Extending applications of AFM to fluidic AFM in single living cell and extracellular vesicle studies

In this article, a review of the application of atomic force microscopy (AFM) for the analyses of extracellular vesicles is presented. This information is then extended to include fluidic Atomic Force Microscopy (fluidic AFM) applications. Fluidic AFM is an offshoot of AFM that combines a microfluidic cantilever with AFM and has enabled the research community to conduct biological, pathological, and pharmacological studies on cells at the single-cell level in a liquid environment. AFM applications involving single cell and extracellular vesicle studies, colloidal force spectroscopy, and single cell adhesion measurements are discussed. In this review, new results are offered, using fluidic AFM, to illustrate (1) the speed with which sequential measurements of adhesion using coated colloid beads can be done, (2) the ability to assess lateral binding forces (LBFs) of endothelial or epithelial cells in a confluent cell monolayer in appropriate physiological environment, and (3) the ease of measurement of vertical binding force (VBFs) of intercellular adhesion between heterogeneous cells. Finally, key applications are discussed that include extracellular vesicle absorption, manipulation of a single living cell by intracellular injection, sampling of cellular fluid from a single living cell, patch clamping, and mass measurements of a single living cell.

Cryo secondary ion mass spectrometry for wood component visualization: a mini review

Various phenomena in living physiological systems are conducted on the hydrated conditions, and in many cases, they do not work in a dry state. Imaging mass spectrometry is one of the direct detection methods scanning the sample surface with some focused and pulsed energy and analysing the sputtered components. However, under the high vacuum conditions required for usual imaging mass spectrometry, the sample surface is rapidly dried. It is difficult for the target cell to survive, and the original situation are lost soon. Here, the combination of a freeze-fixation and a cryo sample stage is a promising method to do mass spectrometry while maintaining the original situation. By rapidly freezing the cells, the momentary situation as a living cell is fixed. The situation in a living cell can be captured as still images by cryo imaging mass spectrometry. This mini-review introduces the outline of imaging mass spectrometry especially for low molecular weight components and recent results for frozen-hydrated samples by cryo secondary ion mass spectrometry.

On the reproductive mechanism of Gram-negative protocells

Protocells are thought to have existed on early Earth before the origin of prokaryotes. These primitive cells are believed to have carried out processes like replication solely based on the physicochemical properties of their cell constituents. Despite considerable efforts, replication of a living cell-driven entirely by laws of physics and chemistry has never been achieved. To test this hypothesis, we transformed extant bacteria into sacks of cytoplasm, incapable of regulating either their morphology or reproductive processes. We then exposed these proxy-protocells (bacterial protoplasts) to presumed Archaean Eon environmental conditions to understand if or how these cells reproduce. Contrary to the current presumption that bacterial protoplasts reproduce in a haphazard manner, under our experimental conditions they reproduced via a multi-stage reproductive cycle, resulting in viable daughter cells. Our observations suggest that this mechanism of reproduction could in fact be well explained from a biophysical perspective. Based on our observations we argue that this method of reproduction is better suited for the environmental conditions of early Earth.

Chemical communication at the synthetic cell/living cell interface

Although the complexity of synthetic cells has continued to increase in recent years, chemical communication between protocell models and living organisms remains a key challenge in bottom-up synthetic biology and bioengineering. In this Review, we discuss how communication channels and modes of signal processing can be established between living cells and cytomimetic agents such as giant unilamellar lipid vesicles, proteinosomes, polysaccharidosomes, polymer-based giant vesicles and membrane-less coacervate micro-droplets. We describe three potential modes of chemical communication in consortia of synthetic and living cells based on mechanisms of distributed communication and signal processing, physical embodiment and nested communication, and network-based contact-dependent communication. We survey the potential for applying synthetic cell/living cell communication systems in biomedicine, including the in situ production of therapeutics and development of new bioreactors. Finally, we present a short summary of our findings.

Active Segregation Dynamics in the Living Cell

In this paper, we bring together our efforts in identifying and understanding nonequilibrium phase segregation driven by active processes in the living cell, with special focus on the segregation of cell membrane components driven by active contractile stresses arising from cortical actomyosin. This also has implications for active segregation dynamics in membraneless regions within the cytoplasm and nucleus (3d). We formulate an active version of the Flory-Huggins theory that incorporates a contribution from fluctuating active stresses. Apart from knitting together some of our past theoretical work in a comprehensive narrative, we highlight some new results, and establish a cor- respondence with recent studies on Active Model B/B+. We point to the many unusual aspects of the dynamics of active phase segregation, such as (i) anomalous growth dynamics, (ii) coarsening accompanied by propulsion and coalescence of domains that exhibit nonreciprocal effects, (iii) seg- regation into mesoscale domains, (iv) emergence of a nonequilibrium phase segregated steady state characterised by strong macroscopic fluctuations (fluctuation dominated phase ordering (FDPO)), and (v) mesoscale segregation even above the equilibrium Tc. Apart from its implications for actively driven segregation of binary fluids, these ideas are at the heart of an Active Emulsion description of the lateral organisation of molecules on the plasma membrane of living cells, whose full molecular elaboration appears elsewhere.