Although you’ve likely seen refraction at work in your daily life – a spoon appearing to bend in a glass of water or the colorful patterns made by oil and water mixtures on pavement after a storm – you might not connect these everyday experiences with the quality of your microscopy image in a lab experiment. Refraction occurs when light crosses an interface between materials with different refractive indices (RI), which describes the speed at which light travels in a material. Light bends, or refracts, according to Snell’s Law, which defines the angle of bending according to the angle of incidence and the degree of RI mismatch. In the path taken by light through a microscope and into a sample, there are many sites of RI mismatch. When rays of light cross a boundary at different angles (for example, in a point scanning confocal microscope, where an excitation beam is focused to a point within a sample), these rays are bent to varying degrees due to their different incident angles (Snell’s Law), and therefore no longer focus to a point. This is called spherical aberration and reduces both resolution and brightness of your microscopy image.
In our paper, we highlight that to minimize spherical aberration, the sample and associated mounting medium RI must be well matched to the RI of the immersion medium, the material that fills the space between sample (or coverslip) and objective lens. For example, high RI mounting media (RI ~1.52) match the RI of the cover glass and immersion oil. Other common mounting media for fluorescence microscopy are often glycerol-based and have varied RIs. These are well matched to glycerol immersion objectives while live samples are best imaged with water or silicone oil objectives. Tissue clearing, reviewed by the same authors here, has introduced a wider variety of mounting media, including glycerol, Dibenzyl ether and Ethyl Cinnamate. Because cleared samples are frequently quite large, many researchers opt for long working distance air objectives to image through these samples. It should be noted, however, that this introduces a large RI mismatch between the air (RI = 1.00) and the high RI clearing media (DBE RI = 1.56) and therefore substantial spherical aberration. Therefore, best practice is to choose an objective optimized for a sample’s particular RI whenever possible.
Unfortunately, a perfect match is not always possible, and the problem is further complicated by appreciating how spherical aberration can alter the Z dimension of a thick sample and confound volumetric measurements. Because converging rays are aberrated by an RI mismatch (for example, low RI immersion medium to high RI mounting medium), the approximate point to which they focus is deeper than expected and the optical “Z-step” is therefore larger than the physical movement of the objective or stage. This produces an axial distortion in the final image. Have you ever created a 3D rendering of your sample and thought it looked a little too much like a pancake? Or complained that your mounting medium is shrinking your sample? This could be due to spherical aberration. Unfortunately, lost axial information cannot be recovered post hoc by correcting optical section spacing in the final image. The best practice is to calculate the proper sampling frequency prior to acquisition based on the known RI mismatch present in the system (if RI mismatch itself cannot be eliminated). In our article, we summarize previous computational methods to calculate appropriate sampling in the event of RI mismatch and present an efficient compromise. We package this correction in a FIJI macro, which can be used prior to imaging to calculate appropriate sampling or after imaging to adjust z-spacing.
Microscopists, at the very least, should explore the refractive index of materials they are using and the objective lenses available to them; the cure to a dim or blurry image may be a simple tweak away.
by Erin Diel
