Immersion Media

Numerical aperture is n sin θ, and sine cannot exceed 1. In air, n = 1.0, so NA cannot reach 1.0 however the lens is built — about 0.95 in practice. That ceiling is trigonometry, not engineering, and no amount of money moves it.

Immersion media exist to attack the other factor. Water gives n = 1.33, glycerol 1.47, immersion oil 1.515, and NA above 1 becomes available: 1.27 for water, 1.4 to 1.49 for oil. There is a second effect working in the same direction — matching the index between coverslip and lens suppresses the refraction at that interface that would otherwise bend the steepest rays away entirely. So the medium both raises the ceiling and lets you actually reach it.

Here is the result that catches people. Take an aqueous live sample and image 50 µm deep into it. Compare a 1.4 NA oil objective against a 1.2 NA water objective. On paper the oil wins comfortably. In practice the water objective is very often better, and the gap widens the deeper you go.

The cause is index mismatch. Oil at 1.515 is matched to glass, but your specimen is essentially water at 1.33. Every ray crossing that boundary refracts, and rays at different angles focus at different depths — spherical aberration, growing roughly in proportion to depth. At the coverslip it is negligible. At 50 µm it has spread the point spread function enough to give back the resolution the higher NA bought, and dimmed the signal along with it. A water objective is matched to the specimen, so it does not accumulate the error.

Which reframes the selection rule. Match the medium to the specimen, not to the lens catalog. Oil is right at the coverslip and for cleared or mounted samples with matched index; water is right for live aqueous work at depth; glycerol sits between and suits many clearing protocols.

Immersion oil is a formulated synthetic with n = 1.515 at 23 °C and dispersion matched to glass. That temperature qualifier is real: index drifts with temperature, so an oil objective calibrated at the bench behaves slightly differently in a 37 °C incubator, and some objectives carry a temperature-specific correction for exactly this. Type A and Type B differ in viscosity; mixing brands can cause haze or separation.

Water is the awkward one, because it evaporates. A long time-lapse on a water-immersion objective can lose its medium mid-experiment, and the failure looks like gradual defocus and dimming rather than anything obviously mechanical. Water dispensers exist for precisely this. Glycerol sits between in index and does not evaporate appreciably, which is part of why cleared-tissue protocols favor it. Silicone oil at 1.40 is close to live cell cytoplasm and holds its match with depth better than either oil or water.

Consider a 1.4 NA oil objective and a 1.2 NA water objective on the same aqueous specimen at 560 nm. Rayleigh at the coverslip: 0.61 × 560 / 1.4 = 244 nm for the oil, 285 nm for the water. The oil leads by 17%.

Move to 50 µm depth and the oil objective's effective performance has degraded through accumulated spherical aberration, while the water objective's is close to unchanged because it is index matched to what it is looking through. The crossover depth depends on the sample, but it is typically tens of micrometers, not hundreds — shallow enough that a great many routine experiments sit on the wrong side of it while using the objective with the better number on the barrel.