Live Cell Imaging Microscopy Research Articles

Low Density Lipoprotein Receptor Variants in the Beta-Propeller Subdomain and Their Functional Impact.

Background: Pathogenic variants in the low density lipoprotein receptor gene are associated with familial hypercholesterolemia. Some of these variants can result in incorrect folding of the LDLR protein, which is then accumulated inside the cell and cannot fulfill its function to internalize LDL particles. We analyzed the functional impact of 10 LDLR variants localized in the beta-propeller of epidermal growth factor precursor homology domain. The experimental part of the work was complemented by a structural analysis on the basis of 3D LDLR protein structure.Methods: T-Rex Chinese hamster ovary cells transfected with the human LDLR gene were used for live cell imaging microscopy, flow cytometry, and qRT-PCR analysis.Results: Our results showed that the analyzed LDLR protein variants can be divided into three groups. (1) The variants buried inside the 3D protein structure expressing proteins accumulated in the endoplasmic reticulum (ER) with no or reduced plasma membrane localization and LDL particle internalization, and associated with an increased gene expression of ER-resident chaperones. (2) The variants localized on the surface of 3D protein structure with slightly reduced LDLR plasma membrane localization and LDL particle internalization, and associated with no increased mRNA level of ER-resident chaperones. (3) The variants localized on the surface of the 3D protein structure but expressing proteins with cell responses similar to the group 1.Conclusion: All analyzed LDLR variants have been evaluated as pathogenic but with different effects on protein localization and function, and expression of genes associated with ER stress.

Live cell imaging of lytic granule motility in anti-ErbB2 CAR NK cells and FcR NK cells plus Herceptin towards ErbB2+ breast cancer cells

Background & Aim Upon encountering a susceptible target, NK cells mediate directed cytotoxicity by exocytosis of lytic effector molecules such as perforin and granzymes. The steps leading to NK cell granule exocytosis are highly regulated. Granule exocytosis is preceded by convergence of granules to the microtubule organizing center (MTOC) and subsequent polarization of the MTOC and granules to the immunological synapse (IS). In case of antibody-dependent cell-mediated cytotoxicity (ADCC), it has been shown that signaling through the Fc receptor is critical to polarize MTOC and granules to the IS with otherwise resistant targets. Methods, Results & Conclusion Here we used spinning disk confocal microscopy for live cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR expressing NK-92 cells (NK-92/5.28.z) and research-grade high affinity FcR expressing NK-92 cells plus Herceptin™ towards ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. Interestingly, unmodified NK-92 cells in combination with MDA-MB-453 cells showed granule convergence to the MTOC, but failed to polarize MTOC and granules to the IS. In contrast, retargeting by either CAR or mAb/FcR towards the ErbB2 antigen on MDA-MB-453 enabled granule polarization to the IS resulting in highly effective cytotoxicity. Granule polarization was rapid in both the CAR and high affinity FcR expressing NK-92 cells after cell-cell contact was initiated. These observations suggest that retargeting of NK-92 cells by either transgenic CAR or high affinity FcR expression in combination with tumor-specific antibodies confers tumor cell lysis by enabling the otherwise impaired MTOC and granule polarization to the IS which resembles the physiological exocytosis cascade observed in naturally occurring ADCC.

Inhibition of Kv10.1 Channels Sensitizes Mitochondria of Cancer Cells to Antimetabolic Agents.

Reprogramming of energy metabolism constitutes one of the hallmarks of cancer and is, therefore, an emerging therapeutic target. We describe here that the potassium channel Kv10.1, which is frequently overexpressed in primary and metastatic cancer, and has been proposed a therapeutic target, participates in metabolic adaptation of cancer cells through regulation of mitochondrial dynamics. We used biochemical and cell biological techniques, live cell imaging and high-resolution microscopy, among other approaches, to study the impact of Kv10.1 on the regulation of mitochondrial stability. Inhibition of Kv10.1 expression or function led to mitochondrial fragmentation, increase in reactive oxygen species and increased autophagy. Cells with endogenous overexpression of Kv10.1 were also more sensitive to mitochondrial metabolism inhibitors than cells with low expression, indicating that they are more dependent on mitochondrial function. Consistently, a combined therapy using functional monoclonal antibodies for Kv10.1 and mitochondrial metabolism inhibitors resulted in enhanced efficacy of the inhibitors. Our data reveal a new mechanism regulated by Kv10.1 in cancer and a novel strategy to overcome drug resistance in cancers with a high expression of Kv10.1.

Galvanotropic Chamber for Controlled Reorientation of Pollen Tube Growth and Simultaneous Confocal Imaging of Intracellular Dynamics.

Successful fertilization and seed set require the pollen tube to grow through several tissues, to change its growth orientation by responding to directional cues, and to ultimately reach the embryo sac and deliver the paternal genetic material. The ability to respond to external directional cues is, therefore, a pivotal feature of pollen tube behavior. In order to study the regulatory mechanisms controlling and mediating pollen tube tropic growth, a robust and reproducible method for the induction of growth reorientation in vitro is required. Here we describe a galvanotropic chamber designed to expose growing pollen tubes to precisely calibrated directional cues triggering reorientation while simultaneously tracking subcellular processes using live cell imaging and confocal laser scanning microscopy.

EPA and DHA attenuate deoxynivalenol-induced intestinal porcine epithelial cell injury and protect barrier function integrity by inhibiting necroptosis signaling pathway.

Deoxynivalenol (DON) is one of the most common mycotoxins that contaminates food or feed and cause intestinal damage. Long-chain n-3 polyunsaturated fatty acids (PUFA) such as EPA and DHA exert beneficial effects on intestinal integrity in animal models and clinical trials. Necroptosis signaling pathway plays a critical role in intestinal cell injury. This study tested the hypothesis that EPA and DHA could alleviate DON-induced injury to intestinal porcine epithelial cells through modulation of the necroptosis signaling pathway. Intestinal porcine epithelial cell 1 (IPEC-1) cells were cultured with or without EPA or DHA (6.25-25μg/mL) in the presence or absence of 0.5μg/mL DON for indicated time points. Cell viability, cell number, lactate dehydrogenase (LDH) activity, cell necrosis, transepithelial electrical resistance (TEER), fluorescein isothiocyanate-labeled dextran4kDa (FD4) flux, tight junction protein distribution, and protein abundance of necroptosis related signals were determined. EPA and DHA promoted cell growth indicated by higher cell viability and cell number, and inhibited cell injury indicated by lower LDH activity in the media. EPA and DHA also improved intestinal barrier function, indicated by higher TEER and lower permeability of FD4 flux as well as increased proportions of tight junction proteins located in the plasma membrane. Moreover, EPA and DHA decreased cell necrosis demonstrated by live cell imaging and transmission electron microscopy. Finally, EPA and DHA downregulated protein expressions of necroptosis related signals including tumor necrosis factor receptor (TNFR1), receptor interacting protein kinase 1 (RIP1), RIP3, phosphorylated mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase family 5 (PGAM5), dynamin-related protein 1 (Drp1), and high mobility group box-1 protein (HMGB1). EPA and DHA also inhibited protein expression of caspase-3 and caspase-8. These results suggest that EPA and DHA prevent DON-induced intestinal cell injury and enhance barrier function, which is associated with inhibition of the necroptosis signaling pathway.

Assessment of doxorubicin-induced remodeling of Ca2+ signaling and associated Ca2+ regulating proteins in MDA-MB-231 breast cancer cells

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 μM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 μM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.

Capturing Stem Cell Behavior Using Intravital and Live Cell Microscopy.

Stem cells maintain tissue homeostasis by driving cellular turnover and regeneration upon damage. They reside within specialized niches that provide the signals required for stem cell maintenance. Stem cells have been identified in many tissues and cancer types, but their behavior within the niche and their reaction to microenvironmental signals were inferred from limited static observations. Recent advances in live imaging techniques, such as live cell imaging and intravital microscopy, have allowed the visualization of stem cell behavior and dynamics over time in their (near) native environment. Through these recent technological advances, it is now evident that stem cells are much more dynamic than previously anticipated, resulting in a model in which stemness is a state that can be gained or lost over time. In this review, we will highlight how live imaging and intravital microscopy have unraveled previously unanticipated stem cell dynamics and plasticity during development, homeostasis, regeneration, and tumor formation.

Microtubule-dependent transport of arenavirus matrix protein demonstrated using live-cell imaging microscopy.

Lassa virus (LASV), belonging to the family Arenaviridae, causes severe haemorrhagic manifestations and is associated with a high mortality rate in humans. Thus, it is classified as a biosafety level (BSL)-4 agent. Since countermeasures for LASV diseases are yet to be developed, it is important to elucidate the molecular mechanisms underlying the life cycle of the virus, including its viral and host cellular protein interactions. These underlying molecular mechanisms may serve as the key for developing novel therapeutic options. Lymphocytic choriomeningitis virus (LCMV), a close relative of LASV, is usually asymptomatic and is categorized as a BSL-2 agent. In the present study, we visualized the transport of viral matrix Z protein in LCMV-infected cells using live-cell imaging microscopy. We demonstrated that the transport of Z protein is mediated by polymerized microtubules. Interestingly, the transport of LASV Z protein showed characteristics similar to those of Z protein in LCMV-infected cells. The live-cell imaging system using LCMV provides an attractive surrogate measure for studying arenavirus matrix protein transport in BSL-2 laboratories. In addition, it could be also utilized to analyze the interactions between viral matrix proteins and the cellular cytoskeleton, as well as to evaluate the antiviral compounds that target the transport of viral matrix proteins.

Synthesis and properties of water-soluble 1,9-dialkyl-substituted BF2 azadipyrromethene fluorophores

Bis-alkylsulfonic acid and polyethylene glycol (PEG)-substituted BF2 azadipyrromethenes have been synthesized by an adaptable and versatile route. Only four synthetic stages were required to produce the penultimate fluorophore compounds, containing either two alcohol or two terminal alkyne substituents. The final synthetic step introduced either sulfonic acid or polyethylene glycol groups to impart aqueous solubility. Sulfonic acid groups were introduced by reaction of the bis-alcohol-substituted fluorophore with sulfur trioxide, and a double Cu(I)-catalyzed cycloaddition reaction between the bis-alkyne fluorophore and methoxypolyethylene glycol azide yielded a neutral bis-pegylated derivative. Both fluorophores exhibited excellent near-infrared (NIR) photophysical properties in methanol and aqueous solutions. Live cell microscopy imaging revealed efficient uptake and intracellular labelling of cells for both fluorophores. Their simple synthesis, with potential for last-step structural modifications, makes the present NIR-active azadipyrromethene derivatives potentially useful as NIR fluorescence imaging probes for live cells.

A co-culture model of the bovine alveolus.

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro- in vivo correlation. We describe here a co-culture model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the co-culture model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their co-culture with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The co-culture model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

Salpingitis Impairs Bovine Tubal Function and Sperm-Oviduct Interaction

Salpingitis is a common cause for subfertility and infertility both in humans and animals. However, the effects of salpingitis on tubal function and reproductive success are largely unknown. Therefore we set out to investigate the effects of inflammation on sperm and oocyte transport and gameto-maternal interaction in the oviduct using the bovine as a model. For this purpose, oviducts revealing mild (n = 45), moderate (n = 55) and severe (n = 45) inflammation were obtained from cows immediately after slaughter and investigated by live cell imaging, histochemistry and scanning electron microscopy. Our studies showed that endometritis was always correlated with salpingitis. Moderate and severe inflammation caused a significant increase in the thickness of tubal folds (p < 0.05). Severe inflammation was characterized by luminal accumulations of mucus and glycoproteins, increased apoptosis, loss of tight junctions and shedding of tubal epithelial cells. The mean ciliary beat frequency (CBF) in the ampulla was significantly reduced as compared to the controls (p < 0.05). The higher the grade of inflammation, the lower was the CBF (p < 0.001). In severe inflammation, spermatozoa were stuck in mucus resulting in decreased sperm motility. Our results imply that tubal inflammation impairs proper tubal function and leads to reduced sperm fertilizing capacity.

Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection*

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process.

A bilayer tissue culture model of the bovine alveolus

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro-in vivo correlation. We describe here a co-culture bilayer model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and the bilayer cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the bilayer model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their culture as a bilayer in conjunction with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The bilayer model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

A Global Method for Non-Rigid Registration of Cell Nuclei in Live Cell Time-Lapse Images.

Non-rigid registration of cell nuclei in time-lapse microscopy images can be achieved through estimating the deformation fields using optical flow methods. In contrast to local optical flow models employed in the existing non-rigid registration methods, we introduce approaches based on a global optical flow model. Our registration model consists of a data fidelity term and a regularization term. We compared different regularizers for the deformation fields and found that a convex quadratic function is more suitable than non-convex ones. To improve the robustness, we propose an adaptive weighting scheme based on the statistics of the noise in fluorescence microscopy images as well as a combined local-global scheme. Moreover, we extend the global method by exploiting high-order image features. The best suitable high-order features are determined through learning two generative image models, namely, fields of experts and convolutional Gaussian restricted Boltzmann machine, whose model formulations are both consistent with the assumption of high-order feature constancy in the registration model. Using multiple data sets of real 2D and 3D live cell microscopy image sequences as well as synthetic image data, we demonstrate that our proposed approach outperforms the previous methods in terms of both registration accuracy and computational efficiency.

Scanning Ion Conductance Microscopy and Atomic Force Microscopy for Live Cell Imaging: A Comparison

Scanning Ion Conductance Microscopy and Atomic Force Microscopy for Live Cell Imaging: A Comparison

Rhodamine-assisted fluorescent strategy for the sensitive and selective in-field mapping of environmental pollutant Hg(II) with potential bioimaging

Mercury is a global pollutant that can damage human brain, central nervous system and endocrine moreover, other biological systems. Its real-time monitoring is a key challenge in the control of mercury emissions or pollution. In this study, a simple and efficient fluorescent sensor (PST) comprising of rhodamine B and 2-amino-5-bromopyridine was developed for the detection of environmental toxicant Hg2+. The newly synthesized sensor displayed a highly selective and sensitive “turn-on” response by the addition of Hg2+ among miscellaneous metal cations (Ag+, Al3+, Ba2+, Ca2+, Cd2+, Fe3+, Fe2+, K+, Li+, Mg2+, Mn2+, Cu2+, Pb2+, Ni2+, Na+ and Zn2+) in CH3CN: H2O (8:2 v/v) system. The 1:1 binding stoichiometry, limit of detection (LOD) 0.63 μM along with binding constant of 1.04 × 105 M−1 was calculated for the complex PST-Hg2+. The reversible nature of the complex was confirmed in the presence of ethylenediaminetetraacetate (EDTA). The significant enhancement of fluorometric and absorption response was attributed to the spirolactam ring opening via photo-induced electron transfer (PET) of xanthene moiety. This spirolactam ring opening was further verified by density functional theory, 1H NMR and Fourier transform infrared spectroscopy (FT-IR). The real-time applicability of the sensor in environmental and biological fields was proved as detection kit on TLC strips and with the help of Laser confocal microscopy for live cell imaging by using HeLa cells. Conclusively, PST can be regarded as accurate ‘two in one’ kit for colorimetric recognition of Hg2+ in the environment and fluorescent cell imaging in live HeLa cells.

Genetic and pharmacological inactivation of apical Na+-K+-2Cl- cotransporter 1 in choroid plexus epithelial cells reveals the physiological function of the cotransporter.

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl- cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl- concentration ([Cl-]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl- measurements in single CPECs of NKCC1+/+ and NKCC1-/- mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1-/- CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1-/- are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 μM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1-/- CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl-]i and CWV needed for CSF secretion.

An In Vitro Model for Conditioning Lesion Effect.

Axons of a peripheral nerve grow faster after an axotomy if it attains a prior injury a few days earlier. This is called conditioning lesion effect (CLE) and very much valued since it may provide new insights into neuron biology and axonal regeneration. There are established in vivo experimental paradigms to study CLE, however, there is a need to have an in vitro conditioning technique where CLE occurs in a maximally controlled environment. Mouse primary sensory neurons were isolated from lumbar 4-5 dorsal root ganglia and incubated at 37°C on a silicon-coated watch glass that prevents cell attachment. After this conditioning period they were transferred to laminin coated culture dishes. Similar cultures were set up with freshly isolated neurons from control animals and from the animals that received a sciatic nerve cut 3days earlier. All preparations were placed on a live cell imaging microscopy providing physiological conditions and photographed for 48h. Axonal regeneration and neuronal survival was assessed. During the conditioning incubation period neurons remained in suspended aggregates and did not grow axons. The regeneration rate of the in vitro conditioned neurons was much higher than the in vivo conditioned and control preparations during the first day of normal incubation. However, higher regeneration rates were compromised by progressive substantial neuronal death in both types of conditioned cultures but not in the control preparations. By using neutralizing antibodies, we demonstrated that activity of endogenous leukemia inhibitory factor is essential for induction of CLE in this model.

On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans.

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C.albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip-growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long-range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C.albicans filamentous growth, we studied the dynamics and organisation of the C.albicans secretory pathway using live cell imaging and three-dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub-apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C.albicans likely facilitates short-range "on-site" secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.

Separating Touching Cells Using Pixel Replicated Elliptical Shape Models.

One of the most important and error-prone tasks in biological image analysis is the segmentation of touching or overlapping cells. Particularly for optical microscopy, including transmitted light and confocal fluorescence microscopy, there is often no consistent discriminative information to separate cells that touch or overlap. It is desired to partition touching foreground pixels into cells using the binary threshold image information only, and optionally incorporating gradient information. The most common approaches for segmenting touching and overlapping cells in these scenarios are based on the watershed transform. We describe a new approach called pixel replication for the task of segmenting elliptical objects that touch or overlap. Pixel replication uses the image Euclidean distance transform in combination with Gaussian mixture models to better exploit practically effective optimization for delineating objects with elliptical decision boundaries. Pixel replication improves significantly on commonly used methods based on watershed transforms, or based on fitting Gaussian mixtures directly to the thresholded image data. Pixel replication works equivalently on both 2-D and 3-D image data, and naturally combines information from multi-channel images. The accuracy of the proposed technique is measured using both the segmentation accuracy on simulated ellipse data and the tracking accuracy on validated stem cell tracking results extracted from hundreds of live-cell microscopy image sequences. Pixel replication is shown to be significantly more accurate compared with other approaches. Variance relationships are derived, allowing a more practically effective Gaussian mixture model to extract cell boundaries for data generated from the threshold image using the uniform elliptical distribution and from the distance transform image using the triangular elliptical distribution.