Sirinakis, G. | bioRxiv Science

bioRxiv2024

User-friendly Oblique Plane Microscopy on a fully functional commercially available microscope base

In this work we present an Oblique Plane Microscope designed to work seamlessly with a commercially available microscope base. To support all the functionality offered by the microscope base, where the position of the objective lens is not fixed, we adopted a two-mirror scanning geometry that can compensate for changes to the position of the objective lens during routine microscope operation. We showed that within the expected displacement range of the 100X, 1.35 NA objective lens away from its designed position, and for most practical applications, there is no significant effect on the resolving power, or the fidelity of the 3D data produced by the microscope. Compared to the more traditional scan-lens/galvo-mirror combination, the two-mirror scanning geometry offers higher light-efficiency and a more compact footprint, which could be beneficial to all OPM designs regardless of the use of a commercial base or not.

biophysics↗

bioRxiv2021

A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 {micro}m. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

cell biology↗