Hi there,
In our lab we do long-term (72-96 hs) live microscopy of yeast cells grown in microfluidic chips, at 30ºC. Using a 40x air objective I usually monitor 50 different positions every 10 minutes.
We’d like to improve resolution using an immersion objective, but which one?
Oil? Does it get gummy, or dries?
Silicone Oil?
Water with a dispenser? (it is expensive and tricky to setup)
Thanks you all in advance,
Pablo
Oil will be fine for at least several days, assuming that you use ‘standard’ immersion oil, such as that provided by the manufacturer of the objective you buy.
I think silicone oil is also fine for several days. However silicone oil objectives are often quite a bit more expensive than oil immersion objectives.
Water could work, I’ve been told that Cargille manufactures a low index oil that can be used with a water immersion objective (it has the same index as water), but I have no personal experience here.
Water immersion objectives are also more expensive than oil. So at least for price and simplicity oil is probably the best choice. However if you need to image deep into a sample say 50+ microns water or silicone might be a better choice.
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In addition to what @Hazen_Babcock said, I would consider using an immersion medium that is refractive index (RI) matched to the material your microfluidic chambre is made of (is it glass or plastic? The data sheet should tell you what the RI is).
The objective (whether x40 or x100 or whatever) must be of an immersion type - don’t just immerse a standard x40 into any liquid medium - and must be designed to work with an immersion medium of the required RI (or very close to it).
You also need to consider working distance of the objective to your sample - so if the chambre is thicker than a standard coverslip (between the sample and the objective) then most standard x100 oil objectives would probably not be suitable.
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Water with a dispenser would probably be the best match to the growth medium, but I don’t have experience with those devices.
We mostly use Zeiss oil, and it doesn’t dry up.
When imaging large areas with oil immersion, one trick that we use is to pre-wet the bottom of the vessel to help with oil distribution.
- Take a sheet of Parafilm or other plastic that allows the oil to bead up.
- Dot an array of small drops of oil covering the “footprint” of the chamber.
- Gently lower the chamber onto the droplets of oil.
- Experiment to get the right size droplet to cling to the chamber and enable smooth objective movement and focusing, without falling off or overloading the objective.
Whatever immersion medium you use, it’s essential to protect your objective lens and the internal optics of the microscope. E.g. use use fabric-covered scrunchies and Kimwipes to shield against oil or water dripping down into the objective barrel and the nosepiece.
Good luck!
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Thank you all for the very useful replies. Thanks for the Cargille tip, there is a 1.300-1.395 ±0.0002 RI oil ( Cat #: 1803X/Y) that might match our system. Our samples are very shallow (8um deep) and chip material is PDMS (RI = 1.43), however cells are maintained in aqueous media.