Single-cell Tracking Research Articles

Single-cell Raman micro-spectroscopy for tracking of carotenoids in cyanobacteria exposed to Mn and Zn doped ferrite nanoparticles

Ferrite nanoparticles are increasingly produced and exploited as adsorbents for environmental pollutants. However, their impact on the aquatic microbiota such as cyanobacteria, are not yet investigated. Targeting the environmental monitoring context, in this paper we explored for the first time if any change in the carotenoid signal from cyanobacteria Coelomoron pussilum (AICB 1012) exposed to non-lethal doses of Mn and Zn doped ferrite nanoparticles (NPs) can be associated with the nano-aggression on single-cell level, using micro-Raman spectroscopy. UV–Vis absorption spectroscopy of the bulk culture and single-cell Raman microscopy showed that the carotenoid signal increases relative to the chlorophyll contribution upon exposure of the cells to the Mn-ferrite NPs throughout the 7 days of the experiment. The red-shift and broadening of the strongest carotenoid Raman band arising from (CC) stretching modes indicates the change of carotenoid profile towards increased amount of β-carotene in answer to the NPs stress. The increase of this band intensity relative to the fluorescence background was also observed in Zn-ferrite NPs treatment. Using a simplified and rapid sample preparation procedure, electron microscopy in both transmission and scanning modes, showed greater coverage of the cells by the stable colloidal AgNPs than by the magnetic ferrite NPs. The latter mostly clumped together rather than adhering to the cells. The combined single-cell micro-Raman tracking of physiological response of the unicellular photosynthetic microorganisms coupled with electron microscopy approach to visualise cell-NPs interaction and the extracellular polymeric substance secretion holds the promise for rapid assessment of the NPs-induced environmental stress acting on the unicellular organisms.

Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry.

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 μm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

CellTracker: an automated toolbox for single-cell segmentation and tracking of time-lapse microscopy images.

Recent advances of long-term time-lapse microscopy have made it easy for researchers to quantify cell behavior and molecular dynamics at single-cell resolution. However, the lack of easy-to-use software tools optimized for customized research is still a major challenge for quantitatively understanding biological processes through microscopy images. Here, we present CellTracker, a highly integrated graphical user interface software, for automated cell segmentation and tracking of time-lapse microscopy images. It covers essential steps in image analysis including project management, image pre-processing, cell segmentation, cell tracking, manually correction and statistical analysis such as the quantification of cell size and fluorescence intensity, etc. Furthermore, CellTracker provides an annotation tool and supports model training from scratch, thus proposing a flexible and scalable solution for customized dataset analysis. CellTracker is an open-source software under the GPL-3.0 license. It is implemented in Python and provides an easy-to-use graphical user interface. The source code, instruction manual and demos can be found at https://github.com/WangLabTHU/CellTracker. Supplementary data are available at Bioinformatics online.

Tracking Longitudinal Population Dynamics of Single Neuronal Calcium Signal Using SCOUT

In vivo calcium imaging enables simultaneous recording of large neuronal ensembles engaged in complex operations. Many experiments require monitoring and identification of cell populations across multiple sessions. Population cell tracking across multiple sessions is complicated by non-rigid transformations induced by cell movement and imaging field shifts. We introduce SCOUT (Single-Cell SpatiOtemporal LongitUdinal Tracking), a fast, robust cell tracking method utilizing multiple cell-cell similarity metrics, probabilistic inference, and an adaptive clustering methodology to perform cell identification across multiple sessions. By comparing SCOUT with current popular cell tracking algorithms on simulated, 1-photon and 2-photon recordings, we empirically show this approach improves cell tracking quality, particularly when recordings exhibit spatial footprint movement between sessions or poor neural extraction quality.

HGG-16. EXOSOME-MEDIATED INTER-CLONAL INTERACTIONS IN PEDIATRIC GBM AND DIPG

Pediatric Glioblastoma (pGBM) and Diffuse Intrinsic Pontine Glioma (DIPG) are highly heterogeneous brain tumors which we demonstrated are comprised by distinct sub-clones interacting in a functional network. Exosomes are known to mediate the crosstalk between tumor and its microenvironment. Based on this, we aimed to investigate the role of exosomes in mediating pGBM and DIPG inter-clonal communication. By using optical barcoding for single cell-tracking, we generated two bulk multicolor patient derived-cell lines (one DIPG H3.3K27M and one pGBM histone WT) from which we obtained two and five single cell-derived clones respectively. The sub-clones demonstrated significantly phenotypic differences in terms of morphology, growth, adhesion, migration and invasion properties. In particular, co-culture experiments, with the two most different clones for both cell-lines, confirmed the cell-cell interaction key role in driving their more aggressive phenotype. Furthermore, we found that pGBM and DIPG sub-clones release exosomes which are actively and differentially up-taken by individual clones. Analysis of the exosomal microRNAs showed a different profile between the two selected clones in each cell-line. In particular, we found a pool of five upregulated microRNAs in 1C5 clone (DIPG cell-line) strongly associated to Wnt-signaling and PI3K-AKT pathway. Similarly, a pool of five upregulated microRNAs for 5E2 clone (pGBM cell-line) were found associated with focal adhesion and PI3K-AKT pathway. Our study may provide novel therapeutic strategies by interfering with the exosome-mediated inter-clonal communication in pGBM and DIPG.

Direct Antimicrobial Susceptibility Testing on Clinical Urine Samples by Optical Tracking of Single Cell Division Events.

With the increasing prevalence of antibiotic resistance, the need to develop antimicrobial susceptibility testing (AST) technologies is urgent. The current challenge has been to perform the antibiotic susceptibility testing in short time, directly with clinical samples, and with antibiotics over a broad dynamic range of clinically relevant concentrations. Here, a technology for point-of-care diagnosis of antimicrobial-resistant bacteria in urinary tract infections, by imaging the clinical urine samples directly with an innovative large volume solution scattering imaging (LVSi) system and analyzing the image sequences with a single-cell division tracking method is developed. The high sensitivity of single-cell division tracking associated with large volume imaging enables rapid antibiotic susceptibility testing directly on the clinical urine samples. The results demonstrate direct detection of bacterial infections in 60clinical urine samples with a 60 min LVSi video, and digital AST of 30 positive clinical samples with 100% categorical agreement with both the clinical culture results and the on-site agar plating validation results. This technology provides opportunities for precise antibiotic prescription and proper treatment of the patient within a single clinic visit.

Propionate Enhances Cell Speed and Persistence to Promote Intestinal Epithelial Turnover and Repair.

Background and AimsGut bacteria-derived short-chain fatty acids (SCFAs) play crucial roles in the maintenance of intestinal homeostasis. However, how SCFAs regulate epithelial turnover and tissue repair remain incompletely understood. In this study, we investigated how the SCFA propionate regulates cell migration to promote epithelial renewal and repair.MethodsMouse small intestinal epithelial cells (MSIE) and human Caco-2 cells were used to determine the effects of SCFAs on gene expression, proliferation, migration, and cell spreading in vitro. Video microscopy and single cell tracking were used to assess cell migration kinetically. 5-bromo-2’-deoxyuridine (BrdU) and hydroxyurea were used to assess the effects of SCFAs on migration in vivo. Lastly, an acute colitis model using dextran sulfate sodium (DSS) was used to examine the effects of SCFAs in vivo.ResultsUsing video microscopy and single cell tracking, we found that propionate promoted intestinal epithelial cell migration by enhancing cell spreading and polarization, which led to increases in both cell speed and persistence. This novel function of propionate was dependent on inhibition of class I histone deacetylases (HDAC) and GPR43 and required signal transducer and activator of transcription 3 (STAT3). Furthermore, using 5-bromo-2’-deoxyuridine (BrdU) and hydroxyurea in vivo, we found that propionate enhanced cell migration up the crypt-villus axis under homeostatic conditions, while also protecting against ulcer formation in experimental colitis.ConclusionOur results demonstrate a mechanism by which propionate stimulates cell migration in an HDAC inhibition, GPR43, and STAT3 dependent manner, and suggest that propionate plays an important role in epithelial migration independent of proliferation.

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development.

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.

Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements

Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of the rotational direction, which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150°. The flagellar helicity remained right-handed with a 1.3 μm pitch and 0.14 μm helix radius, which is consistent with the feature of a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not spread on a semi-solid surface. Based on these results, we concluded that a flagellar polymorphism is essential for spreading in structured environments.

Constrained optimal foraging by marine bacterioplankton on particulate organic matter

Optimal foraging theory provides a framework to understand how organisms balance the benefits of harvesting resources within a patch with the sum of the metabolic, predation, and missed opportunity costs of foraging. Here, we show that, after accounting for the limited environmental information available to microorganisms, optimal foraging theory and, in particular, patch use theory also applies to the behavior of marine bacteria in particle seascapes. Combining modeling and experiments, we find that the marine bacterium Vibrio ordalii optimizes nutrient uptake by rapidly switching between attached and planktonic lifestyles, departing particles when their nutrient concentration is more than hundredfold higher than background. In accordance with predictions from patch use theory, single-cell tracking reveals that bacteria spend less time on nutrient-poor particles and on particles within environments that are rich or in which the travel time between particles is smaller, indicating that bacteria tune the nutrient concentration at detachment to increase their fitness. A mathematical model shows that the observed behavioral switching between exploitation and dispersal is consistent with foraging optimality under limited information, namely, the ability to assess the harvest rate of nutrients leaking from particles by molecular diffusion. This work demonstrates how fundamental principles in behavioral ecology traditionally applied to animals can hold right down to the scale of microorganisms and highlights the exquisite adaptations of marine bacterial foraging. The present study thus provides a blueprint for a mechanistic understanding of bacterial uptake of dissolved organic matter and bacterial production in the ocean-processes that are fundamental to the global carbon cycle.

Lnc GNG12-AS1 knockdown suppresses glioma progression through the AKT/GSK-3β/β-catenin pathway.

Background: Long non-coding RNAs (lncRNAs) are increasingly being regarded as regulators of glioma development. Notably, some studies report that GNG12-AS1 plays important functions and molecular mechanism in breast cancer, but there are no existing studies in glioma.Objective: To analyze the biological functions and potential mechanisms of GNG12-AS1 in glioma.Methods: We detected the expression of GNG12-AS1 in glioma tissues through analyzing TCGA data as well as our clinical samples. We then evaluated cell proliferation through MTT assay and colony formation and cell migration by transwell assay, wound healing assay and single cell tracking assay. After, we analyzed the effects of the AKT/GSK-3β/β-catenin through Western blotting and utilized the β-catenin agonist SKL2001 for the rescue experiment.Results: GNG12-AS1 was highly expressed in glioma tissues. The silence of GNG12-AS1 inhibited the proliferation, migration and epithelial–mesenchymal transition of glioma cells, and reduced the activity of the AKT/GSK-3β/β-catenin pathway. Notably, SKL2001 could reverse cell migration as well as β-catenin expression in glioma cells with lower GNG12-AS1 expression.Conclusions: GNG12-AS1 regulates proliferation and migration of glioma cells through the AKT/GSK-3β/β-catenin signaling and can perhaps be a new target for the treatment of glioma.

Abstract 619: Dynamic protein expression governs cellular PARP inhibitor response and represents a pathway to single cell drug resistance

Abstract Protein expression functions as a biomarker for precision drug therapy to more effectively treat cancer or as a measurement of drug resistance. Expression levels are commonly determined in bulk populations of cells at single time points. However, cells often undergo dynamic expression during therapeutic response that may influence drug resistance. Therefore, how changing expression levels dynamically impact cellular response remains largely unknown, limiting determination of bona fide protein biomarkers while leaving mechanisms of therapeutic resistance underappreciated. Drugs inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP) have been increasingly used to treat various cancers. PARP inhibitors (PARPi) are especially potent in cancers exhibiting deficient homologous recombination, or BRCAness. Yet, acquired PARPi resistance is common and remains a major area of concern, underscoring the importance of discovering how to overcome resistance mechanisms. To determine the impact of dynamic protein expression on PARPi resistance, we have developed a platform approach that dynamically tracks specific protein expression during drug treatment in single cells. We used CAS9-directed plasmid insertion to precisely couple fluorescent protein expression with translation of PARP1 and, combined with longitudinal (days) single cell tracking, correlated PARP1 expression levels with cellular fate during drug exposure. Remarkably, we found that dynamic expression of PARP1 controls cellular fate - division, death, or senescence. PARPi were effective in breast cancer cells when PARP1 expression remained steady. However, cells with similar initial PARP1 expression that significantly increased expression proved resistant. Furthermore, while all PARPi enzymatically inhibit the target protein, the 5 approved drugs display a differential ability to trap PARP1 onto damaged DNA, producing increased potency. We found dynamic PARP1 expression impacted PARPi resistance differently based on drug trapping ability, suggesting a mechanism to select the most effective PARPi for each patient. Guided by a CRISPR knockout screen, we then determined dynamic expression of other important proteins in PARPi response, finding dynamic expression influence in cellular resistance. These results highlight the role of dynamic DNA damage repair in response to PARPi and present potential combination therapy targets. Complementary to dynamic transcriptional profiling of cell lines under drug treatment, our approach displays significant resistance dependence on dynamic protein expression, but provides increased sensitivity through tracking single cells. Overall, our novel approach to measure dynamic protein expression levels highlights previously unknown avenues of cellular resistance. Citation Format: Bianca Fernandez, J Matthew Dubach. Dynamic protein expression governs cellular PARP inhibitor response and represents a pathway to single cell drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 619.

In vivo tracking of single cells with PET.

Positron emission tomography enables the tracking, in a mouse, of an intravenously injected cell radiolabelled with mesoporous silica nanoparticles containing 68Ga.

Salinomycin Treatment Specifically Inhibits Cell Proliferation of Cancer Stem Cells Revealed by Longitudinal Single Cell Tracking in Combination with Fluorescence Microscopy

A cell line derived from a tumor is a heterogeneous mixture of phenotypically different cells. Such cancer cell lines are used extensively in the search for new anticancer drugs and for investigating their mechanisms of action. Most studies today are population-based, implying that small subpopulations of cells, reacting differently to the potential drug go undetected. This is a problem specifically related to the most aggressive single cancer cells in a tumor as they appear to be insensitive to the drugs used today. These cells are not detected in population-based studies when developing new anticancer drugs. Thus, to get a deeper understanding of how all individual cancer cells react to chemotherapeutic drugs, longitudinal tracking of individual cells is needed. Here we have used digital holography for long time imaging and longitudinal tracking of individual JIMT-1 breast cancer cells. To gain further knowledge about the tracked cells, we combined digital holography with fluorescence microscopy. We grouped the JIMT-1 cells into different subpopulations based on expression of CD24 and E-cadherin and analyzed cell proliferation and cell migration for 72 h. We investigated how the cancer stem cell (CSC) targeting drug salinomycin affected the different subpopulations. By uniquely combining digital holography with fluorescence microscopy we show that salinomycin specifically targeted the CD24− subpopulation, i.e., the CSCs, by inhibiting cell proliferation, which was evident already after 24 h of drug treatment. We further found that after salinomycin treatment, the surviving cells were more epithelial-like due to the selection of the CD24+ cells.

Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice.

Transformed astrocytes in the most aggressive form cause glioblastoma, the most common cancer in the central nervous system with high mortality. The physiological electric field by neuronal local field potentials and tissue polarity may guide the infiltration of glioblastoma cells through the electrotaxis process. However, microenvironments with multiplex gradients are difficult to create. In this work, we have developed a hybrid microfluidic platform to study glioblastoma electrotaxis in controlled microenvironments with high throughput quantitative analysis by machine learning-powered single cell tracking software. By equalizing the hydrostatic pressure difference between inlets and outlets of the microchannel, uniform single cells can be seeded reliably inside the microdevice. The electrotaxis of two glioblastoma models, T98G and U-251MG, requires an optimal laminin-containing extracellular matrix and exhibits opposite directional and electro-alignment tendencies. Calcium signaling is a key contributor in glioblastoma pathophysiology but its role in glioblastoma electrotaxis is still an open question. Anodal T98G electrotaxis and cathodal U-251MG electrotaxis require the presence of extracellular calcium cations. U-251MG electrotaxis is dependent on the P/Q-type voltage-gated calcium channel (VGCC) and T98G is dependent on the R-type VGCC. U-251MG electrotaxis and T98G electrotaxis are also mediated by A-type (rapidly inactivating) voltage-gated potassium channels and acid-sensing sodium channels. The involvement of multiple ion channels suggests that the glioblastoma electrotaxis is complex and patient-specific ion channel expression can be critical to develop personalized therapeutics to fight against cancer metastasis. The hybrid microfluidic design and machine learning-powered single cell analysis provide a simple and flexible platform for quantitative investigation of complicated biological systems.

Spatiotemporal dynamics of bacterial collective migration

Collective migration is a highly ordered collective behavior exhibited by species in response to a stimulus. Such behavior can help a population to gain a competitive advantage ahead of environmental changes. However, the process that shapes collective migration is complicated by the involvement of multiple factors, including genetics, phenotypic diversity, and the environment. In the last 50 years, mathematical models, computerized simulations, and quantitative experiments have all contributed to significant progress in the study of spatiotemporal dynamics in bacterial population migration. In this review, we first summarize the fundamental research on single-cell behavior of bacteria and collective migration, then overview recent work on the spatiotemporal dynamics of bacterial population migration and the applications of quantitative synthetic biology in collective migration research. Common bacteria such as Escherichia coli undergo random walks when grown in suspension in bulk liquid, consisting of successions of “runs” and “tumbles”. To find favorable environments for survival, bacteria process chemical stimuli by swimming towards (or away from) the sources of attractants (or repellents). Bacteria evaluate changes in signal concentrations by a well-characterized chemotaxis pathway, and then modulate the tumble frequency accordingly. Increasing levels of a chemoattractant decrease the tumble frequency, allowing the bacteria to approach the source of chemoattractant. Bacteria tend to live in groups in the natural environment and exhibit collective migration. Two forms of bacterial collective migration, chemotactic migration and swarming, have been extensively studied. In a capillary or semisolid medium containing a chemoattractant, the bacterial population consumes the attractant to form a gradient. Such a gradient attracts more bacteria to concentrate at the location of higher attractant concentration, allowing the population to re-establish the gradient. The attractant-consumption loop results in chemotactic collective migration of bacteria. By contrast, on the moist surface of a solid medium, highly compact bacteria swim on a thin layer above the surface to form a swarm. The population growth and collision between bacterial cells pushes the groups to expand and results in swarming. To understand the spatiotemporal dynamics of bacterial collective motion, mathematical models were developed together with single-cell tracking techniques. Based on the classic Keller-Segel model, which describes the macroscopic movement of chemotactic collective migration, recent studies have incorporated the chemotaxis pathway of single cells or have examined the effects of phenotypic diversity. The details of chemotactic collective migration in bacteria are becoming increasingly clear. Using another approach, recent studies of bacterial swarming combined the Vichek model of collective motion with fluid dynamics to reveal coordinated behaviors within the swarm. Furthermore, interactions between bacteria and physical obstacles were found to have significant impacts on bacterial motion. Developments in synthetic biology have provided tools to alter bacterial behavior through synthetic gene circuits. Scientists can then alter bacterial motility quantitatively to better understand bacterial collective migration, or to create collective patterns using specially designed gene circuits. These studies show great potential for the application of synthetic biology in studies of bacterial collective migration.

Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots

Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity.

Whole-body tracking of single cells via positron emission tomography.

In vivo molecular imaging can measure the average kinetics and movement routes of injected cells through the body. Yet owing to the non-specific accumulation of the contrast agent and its efflux from the cells, most such imaging methods suffer from inaccurate estimations of the distribution of the cells. Here, we show that single human breast cancer cells loaded with mesoporous silica nanoparticles concentrating the 68Ga radioisotope and injected in immunodeficient mice can be tracked in real time from the pattern of annihilation photons detected by positron emission tomography, with respect to anatomical landmarks derived from X-ray computed tomography. We show that the cells travelled at an average velocity of 50 mm/s and arrested in the lungs two-to-three seconds after tail-vein injection in the mice, which is consistent with the blood-flow rate. Single-cell tracking could be used to determine the kinetics of cell trafficking and arrest during the earliest phase of the metastatic cascade, the trafficking of immune cells during cancer immunotherapy, and the distribution of cells after transplantation.

Optical coherence tomography and fluorescence microscopy dual-modality imaging for in vivo single-cell tracking with nanowire lasers.

Emerging cell-based therapies such as stem cell therapy and immunotherapy have attracted broad attention in both biological research and clinical practice. However, a long-standing technical gap of cell-based therapies is the difficulty of directly assessing treatment efficacy via tracking therapeutically administered cells. Therefore, imaging techniques to follow the in vivo distribution and migration of cells are greatly needed. Optical coherence tomography (OCT) is a clinically available imaging technology with ultrahigh-resolution and excellent imaging depth. It also shows great potential for in vivo cellular imaging. However, due to the homogeneity of current OCT cell labeling contrast agents (such as gold and polymer nanoparticles), only the distribution of entire cell populations can be observed. Precise tracking of the trajectory of individual single cells is not possible with such conventional contrast agents. Microlasers may provide a route to track unique cell identifiers given their small size, high emission intensities, rich emission spectra, and narrow linewidths. Here, we demonstrate that nanowire lasers internalized by cells provide both OCT and fluorescence signal. In addition, cells can be individually identified by the unique lasing emission spectra of the nanowires that they carry. Furthermore, single cell migration trajectories can be monitored both in vitro and in vivo with OCT and fluorescence microscopy dual-modality imaging system. Our study demonstrates the feasibility of nanowire lasers combined with the dual-modality imaging system for in vivo single cell tracking with a high spatial resolution and identity verification, an approach with great utility for stem cell and immunomodulatory therapies.