing PPE. Check actions to be taken.
21. When the two apertures are aligned, start scanning trying to see fluorescence from a bright sample on the screen. Keep adjusting alignment and MFP screws until alignment is completed.
Preparing the room to normal operation
22. Close the optical path. Before securing all covers and panels, check that the alignment is still ok.
23. Secure all safety panels
24. With the laser ON, shutter OFF and during scanning, verify with the IR viewer that no beam is exciting the enclosed laser path.
25. Remove safety warning on the door and operate equipment as normal.
