Leutenegger, M. | Search bioRxiv

bioRxiv2020

MINSTED fluorescence localization and nanoscopy

We introduce MINSTED, a stimulated-emission-depletion (STED) based fluorescence localization and super-resolution microscopy concept providing spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED donut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background, and the required number of fluorescence detections are low compared to most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1000 detections per fluorophore provide a localization precision of 1-3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement of far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

biophysics↗

bioRxiv2020

A quantitative analysis of the interplay of environment, neighborhood and cell state in 3D spheroids

1Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. Even cells with deregulated signaling can adapt to their environment. To analyze the interplay of environment, neighborhood, and cell state on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging to analyze up to 240 pooled spheroid microtissues. This system allowed us to quantify the contributions of environment, neighborhood, and intracellular state to phenotypic variability in spheroid cells. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. We found that the contributions of cell-intrinsic and environmental factors are hierarchically interdependent. By overexpression of 51 signaling protein constructs in subsets of cells, we identified proteins that have cell-intrinsic and extrinsic effects, exemplifying how cell states depend on the cellular neighborhood in spheroid culture. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.

systems biology↗