Translation speed limitations from drag between immersion medium and coverslip

Hi all,

We are working on an application requiring rapid stage translation between multiple fields of view (FOVs). Recently, we got an interesting suggestion from a reviewer, asking us to “comment on the tolerable speed in lateral and axial directions” from the perspective of drag resistance between the immersion medium and the coverslip.
I thought maybe some of you have thought about this in more depth and for longer, so I am curious about any insights you might have?
Of course, any information that would contribute to our response to the reviewer would be properly acknowledged!

Briefly, here is what I’ve come up so far:

  • intuitively I would think that the translation is less affected in the axial compared to the lateral direction
  • drag should be proportional to the viscosity of the immersion medium, such that lower viscosity results in less drag
  • if the timescales of the drag and viscous behaviour of the immersion medium are much lower than the stage response and translation timescales, then there is a separation of timescales and we can ignore the effect of drag for practical applications

Looking forward to hearing your thoughts!

I’ve definitely experienced issues with stage speed causing problems with oil in incubation scenarios, when the stage is warmed to 37. Here, conventional oils become less viscous, and slowing the stage down can help, this is the opposite of what it sounds like you want (speed!). A silicone objective may be in order…

Part of the answer depends upon the optical set up. If you are using an inverted microscope (like an old Zeiss IM35) and cover-slip bottomed culture dishes - with cell growing directly on the coverslip and the lens underneath, then translation speed is irrelevant. We used such a set up in intracellular injection studies together with 63X and 100X oil immersion lenses.
If you are using a water immersion lens, then I suspect it depend upon working distance - how close lens actually comes to the cells.

On our automated scope, we have a “slosh” delay to allow any well contents movement to stabilize. Turns out it works well to allow the oil to “catch up” with the objective movement. We are moving a few mm/sec but the need to wait a second or two for the oil drop to re position between the objective and sample. Hope that helps, Chris