Fluorophores stability in mounting media developed at LM IMG

Session

Poster Session

Authors

Ivan Novotny (1), Michaela Blazikova (1), Ondrej Horvath (1)

Affiliations

1. Light microscopy, Institute of Molecular Genetics of the Czech Academy of Sciences

Keywords

mounting media

fluorescence microscopy

super-resolution

Abstract text

In the field of light microscopy, particularly in advanced and super-resolution methods, the stability of fluorescence is crucial. This stability is always provided by adequate sample preparation and is defined by the choice of mounting media.

At the Light Microscopy facility of the Institute of Molecular Genetics of the Czech Academy of Sciences (IMG CAS CZ), our research has intensively focused on developing appropriate mounting media to enhance fluorophore stability for advanced imaging techniques. This work originated through our association with Euro-BioImaging and MPI-CBG, and further supported by the Czech-BioImaging methodical grant.

Our efforts have culminated in the development of three dedicated mounting media, marking a significant advancement in our work. These developments have proceeded in collaboration with the ADVI startup, leading to the commercial production of these media through a spinoff collaboration model.

A notable highlight of our achievements is the formulation of a dedicated mounting medium that has shown exceptional results in the 3D STED super-resolution visualization of entire mouse oocytes, underscoring the efficacy of our media in preserving fluorophore stability under demanding imaging conditions [1, 2].

For researchers, it is crucial to compare the stability provided by this new family of mounting media. Therefore, we conducted precise measurements of fluorescence bleaching assays for several of the most common fluorophores in our mounting media, comparing them to high-quality commercial standards.

As a result, we present bleaching curves for the mounting media, obtained through properly defined measurements using a photon-counting quantitative approach in a confocal microscope. The results are not only proof of the high quality of ADVI mounting media but also the assay has given a standard approach in mounting media fluorescence stabilization measurement.

References

[1]

Frolikova M., Blazikova M., Capek M., Chmelova H., Valecka J., Kolackova V., et al. (2023). A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte. bioRxiv, 531472. doi: 10.1101/2023.03.07.531472

[2]

Frolikova M, Sur VP, Novotny I, Blazikova M, Vondrakova J, Simonik O, Ded L, et al. (2023). Juno and CD9 protein network organization in oolemma of mouse oocyte. Front Cell Dev Biol. 11:1110681. doi: 10.3389/fcell.2023.1110681