Question: When using immersion oil, should I be cautious about what type of oil I use? There are many oils on the market, designed for different temperatures, etc., Is this an important consideration?
Thank you!
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Hi Kelly!
The tricky thing here is that while immersion oil is usually marked as having one refractive index, refractive index is actually a wavelength-specific property⌠for example:
How refractive index changes across wavelength is referred to as dispersion, and what really matters is finding an immersion oil with the appropriate refractive index across all the wavelengths you wish to use, at the temperatures you wish to use, with the lenses you with to use (otherwise youâll get aberrations in the image). The combinatorics can get pretty daunting. Microscope manufacturers have detailed info on the optical design of their lenses, and so can suggest/provide oils that are likely to work with their lenses at temperatures you are using. In truth, thereâs no perfect immersion oil for all possible combinations: youâll always see spherical and chromatic aberrations (and perhaps others) as you depart from the operating specifications of the lens, say with depth from the coverslip, or coverslip thickness variability âŚ
But thereâs also nothing fundamentally âwrongâ with trying various oils. The only things to be cautious about, are
- make sure you clean the lens and the coverslip thoroughly between different oils
- know what youâre looking for when changing and evaluating different oils. That is: donât go changing oil without checking that you havenât dramatically increased spherical or chromatic aberration in your sample.
Non-optically, i suppose itâs worth mentioning that some oils can harden or polymerize when left for a while, so if youâre going to start playing around with different oils, you might ask the manufacturer about any known longer-term issues with that formulation.
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Another non-optical thing to consider:
While I always recommend using a #1.5 glass coverslip, there are a quite a few âoptical polymerâ coverslip products on the market these days. If you use these, you should check whether the immersion oil youâve chosen is compatible with the coverslip material.
We tested a panel of immersion oils on polymer coverslips and found that many commonly-used immersion oils damaged the coverslip, sometimes causing cracks and coverslip breakage, within a few hours of contact. The coverslip manufacturer should provide information on recommended oils, but itâs a good idea to test it yourself too.
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This can be an important consideration. The refractive index matching and background fluorescence levels of the oil are important, viscosity is another feature, some oil formulations are thinner and spread around more, which is particularly an issue with timelapse - as is drying. Some oils will start to dry out and become very tacky, like dried honey, after several hours. If you are using an oil that drys be very careful as it is difficult to clean the objective and anything else that the oil gets onto (not that that ever happens). There are excellent objectives that are glycerol, silicone, or even can use multiple immersion liquids - Iâve heard silicone is excellent for long working distance, high NA timelapse as the silicone travels with the objective better and doesnât spread out as much. There are some nice resources out there that have tables of oils and âtypesâ, such as Nikon type B, N, F, etc., you should check them out!
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