Data underlying the publication: Depth-dependent scaling of axial distances in light microscopy

IntroductionData underlying the publication: Depth-dependent scaling of axial distances in light microscopyPublication DOI: https://doi.org/10.1364/OPTICA.520595Repository created by Daan Boltje<br>AbstractIn volume fluorescence microscopy, refractive index matching is essential to minimize aberrations. There are however, common imaging scenarios, where a refractive index mismatch (RIM) between immersion and sample medium cannot be avoided. This RIM leads to an axial deformation in the acquired image data. Over the years, different axial scaling factors have been proposed to correct for this deformation. While some reports have suggested a depth-dependent axial deformation, so far none of the scaling theories has accounted for a depth-dependent, non-linear scaling. Here, we derive an analytical theory based on determining the leading constructive interference band in the objective lens pupil under RIM. We then use this to calculate a depth-dependent re-scaling factor as a function of the numerical aperture (NA), the refractive indices 𝑛₁ and 𝑛₂, and the wavelength 𝜆. We compare our theoretical results with wave-optics calculations and experimental results obtained using a novel measurement scheme for different values of NA and RIM. As a benchmark, we recorded multiple datasets in different RIM conditions, and corrected these using our depth-dependent axial scaling theory. Finally, we present an online web applet that visualizes the depth-dependent axial re-scaling for specific optical setups. In addition, we provide software which will help microscopists to correctly re-scale the axial dimension in their imaging data when working under RIM.<br>General descriptionAll data underlying the manuscript figures are stored in this repository. Experiments have been carried out at the Department of Imaging Physics at Delft University of Technology (Delft, NL) between December 2022 and February 2023.<br>The repository contains images generated by wave-optics calculations, images acquired on a confocal microscope imaging under RIM and image z-stacks of fluorecent beads residing on the coverslip, and sapphire ball. Such measurement is done for different numerical aperture objectives and refractive indices. Refer to the README for more information.<br>