Mesoscale heterogeneity is a critical determinant for spiral pattern formation in developing social amoeba.

Heterogeneity is a critical determinant for multicellular pattern formation. Although the importance of microscale and macroscale heterogeneity at the single-cell and whole-system levels, respectively, has been well accepted, the presence and functions of mesoscale heterogeneity, such as cell clusters with distinct properties, have been poorly recognized. We investigated the biological importance of mesoscale heterogeneity in signal-relaying abilities (excitability) in the self-organization of spiral waves of intercellular communications by studying the self-organized pattern formation in a population of Dictyostelium discoideum cells, a classical signal-relaying system model.

Determination of Capillary Blood TSH and Free Thyroxine Levels Using Digital Immunoassay.

Background: The remote performance of thyroid function blood tests is complicated because it requires blood collection.

Objective: To compare TSH and free thyroxine (FT4) levels between capillary and venous blood and assess the adequacy of measuring each value in capillary blood.

Methods: This prospective intervention study was conducted at Ito Hospital and was based on the clinical research method.

Development of a quantitative thyroid-stimulating hormone assay system for a benchtop digital ELISA desktop analyzer.

Regular checkups for thyroid-stimulating hormone (TSH) levels are essential for the diagnosis of thyroid disease. The enzyme-linked immunosorbent assay (ELISA) technique is a standard method for detecting TSH in the serum or plasma of hospitalized patients. A recently developed next-generation ELISA, the digital immunoassay (d-IA), has facilitated detection of molecules with ultra-high-sensitivity.

Ratiometric Bioluminescent Indicator for Simple and Rapid Diagnosis of Bilirubin.

Bilirubin in human blood is highly important as a general index of one's physical condition because its concentration changes under the influence of several diseases. In particular, in newborns, jaundice is one of the most common diseases involving unconjugated bilirubin (UCBR), causing serious symptoms such as nuclear jaundice and deafness. Therefore, a frequent measurement of the UCBR levels in the blood is important.

Hierarchical Development of Motile Polarity in Durotactic Cells Just Crossing an Elasticity Boundary.

Cellular durotaxis has been extensively studied in the field of mechanobiology. In principle, asymmetric mechanical field of a stiffness gradient generates motile polarity in a cell, which is a driving factor of durotaxis. However, the actual process by which the motile polarity in durotaxis develops is still unclear.

Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging.

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection.

Imaging local brain activity of multiple freely moving mice sharing the same environment.

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Considering recent demand for its applicability to freely moving subjects, especially for animals in a group and socially interacting with each other, here we propose a new method based on a bioluminescent voltage indicator LOTUS-V. Using our fiber-free recording method based on the LOTUS-V, we succeeded in capturing dynamic change of brain activity in freely moving mice.

Spontaneously Blinking Fluorescent Protein for Simple Single Laser Super-Resolution Live Cell Imaging.

Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched off by excitation light illumination and switched on by thermally induced dehydration, resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

Publisher Correction: Alpha-synuclein facilitates to form short unconventional microtubules that have a unique function in the axonal transport.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with Lp-regularized image reconstruction.

Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination.

Biomimetic Chemical Sensing by Fluorescence Signals Using a Virus-like Particle-Based Platform.

The chemical receptors present in living organisms are promising tools for developing biomimetic chemical sensors. However, these receptors require lipid membranes for functioning under physiological conditions, which prevents their utilization in the production of cell-free in vitro chemical sensing systems. Here, we report the development of a cell-free biomimetic sensing platform using virus-like particles (VLPs) with intact ligand-gated Ca channels and genetically encoded Ca indicator (GECI).

Alpha-synuclein facilitates to form short unconventional microtubules that have a unique function in the axonal transport.

Although α-synuclein (αSyn) has been linked to Parkinson's disease (PD), the mechanisms underlying the causative role in PD remain unclear. We previously proposed a model for a transportable microtubule (tMT), in which dynein is anchored to a short tMT by LIS1 followed by the kinesin-dependent anterograde transport; however the mechanisms that produce tMTs have not been determined. Our in vitro investigations of microtubule (MT) dynamics revealed that αSyn facilitates the formation of short MTs and preferentially binds to MTs carrying 14 protofilaments (pfs).

Intracellular trafficking of particles inside endosomal vesicles is regulated by particle size.

Little comparative information is available on the detailed intracellular dynamics (diffusion, active movement, and distribution mechanisms) of nanoparticles (≤100nm) and sub-micron particles (>100nm). Here, we quantitatively examined the intracellular movements of different-sized particles and of the endosomal vesicles containing those particles. We showed that silica nanoparticles of various sizes (30 to 100nm) had greater motility than sub-micron particles in A549 cells.

Tracking the Movement of a Single Prokaryotic Cell in Extreme Environmental Conditions.

Many bacterial species move toward favorable habitats. The flagellum is one of the most important machines required for the motility in solution and is conserved across a wide range of bacteria. The motility machinery is thought to function efficiently with a similar mechanism in a variety of environmental conditions, as many cells with similar machineries have been isolated from harsh environments.

Genetically encoded ratiometric fluorescent thermometer with wide range and rapid response.

Temperature is a fundamental physical parameter that plays an important role in biological reactions and events. Although thermometers developed previously have been used to investigate several important phenomena, such as heterogeneous temperature distribution in a single living cell and heat generation in mitochondria, the development of a thermometer with a sensitivity over a wide temperature range and rapid response is still desired to quantify temperature change in not only homeotherms but also poikilotherms from the cellular level to in vivo. To overcome the weaknesses of the conventional thermometers, such as a limitation of applicable species and a low temporal resolution, owing to the narrow temperature range of sensitivity and the thermometry method, respectively, we developed a genetically encoded ratiometric fluorescent temperature indicator, gTEMP, by using two fluorescent proteins with different temperature sensitivities.

Genetically encoded bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging.

We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging.

Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging.

Unlabelled: Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons.

Simultaneous imaging of multiple cellular events using high-accuracy fluorescence polarization microscopy.

Förster resonance energy transfer (FRET) has been widely used to design indicators for biomolecules. Conventional FRET-based indicators enable quantitative measurements of analyzes by calculating the ratio between donor and acceptor fluorophores. However, such 'hetero-FRET'-based indicators, which use multiple differently colored fluorophores, restrict the simultaneous use of other colors of fluorescent molecules.

Five colour variants of bright luminescent protein for real-time multicolour bioimaging.

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins.

Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength.

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM).

Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging.

The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging.

A guide to use photocontrollable fluorescent proteins and synthetic smart fluorophores for nanoscopy.

Recent advances in nanoscopy, which breaks the diffraction barrier and can visualize structures smaller than the diffraction limit in cells, have encouraged biologists to investigate cellular processes at molecular resolution. Since nanoscopy depends not only on special optics but also on 'smart' photophysical properties of photocontrollable fluorescent probes, including photoactivatability, photoswitchability and repeated blinking, it is important for biologists to understand the advantages and disadvantages of fluorescent probes and to choose appropriate ones for their specific requirements. Here, we summarize the characteristics of currently available fluorescent probes based on both proteins and synthetic compounds applicable to nanoscopy and provide a guideline for selecting optimal probes for specific applications.