Issue with air objectives

[romainbrette]

We now have a 20x air objective. So the objective is not immersed, but the specimen is normally in water. I hadn't realized this, but because refractive indexes are different, this seems to imply (I have to check) that when an immersed object (pipette) is moved down by an amount z, the objective must move by an amount z*(n1/n2) where n1 and n2 are the refractive indexes of the two media (air and water). With air and water, the ratio is about 4/3.

An implication of this is that to change between an air objective and an immersion objective, we cannot just change the magnification; there is in fact an entire 3D matrix change (normally magnification and n1/n2 should suffice but it's not entirely clear). So it appears that the simplest way to deal with this is to redo the calibration entirely for each objective.

Finally, another potential issue is the sensitive to the shape of water surface. That is, if it is curved, the image becomes distorted and this means the change of coordinates between manipulator and camera coordinates is not linear any more... What does it imply for microdroplets for example? And in slices or cultures, how about the fact that the surface of water is typically not exactly flat because of surface tension? Does it introduce distorsions?

[romainbrette]

Additionally, the offset depends on the z position of water surface.

I also realize that this might be a way to measure the position of the manipulator and objective axes relative to gravity (because water surface is horizontal).

[romainbrette]

Another point: there's a magnification of 4/3, independent of water depth. Non-flat water surface implies nonlinear coordinate changes. Flat water surface means linear coordinate changes, but possibly with a rotation (depending on whether it is parallel to the camera plane).

[romainbrette]

I'm confused now about this magnification due to water. It doesn't seem right!

[romainbrette]

Implications for Paramecium experiments

Two types of experiments:

• Tracking: platform calibration might have to be done in water (not clear). It is also very important that the focal plane is horizontal.
• Electrophysiology. When we locate the cell on screen, it is immersed. However, the droplet is very shallow, especially when it dries out. So calibration should be done in air. However, we cannot trust the z position. This means that the z position of the coverslip should be first measured, then the pipette should be placed above, at z+?? (50 um?), then descended, possibly at a fixed ending position. Then when we place the second pipette, the cell is immersed. So here probably the calibration should be done immersed.