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Retinal neurogenesis and lamination: What to become, where to become it and how to move from there!
The vertebrate retina is an important outpost of the central nervous system, responsible for the perception and transmission of visual information. It consists of five different types of neurons that reproducibly laminate into three layers, a process of crucial importance for the organ’s function. Unsurprisingly, impaired fate decisions as well as impaired neuronal migrations and lamination lead to impaired retinal function. However, how processes are coordinated at the cellular and tissue level and how variable or robust retinal formation is, is currently still underexplored. In my lab, we aim to shed light on these questions from different angles, studying on the one hand differentiation phenomena and their variability and on the other hand the downstream migration and lamination phenomena. We use zebrafish as our main model system due to its excellent possibilities for live imaging and quantitative developmental biology. More recently we also started to use human retinal organoids as a comparative system. We further employ cross disciplinary approaches to address these issues combining work of cell and developmental biology, biomechanics, theory and computer science. Together, this allows us to integrate cell with tissue-wide phenomena and generate an appreciation of the reproducibility and variability of events.
Reconstruct cellular dynamics from single cell data
Recent advances of single cell techniques catalyzed quantitative studies on the dynamics of cell phenotypic transitions (CPT) emerging as a new field. However, fixed cell-based approaches have fundamental limits on revealing temporal information, and fluorescence-based live cell imaging approaches are technically challenging for multiplex long-term imaging. To tackle the challenges, we developed an integrated experimental/computational platform for reconstructing single cell phenotypic transition dynamics. Experimentally, we developed a live-cell imaging platform to record the phenotypic transition path of A549 VIM-RFP reporter cell line and unveil parallel paths of epithelial-to-mesenchymal transition (EMT). Computationally, we modified a finite temperature string method to reconstruct the reaction coordinate from the paths, and reconstruct a corresponding quasi-potential, which reveals that the EMT process resembles a barrier-less relaxation process. Our work demonstrates the necessity of extracting dynamical information of phenotypic transitions and the existence of a unified theoretical framework describing transition and relaxation dynamics in systems with and without detailed balance.
Making connections: how epithelial tissues guarantee folding
Tissue folding is a ubiquitous shape change event during development whereby a cell sheet bends into a curved 3D structure. This mechanical process is remarkably robust, and the correct final form is almost always achieved despite internal fluctuations and external perturbations inherent in living systems. While many genetic and molecular strategies that lead to robust development have been established, much less is known about how mechanical patterns and movements are ensured at the population level. I will describe how quantitative imaging, physical modeling and concepts from network science can uncover collective interactions that govern tissue patterning and shape change. Actin and myosin are two important cytoskeletal proteins involved in the force generation and movement of cells. Both parts of this talk will be about the spontaneous organization of actomyosin networks and their role in collective tissue dynamics. First, I will present how out-of-plane curvature can trigger the global alignment of actin fibers and a novel transition from collective to individual cell migration in culture. I will then describe how tissue-scale cytoskeletal patterns can guide tissue folding in the early fruit fly embryo. I will show that actin and myosin organize into a network that spans a domain of the embryo that will fold. Redundancy in this supracellular network encodes the tissue’s intrinsic robustness to mechanical and molecular perturbations during folding.
Do leader cells drive collective behavior in Dictyostelium Discoideum amoeba colonies?
Dictyostelium Discoideum (DD) are a fascinating single-cellular organism. When nutrients are plentiful, the DD cells act as autonomous individuals foraging their local vicinity. At the onset of starvation, a few (<0.1%) cells begin communicating with others by emitting a spike in the chemoattractant protein cyclic-AMP. Nearby cells sense the chemical gradient and respond by moving toward it and emitting a cyclic-AMP spike of their own. Cyclic-AMP activity increases over time, and eventually a spiral wave emerges, attracting hundreds of thousands of cells to an aggregation center. How DD cells go from autonomous individuals to a collective entity remains an open question for more than 60 years--a question whose answer would shed light on the emergence of multi-cellular life. Recently, trans-scale imaging has allowed the ability to sense the cyclic-AMP activity at both cell and colony levels. Using both the images as well as toy simulation models, this research aims to clarify whether the activity at the colony level is in fact initiated by a few cells, which may be deemed "leader" or "pacemaker" cells. In this talk, I will demonstrate the use of information-theoretic techniques to classify leaders and followers based on trajectory data, as well as to infer the domain of interaction of leader cells. We validate the techniques on toy models where leaders and followers are known, and then try to answer the question in real data--do leader cells drive collective behavior in DD colonies?
The life of a mucosalivary droplet: Lessons from synthetic breaths and sneezes
The main transmission mode of the COVID-19 disease is through virus-laden aerosols and droplets generated by expiratory events, such as breathing and sneezing. Patients with respiratory diseases are typically treated with oxygenation devices in hospitals, homes, and other settings where they increase the risk of spreading the disease to caregivers and first responders. Here, I will discuss a systematic study of aerosol and droplet dispersal through the air and their final deposition on surfaces. Through laser and fluorescent imaging techniques, we measure the volumetric spatial-temporal dynamics of droplet dispersal while varying rheological properties of the mucosaliva. We then demonstrate that a standard nose and mouth mask reduces the amount of mucosaliva dispersed by a factor of at least a hundred. Our ongoing collaborations with doctors and respiratory therapists from the Baystate Medical Hospital are developing new guidelines to help mitigate disease spread in a hospital setting.
Light-bacteria interactions
In 1676, using candle light and a small glass sphere as the lens, van Leeuwenhoek discovered the microscopic world of living microorganisms. Today, using lasers, spatial light modulators, digital cameras and computers, we study the statistical and fluid mechanics of microswimmers in ways that were unimaginable only 50 years ago. With light we can image swimming bacteria in 3D, apply controllable force fields or sculpt their 3D environment. In addition to shaping the physical world outside cells we can use light to control the internal state of genetically modified bacteria. I will review our recent work with light-bacteria interactions, going from some fundamental problems in the fluid and statistical mechanics of microswimmers to the use of bacteria as propellers for micro-machines or as a "living" paint controlled by light.
“Understanding the Function and Dynamics of Organelles through Imaging”
Powerful new ways to image the internal structures and complex dynamics of cells are revolutionizing cell biology and bio-medical research. In this talk, I will focus on how emerging fluorescent technologies are increasing spatio-temporal resolution dramatically, permitting simultaneous multispectral imaging of multiple cellular components. In addition, results will be discussed from whole cell milling using Focused Ion Beam Electron Microscopy (FIB-SEM), which reconstructs the entire cell volume at 4 voxel resolution. Using these tools, it is now possible to begin constructing an “organelle interactome”, describing the interrelationships of different cellular organelles as they carry out critical functions. The same tools are also revealing new properties of organelles and their trafficking pathways, and how disruptions of their normal functions due to genetic mutations may contribute to important diseases.
Holographic control of neuronal circuits
Genetic targeting of neuronal cells with activity reporters (calcium or voltage indicators) has initiated the paradigmatic transition whereby photons have replaced electrons for reading large-scale brain activities at cellular resolution. This has alleviated the limitations of single cell or extracellular electrophysiological probing, which only give access to the activity of at best a few neurons simultaneously and to population activity of unresolved cellular origin, respectively. In parallel, optogenetics has demonstrated that targeting neuronal cells with photosensitive microbial opsins, enables the transduction of photons into electrical currents of opposite polarities thus writing, through activation or inhibition, neuronal signals in a non-invasive way. These progresses have in turn stimulated the development of sophisticated optical methods to increase spatial and temporal resolution, light penetration depth and imaging volume. Today, nonlinear microscopy, combined with spatio-temporal wave front shaping, endoscopic probes engineering or multi scan heads design, enable in vivo in depth, simultaneous recording of thousands of cells in mm 3 volumes at single-spike precision and single-cell resolution. Joint progress in opsin engineering, wave front shaping and laser development have provided the methodology, that we named circuits optogenetics, to control single or multiple target activity independently in space and time with single- neuron and single-spike precision, at large depths. Here, we will review the most significant breakthroughs of the past years, which enable reading and writing neuronal activity at the relevant spatiotemporal scale for brain circuits manipulation, with particular emphasis on the most recent advances in circuit optogenetics.
Spinners, not swimmers: how sperm flagella fooled us for 350 years - now in 3D!
In the 17th century, Antonie van Leeuwenhoek used one of the earliest microscopes to see how sperm swim. He described the sperm as a “living animalcule” with a “tail, which, when swimming, lashes with a snakelike movement, like eels in water”. Strikingly, this perception of how sperm moves has not changed since. Indeed, anyone today with a modern microscope would make the same observation: sperm swim forward by wiggling their tail symmetrically side-to-side. Our new research using 3D microscopy shows that we have all been victims of a sperm deception, an illusion. Only now we can see that for 350 years we have been wrong about how sperm actually swims.
Keynote talk: Imaging Interacting Organelles to Understand Metabolic Homeostasis
Powerful new ways to image the internal structures and complex dynamics of cells are revolutionizing cell biology and bio-medical research. In this talk, I will focus on how emerging fluorescent technologies are increasing spatio-temporal resolution dramatically, permitting simultaneous multispectral imaging of multiple cellular components. In addition, results will be discussed from whole cell milling using Focused Ion Beam Electron Microscopy (FIB-SEM), which reconstructs the entire cell volume at 4 voxel resolution. Using these tools, it is now possible to begin constructing an “organelle interactome”, describing the interrelationships of different cellular organelles as they carry out critical functions. The same tools are also revealing new properties of organelles and their trafficking pathways, and how disruptions of their normal functions due to genetic mutations may contribute to important diseases.
Dynamics of microbiota communities during physical perturbation
The consortium of microbes living in and on our bodies is intimately connected with human biology and deeply influenced by physical forces. Despite incredible gains in describing this community, and emerging knowledge of the mechanisms linking it to human health, understanding the basic physical properties and responses of this ecosystem has been comparatively neglected. Most diseases have significant physical effects on the gut; diarrhea alters osmolality, fever and cancer increase temperature, and bowel diseases affect pH. Furthermore, the gut itself is comprised of localized niches that differ significantly in their physical environment, and are inhabited by different commensal microbes. Understanding the impact of common physical factors is necessary for engineering robust microbiota members and communities; however, our knowledge of how they affect the gut ecosystem is poor. We are investigating how changes in osmolality affect the host and the microbial community and lead to mechanical shifts in the cellular environment. Osmotic perturbation is extremely prevalent in humans, caused by the use of laxatives, lactose intolerance, or celiac disease. In our studies we monitored osmotic shock to the microbiota using a comprehensive and novel approach, which combined in vivo experiments to imaging, physical measurements, computational analysis and highly controlled microfluidic experiments. By bridging several disciplines, we developed a mechanistic understanding of the processes involved in osmotic diarrhea, linking single-cell biophysical changes to large-scale community dynamics. Our results indicate that physical perturbations can profoundly and permanently change the competitive and ecological landscape of the gut, and affect the cell wall of bacteria differentially, depending on their mechanical characteristics.
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