Optogenetic Stimulation and Cell Localisation in Three-Dimensional Cell Structures

Disturbances in the signal transmission are a main cause for heart arrhythmia and deadly ventricular fibrillation. However, the processes generating and interrupting these events are not fully understood yet. Optogenetics – a set of methods encompassing genetic and optical tools for the light-mediated control of cell characteristics – is a promising approach to induce, observe and control irregular signal transmission in in-vitro experiments in a spatially and temporally targeted manner for a deeper phenomenological understanding. The goal of this project is to provide the necessary tools for understanding signal transmission in three-dimensional in vitro cardiomyocyte cell structures. To this end, several approaches will be followed. First, three-dimensional optogenetic stimulation will be realized using non-linear two-photon processes to achieve cell-sized optical confinement in deep tissue (hundrets of microns). This approach will be extended using the method of temporal focusing to achieve a simultaneous stimulation of larger cell patches or volumes, guaranteeing the necessary temporal resolution for a 3d control of excitation waves in cardiac tissue.

© Felix Schmieder

Schematic of setup for simultaneous 3d targeted single cell stimulation and single shot 3d localization microscopy.

© Lars Büttner

Principle of 3d localization microscopy with point spread function engineering. Single image points are converted to double spots using a special phase mask in fourier space. The depth of the light source can be estimated from the orientation angle ψ between the double spots which changes along the optical axis.  

To observe excited contraction waves in three dimensions without scanning for high temporal resolution, we will apply 3d localization microscopy and tracking of fluorescently labeled cell nuclei using the approach of point spread function engineering. Here, we will initially employ the method of the double helix point spread function, which is quite established in localization microscopy and flow measurement. Using deep neural networks for location deconvolution, we will investigate limitations regarding achievable observation depth in relation to scattering/fluorescent particle density.

Staff:               F. Schmieder, R. Wendland, L. Büttner

Period:            10/2023 – 10/2026

Partner:           Olaf Bergmann, Research Group Cell-based Model Systems, Department of Pharmacology and Toxicology, University Medical Center Goettingen