Imaging Methods
Fiber bundle & adaptor parts list
Instructions for adaptor construction, use, and imaging:
Adaptor construction
1. Print and post-process the appropriate .stl files (Fiber_xxx.stl, base_xxx.stl) on the “Projet 3510 HD Plus” 3D print.
2. Take the “Fiber” component and, by hand, tap the side hole with the “General Purpose Plug Chamfer Tap TIN Coated HSS000-120 Thrd Size, 9/64" Thrd Length”, and clear out center length-wise with the “Uncoated High-Speed Steel Drill Bit, 68 Gauge Size, 1-3/8" Overall Length” drill bit (the thinner drill bit).
3. Using the T1 screwdriver, lightly screw in one of the “Button Head Torx Screws 18-8 Stainless Steel, 000-120 Thread, 1/16" Long, Packs of 100” (the shorter of the two screws).
4. Take the “Base” component and tap the side holes again with the same tap. Clear out the center length-wise with “Uncoated High-Speed Steel Drill Bit, 62 Gauge Size, 1-1/2" Overall Length” (the thicker of the two drill bits).
5. Using the T1 screwdriver, lightly screw in two of the “Button Head Torx Screws 18-8 Stainless Steel, 000-120 Thread, 3/32" Long, Packs of 100” (the longer of the two screws.
6. Repeat the previous steps for as many desired pieces (one Fiber component for each fiber tip, and one Base for each GRIN lens/mouse). We recommend making extras of the bases because they are handled the most and will wear out over time.
7. Place all components in a tube with 70% ethanol and shake. Take out each component and one-by-one use a can of compressed air to clear out debris from all holes.
Adaptor use and imaging
1. Take the “Fiber” adaptor component. Unscrew the screw so that it is only barely in (the center of the component length-wise should be totally open and clear).
2. Very gently insert a fiber bundle tip through the component until the tip is just barely below the edge of the adaptor. Just like you don’t want the GRIN lens poking out of the end of its metal sleeve, you don’t want the fiber bundle tip poking out of the edge of the adaptor. Used this way, the adaptor is designed to protect the fiber tip from scratching against the lens below it.
3. Once in place, very gently tighten the screw until you feel the slightest bit of resistance. You just want the screw to hold the fiber in place within the adaptor. Screw too tight and you risk cracking the fiber.
4. Repeat 1-3 for each fiber. These can be left on the fiber bundle permanently.
*If fibers are already prepped, you can begin following instructions here.
5. Turn on the camera and wait for the light to stop blinking, then turn on the computer.
6. Turn on the laser. A power of 0.25-0.5 mW at each fiber tip is typically enough to get clear enough images (verify with a power meter if first time), but feel free to adjust if contrast is too low.
7. If it is the first-time imaging in a while, you want to ensure that the laser is uniformly illuminating across the three fiber bundles and you may want to adjust the laser alignment. To do this, remove part CXY1 (see “Fiber bundle design”) from in front of the objective, and project the laser onto a dark surface or wall (be careful). Turn the knobs on part KCB1/M to adjust the laser. The goal is to see symmetric, concentric circular spots on the surface. Return part CXY1 to be in front of the objective.
8. Start the imaging software (PVCAM). Below are the settings we use to get a framerate of 34 FPS. Adjust exposure time as needed for different framerates. I choose to bin pixels 2x2 because we typically get fine enough resolution and I want to maximize SNR while minimizing power delivered, but feel free to not use.
9. Make sure the fiber bundles are in focus. Adjust CXY1 horizontally until all edges are sharply focused. (Once assembled, you should not adjust the tube lens to focus).
10. Head-fix a mouse. Carefully clean the top of each lens with 70% ethanol.
11. Take the “Base” adaptor component. You will notice it has two openings along its long-dimension, one opening being wider than the other. Place the narrower side down and slide it onto the GRIN lens atop the mouse’s head. Tighten the bottom screw to secure the adaptor in place.
12. Take a fiber tip with “Fiber” adaptor already attached, and carefully place into the larger opening that is facing up in the “Base” adaptor.
13. Begin imaging, making sure you have a clear view of the monitor while you can still face the mouse with your body/hands.
14. Adjust the z-axis until cells are in focus. One hand will be holding the fiber tip and moving it up and down carefully within the adaptor. The other hand will be holding the T1 screwdriver which will gently tighten the top screw on the base adaptor once you are in focus, to hold the adaptor all together. This will take some practice, but making small, slow movements is the key.
15. Note that tightening the screw will shift your X/Y field of view slightly. The adaptor is designed to mostly eliminate the need to make any X/Y adjustments, but it is not perfect. To change X/Y, you can rotate the fiber adaptor relative to the base before tightening the screw. If recording across multiple sessions, it helps to have a previous reference session open to make sure your field of view is consistent.
16. Repeat 11-12 for each GRIN lens.
17. Once all lenses are in focus, take a sample video of at least 1 minute and verify that you are seeing clear, in-focus transients in each region, before proceeding with behavior.
Live imaging for alignment:
Snapping a test video:
