Live cell imaging

Summary: Live imaging studies give unparalleled insight into dynamic single cell behaviours and fate decisions. However, the challenge of reliably tracking single cells over long periods of time limits both the throughput and ease with which such studies can be performed. Here, we present NucliTrack, a cross platform solution for automatically segmenting, tracking and extracting features from fluorescently-labelled nuclei. NucliTrack performs similarly to other state-of-the-art cell tracking algorithms, but NucliTrack's interactive, graphical interface makes it significantly more user friendly...

Fluorescence is an essential imaging modality for labelling and visualising cells and sub-cellular structures. Multicolour labelling is especially challenging due to differences in physicochemical and photophysical behaviour of structurally unrelated fluorophores in the heterogeneous environments found in sub-cellular compartments. Herein, we report the conjugation of three azide-bearing BODIPYs with similar core structures but widely different emission wavelengths (green, red and NIR) to tyrosine residues of a model globular protein (BSA) via a common linking methodology...

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors...

Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pHresponsive, fully-reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH...

A multi-block fluorescent amphiphilic polyurethane copolymer (TPE-PU), self-assembling into hairy, water-soluble micelles, is used as a subcellular microfilament probe in living cells.

Transplantation of a single hematopoietic stem cell is an important method for its functional characterization, but the standard transplantation protocol relies on cell homing to the bone marrow after intravenous injection. Here, we present a method to transplant single cells directly into the bone marrow of live mice. We developed an optical platform that integrates a multiphoton microscope with a laser ablation unit for microsurgery and an optical tweezer for cell micromanipulation. These tools allow image-guided single cell transplantation with high spatial control...

The molecular and genetic networks underlying the determination of floral organ identity are well studied, but much less is known about how the flower is partitioned into four developmentally distinct whorls. The SUPERMAN gene is required for proper specification of the boundary between stamens in whorl 3 and carpels in whorl 4, as superman mutants exhibit supernumerary stamens but usually lack carpels. However, it has remained unclear whether extra stamens in superman mutants originate from an organ identity change in whorl 4 or the overproliferation of whorl 3...

From March 27-29 2017, the RIKEN Center for Developmental Biology held a symposium entitled 'Towards Understanding Human Development, Heredity, and Evolution' in Kobe, Japan. Recent advances in technologies including stem cell culture, live imaging, single-cell approaches, next-generation sequencing and genome editing have led to an expansion in our knowledge of human development. Organized by Yoshiya Kawaguchi, Mitinori Saitou, Mototsugu Eiraku, Tomoya Kitajima, Fumio Matsuzaki, Takashi Tsuji and Edith Heard, the symposium covered a broad range of topics including human germline development, epigenetics, organogenesis and evolution...

A new carbazole-azine based fluorescent sensor was synthesized and characterized. The selectivity of the sensor for Cu(2+) over other counter ions in a dimethyl sulfoxide/H2 O mixture was shown through enhancement in fluorescence - an off to on transformation. The specificity of the probe towards Cu(2+) was evident in ultraviolet/visible, fluorescence, Fourier transform infrared and mass studies. Application of the probe in the cell imaging and cytotoxicity of living cells is illustrated.

PURPOSE: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments. PROCEDURES: The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively...

Metabolic reprogramming is widely considered a hallmark of cancer, and understanding metabolic dynamics described by the conversion rates or "fluxes" of metabolites can shed light onto biological processes of tumorigenesis and response to therapy. For real-time analysis of metabolic flux in intact cells or organisms, magnetic resonance (MR) spectroscopy and imaging methods have been developed in conjunction with hyperpolarization of nuclear spins. These approaches enable noninvasive monitoring of tumor progression and treatment efficacy and are being tested in multiple clinical trials...

Bending of 2D cell sheets is a fundamental morphogenetic mechanism during animal development and reproduction. A critical player driving cell shape during tissue bending is the actin cytoskeleton. Much of our current knowledge about actin dynamics in whole organisms stems from studies of embryonic development in bilaterian model organisms. Here, we have analysed actin-based processes during asexual bud evagination in the simple metazoan Hydra We created transgenic Hydra strains stably expressing the actin marker Lifeact-GFP in either ectodermal or endodermal epitheliomuscular cells...

Carbon quantum dots (CQDs) have attracted tremendous attention for their prominent fluorescence, excellent stability, and outstanding biocompatibility. Herein, we report a facile method to prepare CQDs from walnut shells, which mainly consist of natural cellulose. After carbonization of the walnut shells and acid treatments, zigzag and armchair edges of CQDs with an average size of 3.4nm were revealed by the high-resolution transmission electron microscopy. Consistent with the (100) planes of graphitic carbon, the lattice spacing distance of these CQDs was 0...

Described here is a localized H2 O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2 O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2 O2 generated by the activated DAAO...

Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker - a custom software for automatic tracking of diverse moving objects usable on various microscope setups...

Drug-induced liver injury (DILI) is considered a serious problem related to public health, due to its unpredictability and acute response. The level of peroxynitrite (ONOO-) generated in liver has long been regarded as a biomarker for the prediction and measurement of DILI. Herein we present two reaction based fluorescent probes (Naph-ONOO- and Rhod-ONOO-) for ONOO- through a novel and universally applicable mechanism: ONOO- mediated deprotection of -keto caged fluorophores. Among them, Rhod-ONOO- can selectively accumulate and react in mitochondria, one of the main sources of ONOO-, with a substantial lower nano-molar sensitivity of 43 nM...

Conjugated polymers have great potential applications in bioimaging. However, the aggregation of conjugated polymers driven by electrostatic and hydrophobic interactions in aqueous media results in the reduction of photoluminescence quantum efficiency. In present work we synthesized a carboxylate gemini surfactant (SDHC) to adjust the aggregation behaviors and fluorescence properties of conjugated polymers (anionic MPS-PPV and cationic PMNT). This gemini surfactant shows very low critical micellar concentration (CMC) in aqueous solution and forms vesicles above CMC...

Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types...

Cell movement and intercellular signaling occur simultaneously to organize morphogenesis during embryonic development. Cell movement can cause relative positional changes between neighboring cells. When intercellular signals are local such cell mixing may affect signaling, changing the flow of information in developing tissues. Little is known about the effect of cell mixing on intercellular signaling in collective cellular behaviors and methods to quantify its impact are lacking. Here we discuss how to determine the impact of cell mixing on cell signaling drawing an example from vertebrate embryogenesis: the segmentation clock, a collective rhythm of interacting genetic oscillators...

We report a direct imaging tool, HGEu001, for primary cilia in living cells, which is specific, and based on the UV light or near infrared laser (via two-photon excitation) induced long-lived europium luminescence.