Timelapse microscopy and incubation (HEPES)? - (Apr/24/2012 )

Hi friendly people,

I'm in an engineering lab and we're gearing up for some timelapse epithelial culture experiments, so forgive me if these questions are basic! Our lab budget for biology is not very large, so I was intending to build my own stage-top incubator, but I had a few parameter questions...

We'd like to run 16-48 hour timelapse with 10X Phase.

1. CO2 levels during timelapse: To save costs, I was planning to use a HEPES buffer for ~24 hrs. I can replace medium during the run if necessary. Is there a problem with using HEPES, or is there a better buffer?

2. How much does relative humidity matter? A lot of expensive stage-top incubation systems try to hit a 75% humidity set-point, but I wasn't sure if they mainly want to control condensation or if there's some biological advantage (beyond just evaporation of medium).

3. Anyone have any experience building incubators? I'm thinking of either enclosing the whole scope in a plastic box and running a little heater+controller, or building a little box that sits on top of my sample and keeps it warm, while still allowing light through the top and bottom.

Thanks for the help!

D

HEPES is fine for this sort of thing. You can get HEPES buffered medium quite easily from Invitrogen or Sigma.

The relative humidity thing is mostly to prevent the medium from evaporating, this is to prevent osmotic stress on the cells.

I don;t have any experience with building them, but I have seen both systems for scopes, with both seeming to work equally well. If you could build a box that only encloses the stage and objectives (still allowing changing of objectives?) it would probably be more user friendly, rather than having something that you need to open up to look at the samples. Note that high humidity encourages growth of fungi, which may be a problem for lenses and electronics aren't a fan of humidity generally!