To the right you see an example of spherical aberration in an image due to a mismatch in refractive indices. This example is explored in more detail at the bottom of this page. Spherical aberration is a common issue in microscopy that is especially hard to correct for images with a large Z-range. Luckily the unique spherical aberration in Huygens can correct for this easily.
Spherical aberration in microscopy
Two forms of spherical aberration are relevant in light microscopy. The most described form is SA due to the lens shape. The other form is SA due to a mismatch in refractive indices between sample and the lens immersion medium. To learn more about refraction and refractive indices, please read our refractive indices page.
Lens spherical aberration
Spherical aberration occurs in all lens-based systems due to the spherical shape of the lens. Light entering near the edges is refracted more strongly than light near the optical axis, causing rays from the same source to focus at different planes. This results in a sharp center but blurry edges. However, modern microscopes typically include corrective elements to minimize this effect.
Refractive index mismatch
In widefield and confocal microscopy, spherical aberration mainly arises from mismatched refractive index (RI) between the lens immersion medium and the sample embedding medium. This mismatch causes light to refract differently depending on the angle of incidence (light refraction), leading rays from a single point to focus at different planes. The focal shift increases with the depth of focus in the sample.
Correcting spherical aberration with Huygens deconvolution
Spherical aberration due to refractive index mismatches can be automatically corrected for by performing Huygens deconvolution. Spherical aberration is automatically corrected for if the following two conditions are met:
Setting coverslip position in Huygens
If your image suffers from a Refractive Index mismatch, make sure to check that the imaging direction and coverslip position are set as they were during acquisition of the image in the microscopy parameters before doing deconvolution. If you are not sure what the orientation of your dataset is, you can always guess it from the asymmetry of the PSF of the cones of light. In the image above showing the PSF distortion, the smooth side of the PSF (bottom of the image) is at the side of the coverslip. For more information see also Point-Spread-Function.
Avoid SA correction in Huygens
To switch the SA correction off (because despite the refractive index mismatch you have other physical correctors in your setup) just set the image Microscopic Parameters to have matching lens refractive index and Medium Refractive Index prior to Deconvolution. If the Numerical Aperture is larger than either of the refractive indices, make sure you use set both RIs to the lowest RI, to allow the software to correctly account for total internal reflection.
Air lenses
Air lenses require special consideration: they may be corrected for spherical aberration and they may behave as, e.g., glycerine lenses, and therefore you will not expect problems if you use glycerol as embedding medium. You can then think about deactivating the SA correction as explained in the previous paragraph. However, this only seems to work well for imaging close to the coverslip. Deeper imaging positions still benefit greatly from the spherical aberration correction by Huygens. Read more in Air Lens Correction for considerations when using air lenses.
RI mismatch distorts PSF
If this is the case for your image, you can work around it by keeping the Z-range of the data as small as possible and measure only close to the coverslip. To resolve the issue the lens immersion medium can be matched to the RI of the sample embedding medium (RI ~ 1.33).
The PSF distortions due to RI mismatches for different imaging depths are shown in more detail on our Point-Spread-Function page.
Practical example
To illustrate the effects of a RI mismatch and the resulting spherical aberration, consider the following case study. Here, active zones of synapes in adult Drosophila brains are visualized by endogeneous expression of GFP labeled Bruchpilot (Brp). Data was acquired and provided by Tory Hermans, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.
If we make a XZ MIP projection of sections of these images you can clearly see the effects of the RI mismatch. The raw data (left image) displays elongated structures due to the fishtank effect and RI mismatch induced spherical aberration causes objects to appear blurred and out-of-focus. This blurring is stronger at the top of the image. When deconvolving this image without properly setting the mismatch between the lens refractive index and sample refractive index, the deconvolved result will show similar spherical aberration. When deconvolving with the (proper) values for the refractive indices, depth dependend spherical aberration will be taken into account for deconvolution, thus correcting the problems in the image.
Related
Refractive Index Point-Spread-Function Parameter variation
References
Diel, E.E., Lichtman, J.W. & Richardson, D.S. Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy. Nat Protoc 15, 2773–2784 (2020). https://doi.org/10.1038/s41596-020-0360-2
Last updated: June 2024