For high resolving power, the numerical aperture (NA) of an objective needs to be greater than 1. This also requires an immersion medium with a refractive index greater than 1, i.e. other than air. Common immersion media are oil, water and glycerol. The immersion medium that has to be used with a certain objective is indicated on the objective.
DIN/ISO standard immersion oil
Any other or more than one immersion media
All optically relevant elements (immersion medium, coverglass, sample) in front of the front lens of the objective have a major influence on the image quality. Ideally, the refractive index throughout all these optical layers should match the refractive indices the objective has been designed for. In reality, this is hardly possible as samples are often inhomogeneous, coverglass thickness is not that precise and the temperature changes during image acquisition. These factors have to be kept in mind when choosing an objective and immersion medium for a certain application.
Refractive indices of some important immersion media
1.33 - 1.38
Type F oil
1.45 (21°C) - 1.46 (37°C) for Leica immersion liquid type G
100% PBS pH 8,9
The higher the numerical aperture of the objective and the deeper the structures of interest inside the sample, the more important it is to match the refractive indices of the sample and the immersion medium. Different refractive indices lead to spherical aberrations and geometrical distortions of the structures. This results in a loss of contrast and definition, as well as structures appearing compressed or stretched.
The refractive index of the Leica immersion oil type N and type F is 1.518 (at 23°C and 546 nm), i.e. the same as standard crown glass (n=1.518). For multicolor imaging, the dispersion of the immersion oil is also important. It is often referred to as the Abbe number and should match the Abbe number the objective was designed for otherwise, chromatic aberrations will occur. For example, Leica immersion oil type N has an Abbe number of 42.1 while immersion oil type F has an Abbe number of 46 which is optimal for fluorescence imaging. Type N is not suitable for fluorescence imaging.
Oil immersion objectives are ideal for samples that are in a medium that matches the refractive index of oil, i.e. classically fixed specimens embedded in resin, Canada balm or glycerol-gelatine, or are imaged close to the coverglass, i.e. less than a few µm. Further away from the coverglass the image brightness and resolution deteriorates quickly if the refractive indices are mismatched.
For live cell imaging, i.e. imaging in an aqueous sample, we strongly recommend the use of water or glycerol immersion.
Oil immersion objectives with extremely high apertures only deliver their full optical performance in a relatively narrow temperature interval, as the refractive index of immersion oil greatly depends on the temperature. The larger the free working distance the thicker the oil layer, and therefore the larger is the impact of the temperature-related aberrations on the image quality. This temperature effect changes linearly with the free working distance but depends on the NA to the power of four.
For experiments at temperatures deviating from room temperature, water immersion is recommend as the refractive index of water is significantly less temperature dependent and can be compensated because many water immersion objectives have correction collars.
Water immersion objectives are optimal for observing living cells in aqueous media as the refractive indices of the immersion medium and the sample are a closer match than for example immersion oil. However, water quickly evaporates at 37°C. The Leica Water Immersion Micro Dispenser automatically adds water during a running experiment to provide stable water immersion.
Today, most fixed samples like cultured cells, tissue slices of thick specimen as well as whole mount embryos are mounted in Mowiol, Vectashield or similar mixtures containing water and glycerol plus various chemicals, e.g. antifade and preservation substances. These media have refractive indices close to that of a 80%/20% glycerol/water mixture (n=1,45). Glycerol objectives are excellent for samples mounted in any medium with a refractive index close to 1.45 – 1.46.
Leica glycerol objectives offer a correction collar to adjust the optics to varying refractive indices coming from changes of the composition of the mounting medium, or from variations of coverglass thickness or temperature. Application Letter No. 17 (April 2004) gives a very detailed description of glycerol objectives and the effects of refractive index mismatches.
Objective for use with glycerol immersion:
HC PL APO 63x/1.30 GLYC CORR CS2
Most multi-immersion (IMM) objectives can be used for glycerol as well as water and oil immersion as described below.
Objectives suitable for the use with multiple immersion liquids, i.e. oil, water and glycerol, can be used with the optimal immersion medium for a large variety of samples.
HC PL APO 10x/0.40 IMM CS
HC PL APO 20x/0.75 IMM CS2