Biology subjects

Mahecic, D.

Publications and source records attributed to Mahecic, D..

4 recordsLinked to original sources

bioRxiv2021

Event-driven acquisition for content-enriched microscopy

A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition (EDA) framework, in which neural network-based recognition of specific biological events triggers real-time control in an instant structured illumination microscope (iSIM). Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because EDA allows the microscope to respond specifically to complex biologi-cal events, it acquires data enriched in relevant content.

cell biology↗

bioRxiv2020

Distinct molecular signatures of fission predict mitochondrial degradation or proliferation

Mitochondrial fission is a highly regulated process which, when disrupted, can alter metabolism, proliferation and apoptosis1-3. The downstream effects have implications for many diseases, from neurodegeneration4-6 to cardiovascular disease7,8 and cancer9,10. Key components of the fission machinery have been identified: constriction sites are initiated by the endoplasmic reticulum (ER)11 and actin12 before dynamin-related protein 1 (Drp1)13 is recruited to the outer mitochondrial membrane via adaptor proteins14-17, where it drives constriction and scission of the membrane18. In the life cycle of mitochondria, fission is important for the biogenesis of new mitochondria as well as the clearance of dysfunctional mitochondria via mitophagy3,19. Global regulation of fission on the cellular level is insufficient to explain how fate decisions are made at the single organelle level, so it is unknown how those dual functions arise, blocking progress in developing therapies that target mitochondrial activity. However, systematically studying mitochondrial division to uncover fate determinants is challenging, since fission is unpredictable, and mitochondrial morphology is extremely heterogeneous. Furthermore, because their ultrastructure lies below the diffraction limit, the dynamic organization of mitochondria and their interaction partners are hard to study at the single organelle level. We used live-cell structured illumination microscopy (SIM) and instant SIM20 for fast multi-colour acquisition of mitochondrial dynamics in Cos-7 cells and mouse cardiomyocytes. We analysed hundreds of fission events, and discovered two functionally and mechanistically distinct types of fission. Mitochondria divide peripherally to shed damaged material into smaller daughter mitochondria that subsequently undergo mitophagy, whereas healthy mitochondria proliferate via midzone division. Both types are Drp1-mediated, but they rely on different membrane adaptors to recruit Drp1, and ER and actin mediated pre-constriction is only involved in midzone fission.

cell biology↗

bioRxiv2020

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Super-resolution microscopies, which allow features below the diffraction limit to be resolved, have become an established tool in biological research. However, imaging throughput remains a major bottleneck in using them for quantitative biology, which requires large datasets to overcome the noise of the imaging itself and to capture the variability inherent to biological processes. Here, we develop a multi-focal flat illumination for field independent imaging (mfFIFI) module, and integrate it into an instant structured illumination microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 {micro}m2 without compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching multiple adjacent images together to perform fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at an effective resolution of [~]35 nm. We apply classification and particle averaging to these large datasets, allowing us to map the 3D organization of post-translational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

biophysics↗

bioRxiv2018

Membrane bending energy and tension govern mitochondrial division

During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide, or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We captured the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discovered that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide.

biophysics↗