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Spherical aberration (SA) occurs when light rays entering at different points of a spherical lens are not focused to the same point of the optical axis. SA that occurs inside the lens elements of a fluorescence microscope is well understood and corrected for. However, SA is also induced when light passes through an interface of refractive index (RI)-mismatched substances (i.e., a discrepancy between the RI of the immersion medium and the RI of the sample). SA due to RI mismatches has many deleterious effects on imaging. Perhaps most important for 3D imaging is that the distance the image plane moves in a sample is not equivalent to the distance traveled by an objective (or stage) during z-stack acquisition. This non-uniform translation along the z axis gives rise to artifactually elongated images (if the objective is immersed in a medium with a higher RI than that of the sample) or compressed images (if the objective is immersed in a medium with a lower RI than that of the sample) and alters the optimal axial sampling rate. In this tutorial, we describe why this distortion occurs, how it impacts quantitative measurements and axial resolution, and what can be done to avoid SA and thereby prevent distorted images. In addition, this tutorial aims to better inform researchers of how to correct RI mismatch–induced axial distortions and provides a practical ImageJ/Fiji-based tool to reduce the prevalence of volumetric measurement errors and lost axial resolution.

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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

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The idea for calculating axial distortion correction factors as described in this tutorial was conceived by D.S.R. and J.W.L. E.E.D. and D.S.R. carried out experiments and analyzed data. D.S.R., J.W.L. and E.E.D. wrote the manuscript. All authors contributed to editing the final manuscript.

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The Laser Alignment Base pivots smoothly on the plumb point for easy and fast alignment. With its adjustable legs, this laser accessory accommodates slightly ...

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Some optical elements are also available with non-spherical surfaces and are then called aspheric optics (or sometimes aspherical optics).

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All necessary code and instructions for running the axial correction macro are provided in the Supplementary Software and Box 1.

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Convex Lenses · When an object is placed at infinity, the real image is formed at the focus. · When an object is placed beyond the centre of curvature, the real ...

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Vimba SDK has many pre-compiled examples in C and Python that we can use to test the camera and different configuration options. The C or Python APIs can be ...

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We thank S. Piccinotti and L. Rubin for providing organoid samples. We thank the Harvard Center for Biological Imaging for infrastructure and support. J.W.L. was supported by the following funding sources: National Institutes of Health grants P50 MH094271, U24 NS109102, and U19 NS104653 and Department of Defense MURI award GG008784.

Model, M. A., Fang, J., Yuvaraj, P., Chen, Y. & Zhang Newby, B. M. 3D deconvolution of spherically aberrated images using commercial software. J. Microsc. 241, 94–100 (2011).

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Ghosh, S. & Preza, C. Fluorescence microscopy point spread function model accounting for aberrations due to refractive index variability within a specimen. J. Biomed. Opt. 20, 75003 (2015).

Peer review information Nature Protocols thanks Chrysanthe Preza and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Schmidt, N. C., Kahms, M., Huve, J. & Klingauf, J. Intrinsic refractive index matched 3D dSTORM with two objectives: comparison of detection techniques. Sci. Rep. 8, 13343 (2018).

Jul 18, 2022 — Tints are applied to a lens after the anti-reflective coating would have to be applied. If an anti-reflective coating is on the lens, the tint ...

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Our Kiralux® and Quantalux® cameras can be used in microscopy, materials inspection, and other demanding quantitative imaging applications. We also offer a ...

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