Jacobs:Confocal Imaging of Flow
Materials for IMCD Culture
- Growth Media: DMEM/F12, 10% FBS, 1% PS, 200ug/ml Geneticin for selection
- Serum Starve Media: change to 0.1% FBS
- Coat the glass bottom of a 35mm glass bottom dish with fibronectin (1:100 in PBS) for 1 hour
- Seed 3k cells in up to 500ul of growth media onto the glass part of a small glass-bottom dish and maintain in culture in for 2 days
- Cells will reach ~80% confluency
- After the cells have adhered (1 hour after seeding) you may add another 1ml of growth media
- Wash with warm PBS and add 1.5ml of serum starve media and culture for 3 days
- Microscope MP off; activate resonance scanner
- Use the 100x oil objective
- Argon: set to 25 to 30%
- Image eGFP: excitation 488/emission 509
- Set 488 laser to ~34%
- Emission spectrum from: 500 to 600
- Set Frequency: 512 x 512
- Line Averaging: 4 is sufficient, 8 is really good
- Use x, y, and z controls to focus on cells and find cilia.
- Once you have focused on a cilium in xyz, it is best to zoom in to it so you can line up the plane of focus for imaging at xzy. Then switch to xzy. If the cilium is not parallel to the xz plane (dashed line), then rotate the field of view until it is parallel.
- To image the entire cilium, make a xyz or xzy stack. It’s easiest to manually type in the z or y position, respectively, guessing at 0.1 um increments to find the edges of the cilium. Once you found one edge, click the begin arrow. Continue finding the other edge and then click the end arrow to complete the stack. A slice thickness of 0.1 to 0.2 um is ideal to capture the cilium (~400nm in diameter).