Immersion media refractive index

Probes & Prep
1

Hello all. Can I have your opinions about choosing the ‘right’ immersion media and lens for imaging?
The coverslip (~170um) has a RI of 1.51. The specimen can be live cells RI 1.33 in media of RI 1.33, live tissue RI 1.46, or fixed cells RI 1.33 in mounting media RI 1.44-1.47.

Can you comment on the following scenarios?

For live monolayer cells (~10um), is it better
to 1. use water lens to match the RI of media and cell
or 2. use oil to match the RI of the dish coverslip
or 3. use water dipping lens in the upright setup?

For fixed monolayer cells (~10um) in mounting media, is it better
to 1. use water lens to match the cell RI
or 2. use glycerol or silicone oil to match the RI of mounting media
or 3. use oil to match the RI of coverslip?

In the above cases, considering the sample is sitting right on the coverslip, the thickness of coverslip is dominant compared to the thickness of sample. Would it make sense to always use oil and oil lens to match coverslip, minimizing refraction?

For fixed tissues (~100um) in mounting media, is it better
to 1.use glycerol or silicone oil to match the RI of mounting media and tissue
or 2. use oil to match the RI of coverslip?
Now the tissue thickness becomes dominant than the coverslip. Would it make more sense to use glycerol lens?

For intravital multiphoton imaging directly without dorsal chamber, is it better
to 1. use water dipping lens
or 2. use glycerol dipping lens?

For intravital multiphoton imaging with a dorsal chamber, is it better
to 1. use water dipping lens
or 2. use glycerol dipping lens
or 3. use oil to match the coverslip RI?
In this case, depending on how deep I can image into the tissue, either coverslip or the sample becomes dominant. Shall I choose the immersion media based on how deep I need to image?

My apologies in advance if I missed some more important considerations or this has been discussed in the past. Please share your thoughts!

2

Regarding the first two scenarios, water and silicon lens are designed to correct for the coverslip in the imaging path. These lens will often have an adjustment collar on the objective which allows them to work correctly with a range of coverslip thicknesses. The presence of a coverslip won’t automatically make them perform worse than an oil objective for ~10um samples.

I think in general you want to the objective that best matches the average RI of the sample. However there are a lot of application specific issues. Oil objectives for example have the highest NA so if your sample is within a few microns of the coverslip they will give you the best picture. Water and Silicon objectives are often a lot more expensive than the equivalent oil objective. Water also evaporates so if you are doing long term imaging this might be another issue.

3

Thank you for your inputs. The water/silicon lens is designed to correct the coverslip for spherical aberration. But their lower RI causes the emission light exit out of the coverslip at a larger angle, into the immersion media. Eventually less light goes into the objective than their oil counterpart. In this sense, I agree oil lens is better for sample within only a few microns of the coverslip. For thicker samples, water/glycerol that better matches the average RI might be more suitable. Thanks again for sharing.