Live Plant Cell Tracking: Fiji plugin to analyze cell proliferation dynamics and understand morphogenesis.

Arabidopsis (Arabidopsis thaliana) primary and lateral roots (LRs) are well suited for 3D and 4D microscopy, and their development provides an ideal system for studying morphogenesis and cell proliferation dynamics. With fast-advancing microscopy techniques used for live-imaging, whole tissue data are increasingly available, yet present the great challenge of analyzing complex interactions within cell populations. We developed a plugin "Live Plant Cell Tracking" (LiPlaCeT) coupled to the publicly available ImageJ image analysis program and generated a pipeline that allows, with the aid of LiPlaCeT, 4D cell tracking and lineage analysis of populations of dividing and growing cells. The LiPlaCeT plugin contains ad hoc ergonomic curating tools, making it very simple to use for manual cell tracking, especially when the signal-to-noise ratio of images is low or variable in time or 3D space and when automated methods may fail. Performing time-lapse experiments and using cell-tracking data extracted with the assistance of LiPlaCeT, we accomplished deep analyses of cell proliferation and clonal relations in the whole developing LR primordia and constructed genealogical trees. We also used cell-tracking data for endodermis cells of the root apical meristem (RAM) and performed automated analyses of cell population dynamics using ParaView software (also publicly available). Using the RAM as an example, we also showed how LiPlaCeT can be used to generate information at the whole-tissue level regarding cell length, cell position, cell growth rate, cell displacement rate, and proliferation activity. The pipeline will be useful in live-imaging studies of roots and other plant organs to understand complex interactions within proliferating and growing cell populations. The plugin includes a step-by-step user manual and a dataset example that are available at https://www.ibt.unam.mx/documentos/diversos/LiPlaCeT.zip.

High-complexity extracellular barcoding using a viral hemagglutinin.

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.

Recent Advances in Molecular Imaging with Gold Nanoparticles.

Gold nanoparticles (AuNP) have been extensively developed as contrast agents, theranostic platforms, and probes for molecular imaging. This popularity has yielded a large number of AuNP designs that vary in size, shape, surface functionalization, and assembly, to match very closely the requirements for various imaging applications. Hence, AuNP based probes for molecular imaging allow the use of computed tomography (CT), fluorescence, and other forms of optical imaging, photoacoustic imaging (PAI), and magnetic resonance imaging (MRI), and other newer techniques. The unique physicochemical properties, biocompatibility, and highly developed chemistry of AuNP have facilitated breakthroughs in molecular imaging that allow the detection and imaging of physiological processes with high sensitivity and spatial resolution. In this Review, we summarize the recent advances in molecular imaging achieved using novel AuNP structures, cell tracking using AuNP, targeted AuNP for cancer imaging, and activatable AuNP probes. Finally, the perspectives and current limitations for the clinical translation of AuNP based probes are discussed.

Preclinical 19F MRI cell tracking at 3 Tesla.

PURPOSE: To develop methods for fluorine-19 (19F) MRI cell tracking in mice on a 3 Tesla clinical scanner. Compared to iron-based cell tracking, 19F MRI has lower sensitivity and, consequently, preclinical 19F cell tracking has only been performed at relatively high magnetic field strengths (> 3 T). Here, we focus on using 19F MRI to detect macrophages in tumors; macrophage density is an indication of tumor aggressiveness and, therefore, 19F MRI could be used as an imaging biomarker. METHODS: Perfluorocarbon (PFC)-labeled macrophages were imaged at 3 T and NMR spectroscopy was performed to validate 19F spin quantification. In vivo 19F MRI was performed on tumor-bearing mice, post-PFC at both 9.4 T and 3 T. 3 T MRI utilized varying NEX and 19F images were analyzed two different ways for 19F quantification. RESULTS: As few as 25,000 cells could be detected as cell pellets at 3 T. 19F quantification in cell pellets by 3 T MRI agreed with NMR spectroscopy. 19F signal was observed in the liver, spleen and tumor in all mice at 9.4 T and 3 T and there was no significant difference in 19F spin quantification. CONCLUSION: This study demonstrates the ability to detect and quantify 19F signal in murine tumors using 19F MRI at 3 T.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture Part 2: Image sequence analysis based evaluation and biological application.

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer unique flow characteristics and support enhanced fluidic interaction for improved immunoaffinity based cell capture. The performance of the microdevice was evaluated by an image sequence analysis based in-house developed single-cell tracking system. Individual cell tracking allowed in-depth analysis of the cell-chip surface interaction mechanism from hydrodynamic point of view. Simulation results were validated by using the hybrid device and the optimized surface functionalization procedure. Finally, the cell capture capability of this new generation microdevice was demonstrated by efficiently arresting cells from a HT29 cell-line suspension.

Analysis of live cell images: Methods, tools and opportunities.

Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits.

Development of an optical system for the non-invasive tracking of stem cell growth on microcarriers.

The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non-invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi-illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12-day culture noted, prior to the onset of aggregation. The developed image acquisition system and post-processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. Biotechnol. Bioeng. 2017;114: 2032-2042. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Current State-of-the-Art 3D Tissue Models and Their Compatibility with Live Cell Imaging.

Mammalian cells grow within a complex three-dimensional (3D) microenvironment where multiple cells are organized and surrounded by extracellular matrix (ECM). The quantity and types of ECM components, alongside cell-to-cell and cell-to-matrix interactions dictate cellular differentiation, proliferation and function in vivo. To mimic natural cellular activities, various 3D tissue culture models have been established to replace conventional two dimensional (2D) culture environments. Allowing for both characterization and visualization of cellular activities within possibly bulky 3D tissue models presents considerable challenges due to the increased thickness and subsequent light scattering features of such 3D models. In this chapter, state-of-the-art methodologies used to establish 3D tissue models are discussed, first with a focus on both scaffold-free and scaffold-based 3D tissue model formation. Following on, multiple 3D live cell imaging systems, mainly optical imaging modalities, are introduced. Their advantages and disadvantages are discussed, with the aim of stimulating more research in this highly demanding research area.

Quantitative Live Cell FLIM Imaging in Three Dimensions.

In this chapter, the concept of fluorescence lifetime and its utility in quantitative live cell imaging will be introduced, along with methods to record and analyze FLIM data. Relevant applications in 3D tissue and live cell imaging, including multiplexed FLIM detection, will also be detailed.

Three-Dimensional Tissue Models and Available Probes for Multi-Parametric Live Cell Microscopy: A Brief Overview.

In recent years, the advances in tissue engineering and regenerative medicine have resulted in introduction of novel 3D tissue models, materials and methods to the regular practice of cell biologists, material scientists and specialists from related areas. 3D tissue models allow mimicking in vivo cell and tissue organization. However, the efficient work in three dimensions has significant challenges, such as compatibility with conventional cell biology methods, live cell imaging and quantification readouts. Here, we briefly discuss the applicability of 3D tissue models to different live cell microscopy modalities and the available range of fluo- and phosphorescent probes and sensors allowing for multi-parametric imaging.

Evaluation of a BGO-Based PET System for Single-Cell Tracking Performance by Simulation and Phantom Studies.

A recent method based on positron emission was reported for tracking moving point sources using the Inveon PET system. However, the effect of scanner background noise was not further explored. Here, we evaluate tracking with the Genisys4, a bismuth germanate-based PET system, which has no significant intrinsic background and may be better suited to tracking lower and/or faster activity sources. Position-dependent sensitivity of the Genisys4 was simulated in Geant4 Application for Tomographic Emission (GATE) using a static (18)F point source. Trajectories of helically moving point sources with varying activity and rotation speed were reconstructed from list-mode data as described previously. Simulations showed that the Inveon's ability to track sources within 2 mm of localization error is limited to objects with a velocity-to-activity ratio < 0.13 mm/decay, compared to < 0.29 mm/decay for the Genisys4. Tracking with the Genisys4 was then validated using a physical phantom of helically moving [(18)F] fluorodeoxyglucose-in-oil droplets (< 0.24 mm diameter, 139-296 Bq), yielding < 1 mm localization error under the tested conditions, with good agreement between simulated sensitivity and measured activity (Pearson correlation R = .64, P << .05 in a representative example). We have investigated the tracking performance with the Genisys4, and results suggest the feasibility of tracking low activity, point source-like objects with this system.

flowClean: Automated identification and removal of fluorescence anomalies in flow cytometry data.

Modern flow cytometry systems can be coupled to plate readers for high-throughput acquisition. These systems allow hundreds of samples to be analyzed in a single day. Quality control of the data remains challenging, however, and is further complicated when a large number of parameters is measured in an experiment. Our examination of 29,228 publicly available FCS files from laboratories worldwide indicates 13.7% have a fluorescence anomaly. In particular, fluorescence measurements for a sample over the collection time may not remain stable due to fluctuations in fluid dynamics; the impact of instabilities may differ between samples and among parameters. Therefore, we hypothesized that tracking cell populations (which represent a summary of all parameters) in centered log ratio space would provide a sensitive and consistent method of quality control. Here, we present flowClean, an algorithm to track subset frequency changes within a sample during acquisition, and flag time periods with fluorescence perturbations leading to the emergence of false populations. Aberrant time periods are reported as a new parameter and added to a revised data file, allowing users to easily review and exclude those events from further analysis. We apply this method to proof-of-concept datasets and also to a subset of data from a recent vaccine trial. The algorithm flags events that are suspicious by visual inspection, as well as those showing more subtle effects that might not be consistently flagged by investigators reviewing the data manually, and out-performs the current state-of-the-art. flowClean is available as an R package on Bioconductor, as a module on the free-to-use GenePattern web server, and as a plugin for FlowJo X. © 2016 International Society for Advancement of Cytometry.

Microfluidic Devices for Terahertz Spectroscopy of Live Cells Toward Lab-on-a-Chip Applications.

THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 °C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells.

Wide-field three-dimensional optical imaging using temporal focusing for holographically trapped microparticles.

A contemporary challenge across the natural sciences is the simultaneous optical imaging or stimulation of small numbers of cells or colloidal particles organized into arbitrary geometries. We demonstrate the use of temporal focusing with holographic optical tweezers in order to achieve depth-resolved two-photon imaging of trapped objects arranged in arbitrary three-dimensional (3D) geometries using a single objective. Trapping allows for the independent position control of multiple objects by holographic beam shaping. Temporal focusing of ultrashort pulses provides the wide-field two-photon depth-selective activation of fluorescent samples. We demonstrate the wide-field depth-resolved illumination of both trapped fluorescent beads and trapped HL60 cells in suspension with full 3D positioning control. These approaches are compatible with implementation through scattering media and can be beneficial for emergent studies in colloidal science and particularly optogenetics, offering targeted photoactivation over a wide area with micrometer-precision depth control.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

Acquisition of cross-polarized diffraction images and study of blurring effect by one time-delay-integration camera.

Blurred diffraction images acquired from flowing particles affect the measurement of fringe patterns and subsequent analysis. An imaging unit with one time-delay-integration (TDI) camera has been developed to acquire two cross-polarized diffraction images. It was shown that selected elements of Mueller matrix of single scatters can be imaged with pixel matching precision in this configuration. With the TDI camera, the effect of blurring on imaging of scattered light propagating along the side directions was found to be much more significant for biological cells than microspheres. Despite blurring, classification of MCF-7 and K562 cells is feasible since the effect has similar influence on extracted image parameters. Furthermore, image blurring can be useful for analysis of the correlations among texture parameters for characterization of diffraction images from single cells. The results demonstrate that with one TDI camera the polarization diffraction imaging flow cytometry can be significantly improved and angular distribution of selected Mueller matrix elements can be accurately measured for rapid and morphology-based assay of particles and cells without fluorescent labeling.

Amino-polyvinyl alcohol coated superparamagnetic iron oxide nanoparticles are suitable for monitoring of human mesenchymal stromal cells in vivo.

Mesenchymal stromal cells (MSCs) are promising candidates in regenerative cell-therapies. However, optimizing their number and route of delivery remains a critical issue, which can be addressed by monitoring the MSCs' bio-distribution in vivo using super-paramagnetic iron-oxide nanoparticles (SPIONs). In this study, amino-polyvinyl alcohol coated (A-PVA) SPIONs are introduced for cell-labeling and visualization by magnetic resonance imaging (MRI) of human MSCs. Size and surface charge of A-PVA-SPIONs differ depending on their solvent. Under MSC-labeling conditions, A-PVA-SPIONs have a hydrodynamic diameter of 42 ± 2 nm and a negative Zeta potential of 25 ± 5 mV, which enable efficient internalization by MSCs without the need to use transfection agents. Transmission X-ray microscopy localizes A-PVA-SPIONs in intracellular vesicles and as cytosolic single particles. After identifying non-interfering cell-assays and determining the delivered and cellular dose, in addition to the administered dose, A-PVA-SPIONs are found to be non-toxic to MSCs and non-destructive towards their multi-lineage differentiation potential. Surprisingly, MSC migration is increased. In MRI, A-PVA-SPION-labeled MSCs are successfully visualized in vitro and in vivo. In conclusion, A-PVA-SPIONs have no unfavorable influences on MSCs, although it becomes evident how sensitive their functional behavior is towards SPION-labeling. And A-PVA-SPIONs allow MSC-monitoring in vivo.

Rhodamine-modified upconversion nanophosphors for ratiometric detection of hypochlorous acid in aqueous solution and living cells.

Hypochlorous acid (HOCl), a reactive oxygen species (ROS) produced by myeloperoxidase (MPO) enzyme-mediated peroxidation of chloride ions, acts as a key microbicidal agent in immune systems. However, misregulated production of HOCl could damage host tissues and cause many inflammation-related diseases. Due to its biological importance, many efforts have been focused on developing fluorescent probes to image HOCl in living system. Compared with those conventional fluorescent probes, up-conversion luminescence (UCL) detection system has been proven to exhibit a lot of advantages including no photo-bleaching, higher light penetration depth, no autofluorescence and less damage to biosamples. Herein, we report a novel water-soluble organic-nano detection system based on rhodamine-modified UCNPs for UCL-sensing HOCl. Upon the interaction with HOCl, the green UCL emission intensity in the detection system were gradually decreased, but the emissions in the NIR region almost have no change, which is very important for the ratiometric UCL detection of HOCl in aqueous solution. More importantly, RBH1-UCNPs could be used for the ratiometric UCL visualization of HOCl released by MPO-mediated peroxidation of chloride ions in living cells. This organic-nano system could be further developed into a novel next-generation imaging technique for bio-imaging HOCl in living system without background noise.

Imaging blood cells through scattering biological tissue using speckle scanning microscopy.

We demonstrate imaging of blood cells enclosed in chicken skin tissue using speckle scanning microscopy (SSM). Clear images of multiple cells were obtained with subcellular resolution and good image fidelity, provided that the object dimension was smaller than the maximum scanning range of the speckle pattern. These results point to the potential and the challenges of using SSM technique for biological imaging.