Distinct mobility patterns of BRCA2 molecules at DNA damage sites.

BRCA2 is an essential tumor suppressor protein involved in promoting faithful repair of DNA lesions. The activity of BRCA2 needs to be tuned precisely to be active when and where it is needed. Here, we quantified the spatio-temporal dynamics of BRCA2 in living cells using aberration-corrected multifocal microscopy (acMFM). Using multicolor imaging to identify DNA damage sites, we were able to quantify its dynamic motion patterns in the nucleus and at DNA damage sites. While a large fraction of BRCA2 molecules localized near DNA damage sites appear immobile, an additional fraction of molecules exhibits subdiffusive motion, providing a potential mechanism to retain an increased number of molecules at DNA lesions. Super-resolution microscopy revealed inhomogeneous localization of BRCA2 relative to other DNA repair factors at sites of DNA damage. This suggests the presence of multiple nanoscale compartments in the chromatin surrounding the DNA lesion, which could play an important role in the contribution of BRCA2 to the regulation of the repair process.

Label-free fluorescence lifetime imaging for the assessment of cell viability in living tumor fragments.

Significance: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM). Aim: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death. Approach: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays. Results: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected. Conclusions: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.

Assessing cell viability with dynamic optical coherence microscopy.

Assessing cell viability is important in many fields of research. Current optical methods to assess cell viability typically involve fluorescent dyes, which are often less reliable and have poor permeability in primary tissues. Dynamic optical coherence microscopy (dOCM) is an emerging tool that provides label-free contrast reflecting changes in cellular metabolism. In this work, we compare the live contrast obtained from dOCM to viability dyes, and for the first time to our knowledge, demonstrate that dOCM can distinguish live cells from dead cells in murine syngeneic tumors. We further demonstrate a strong correlation between dOCM live contrast and optical redox ratio by metabolic imaging in primary mouse liver tissue. The dOCM technique opens a new avenue to apply label-free imaging to assess the effects of immuno-oncology agents, targeted therapies, chemotherapy, and cell therapies using live tumor tissues.

Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases.

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

Biomechanical Strength of a Novel Tendon Splicing Open Book Technique Compared to the Pulvertaft Method Using Unembalmed Human Cadaveric Tissue.

Background: Extensor pollicis longus (EPL) tendon rupture is a known complication of distal radius fractures. The Pulvertaft graft technique is currently used for tendon transfer of extensor indicis proprious (EIP) to EPL. This technique can produce unwanted tissue bulkiness and cosmetic concerns as well as hinder tendon gliding. A novel "open book" technique has been proposed, but relevant biomechanical data are limited. We designed a study to examine the biomechanical behaviours of the "open book" versus Pulvertaft techniques. Methods: Twenty matched forearm-wrist-hand samples were harvested from 10 fresh frozen cadavers (2 female, 8 male) with a mean age of 61.7 (±19.25) years. The EIP was transferred to EPL using the Pulvertaft versus "open book" techniques for each matched pair (sides randomly assigned). The repaired tendon segments were mechanically loaded using a Materials Testing System to examine graft biomechanical behaviours. Results: Mann-Whitney U test outcomes demonstrated that there was no significant difference between "open book" versus Pulvertaft techniques for peak load, load at yield, elongation at yield, or repair width. The "open book" technique demonstrated a significantly lower elongation at peak load and repair thickness, as well as significantly higher stiffness when compared with the Pulvertaft technique. Conclusions: Our findings support the use of the "open book" technique, producing comparable biomechanical behaviours compared to the Pulvertaft technique. Incorporating the "open book" technique potentially requires smaller repair volume, producing size and appearance that is more anatomic when compared with the Pulvertaft.

Contexte: La rupture du tendon du long extenseur du pouce (EPL - Extensor pollicis longus) est une complication connue des fractures distales du radius. La technique de greffe de Pulvertaft est actuellement utilisée pour un transfert tendineux de l'extenseur propre de l'index (EIP - extensor indicis proprious) à l'EPL. Cette technique peut produire une masse de tissu volumineuse non désirée et des préoccupations cosmétiques, mais aussi gêner le glissement du tendon. Une nouvelle technique en « livre ouvert ¼ a été proposée, mais les données biomécaniques pertinentes sont limitées. Nous avons conçu une étude visant à examiner les comportements biomécaniques de la technique « en livre ouvert ¼ comparativement à la technique de Pulvertaft. Méthodes: Vingt échantillons appariés avant-bras-poignet-main ont été prélevés sur 10 cadavres récemment congelés (2 femmes, 8 hommes) d'âge moyen 61,7 (± 19,25) ans. Les EIP ont été transférés aux EPL en utilisant la technique de Pulvertaft et la technique en livre ouvert pour chaque paire appariée (l'affectation du côté à chaque technique a été faite de manière aléatoire). Les segments tendineux réparés ont été chargés mécaniquement au moyen d'un système pour tester les matériaux afin d'étudier les comportements biomécaniques des greffes. Résultats: Les résultats du test U de Mann-Whitney ont montré qu'il n'y avait pas de différence significative entre la technique « en livre ouvert ¼ et la technique de Pulvertaft pour la charge maximum, la charge à la limite apparente d'élasticité, l'allongement à la limite apparente d'élasticité ou la largeur de réparation. La technique « en livre ouvert ¼ a démontré un allongement à la charge maximum et une épaisseur de réparation significativement moindres, ainsi qu'une significativement plus grande raideur, comparativement à la technique de Pulvertaft. Conclusions: Nos constatations étayent l'utilisation de la technique « en livre ouvert ¼ qui procure des comportements biomécaniques comparables à ceux de la technique de Pulvertaft. L'incorporation de la technique « en livre ouvert ¼ requiert potentiellement un plus petit volume de réparation, aboutissant à des dimensions et à un aspect plus anatomiques par rapport à la technique de Pulvertaft.

Novel Machine Vision Image Guidance System Significantly Reduces Procedural Time and Radiation Exposure Compared With 2-dimensional Fluoroscopy-based Guidance in Pediatric Deformity Surgery.

BACKGROUND: Intraoperative 2-dimensional (2D) fluoroscopy imaging has been commonly adopted for guidance during complex pediatric spinal deformity correction. Despite the benefits, fluoroscopy imaging emits harmful ionizing radiation, which has been well-established to have deleterious effects on the surgeon and operating room staff. This study investigated the difference in intraoperative fluoroscopy time and radiation exposure during pediatric spine surgery between 2D fluoroscopy-based navigation and a novel machine vision navigation system [machine vision image guidance system (MvIGS)]. METHODS: This retrospective chart review was conducted at a pediatric hospital with patients who underwent posterior spinal fusion for spinal deformity correction from 2018 to 2021. Patient allocation to the navigation modality was determined by the date of their surgery and the date of implementation of the MvIGS. Both modalities were the standard of care. Intraoperative radiation exposure was collected from the fluoroscopy system reports. RESULTS: A total of 1442 pedicle screws were placed in 77 children: 714 using MvIGS and 728 using 2D fluoroscopy. There were no significant differences in the male-to-female ratio, age range, body mass index, distribution of spinal pathologies, number of levels operated on, types of levels operated on, and the number of pedicle screws implanted. Total intraoperative fluoroscopy time was significantly reduced in cases utilizing MvIGS (18.6 ± 6.3 s) compared with 2D fluoroscopy (58.5 ± 19.0 s) ( P < 0.001). This represents a relative reduction of 68%. Intraoperative radiation dose area product and cumulative air kerma were reduced by 66% (0.69 ± 0.62 vs 2.0 ± 2.1 Gycm 2 , P < 0.001) and 66% (3.4 ± 3.2 vs 9.9 ± 10.5 mGy, P < 0.001) respectively. The length of stay displayed a decreasing trend with MVIGS, and the operative time was significantly reduced in MvIGS compared with 2D fluoroscopy for an average of 63.6 minutes (294.5 ± 15.5 vs 358.1 ± 60.6 min, P < 0.001). CONCLUSION: In pediatric spinal deformity correction surgery, MvIGS was able to significantly reduce intraoperative fluoroscopy time, intraoperative radiation exposure, and total surgical time, compared with traditional fluoroscopy methods. MvIGS reduced the operative time by 63.6 minutes and reduced intraoperative radiation exposure by 66%, which may play an important role in reducing the risks to the surgeon and operating room staff associated with radiation in spinal surgery procedures. LEVEL OF EVIDENCE: Level III; retrospective comparative study.

Binding partners regulate unfolding of myosin VI to activate the molecular motor.

Myosin VI is the only minus-end actin motor and it is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single-molecule imaging to investigate the impact of binding partners upon myosin VI molecular organization and dynamics.

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

Myosin VI regulates the spatial organisation of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

Factors Affecting Slip Progression After In Situ Screw Fixation of Stable Slipped Capital Femoral Epiphysis.

BACKGROUND: Slip progression after in situ fixation of slipped capital femoral epiphysis (SCFE) has been reported as occurring in up to 20% of patients. We review SCFE treated with in situ single screw fixation performed at 2 hospitals over a 15-year period to determine the factors associated with slip progression. METHODS: This case-control study reviews SCFE treated with in situ single cannulated screw fixation with minimum follow up of 1 year and full closure of the affected physis. Slip progression (failure) was defined as worsening of the Southwick slip angle of 10 or more degrees or revision surgery for symptomatic slip progression. Univariate and multivariate analyses were performed comparing success and failure groups for patient characteristics, screw type and position, and radiographic measurements. RESULTS: Ninety three patients with 108 slips met all criteria, with 15 hips (14%) classified as having slip progression (failure). All failures had 3 threads or fewer across the physis. Five hips had 2 threads across the physis, and 4 of the 5 were classified as failures. Lower modified Oxford bone scores were found in the failure group, though the difference was small (0.9, P=0.013). Failure was also associated with partially threaded screws (P=0.001). Failed hips were associated with lower initial Southwick angles (32.8 degrees) than successful hips (40.4 degrees) (P=0.047). In the stepwise model for multivariate regression, 4 factors were identified as significant, with lower initial number of threads (P<0.0001), mild initial Southwick category (P=0.0050), male sex (P=0.0061), and partially threaded screw type (P=0.0116) predicting failure. CONCLUSION: This study is the largest to date evaluating risk factors for slip progression after SCFE fixation, and the first to consider revision surgery for symptomatic slip progression. For stable SCFE, we demonstrate that 4 threads across the physis with a fully threaded screw of 6.5 mm diameter or greater was sufficient to avoid slip progression. We provide a risk stratification for progression of slip showing that in some cases 3 threads across the physis may be sufficient. LEVEL OF EVIDENCE: Level III-case-control study.

Pathological analysis of periprosthetic soft tissue and modes of failure in revision total joint arthroplasty patients.

OBJECTIVES: Implant failure leading to revision total joint arthroplasty can occur through a variety of different mechanisms which are typically associated with a soft tissue response adjacent to the implant that provide insight into the underlying etiology of implant failure. The objective of this study was to elucidate mechanisms of implant failure as they relate to histological classification and findings of adjacent periprosthetic tissue. METHODS: Histological analysis of soft tissue adjacent to the implant was performed in 99 patients with an average age of 64 years old and grouped into four categories based on the study conducted by Morawietz et al.:Type I (N = 47)Wear particle induced typeType II (N = 7)Infectious typeType III (N = 19)Combined type I and IIType IV (N = 26)Indeterminant typeModes of failure were categorized into five groupings based on the study conducted by Callies et al.: Instability (N = 35), Aseptic Loosening (N = 24), Hardware and/or Mechanical Failure (N = 15), Septic (N = 13), and Other failures (N = 12). We calculated odds ratios and conducted regression analysis to assess the relationship between modes of failure and histological findings as well as modes of failure and comorbidities. RESULTS: Hardware/mechanical failure was independently correlated with histological findings of anucleate protein debris, histiocytes, Staphylococcus epidermidis, and synovitis. Furthermore, hardware/mechanical failure was independently correlated with osteosarcoma as a co-morbidity. Septic failure was associated with histological findings of Enterococcus, granulation tissue, and tissue necrosis as well as comorbidities of Crohn's disease, deep venous thrombosis, lung disease, and rheumatoid arthritis. Infection was 5.8 times more likely to be associated with Type II histology. Aseptic loosening was associated with histologic findings of synovitis. CONCLUSION: Our findings support the existing literature on periprosthetic tissue analysis in revision total joint arthroplasty which may improve surgeon understanding of the patholophysiological mechanisms that contribute to implant failure and revision surgery.

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

3D Image Analysis of the Complete Ventricular-Subventricular Zone Stem Cell Niche Reveals Significant Vasculature Changes and Progenitor Deficits in Males Versus Females with Aging.

With age, neural stem cell (NSC) function in the adult ventricular-subventricular zone (V-SVZ) declines, reducing memory and cognitive function in males; however, the impact on females is not well understood. To obtain a global view of how age and sex impact the mouse V-SVZ, we constructed 3D montages after multiplex immunostaining, and used computer-based 3D image analysis to quantify data across the entire niche at 2, 18, and 22 months. We discovered dramatic sex differences in the aging of the V-SVZ niche vasculature, which regulates NSC activity: females showed increased diameter but decreased vessel density with age, while males showed decreased diameter and increased tortuosity and vessel density. Accompanying these vascular changes, males showed significant decline in NSC numbers, progenitor cell proliferation, and more disorganized migrating neuroblast chains with age; however, females did not. By examining the entire 3D niche, we found significant sex differences, with females being relatively spared through very old age.

Actin cables and comet tails organize mitochondrial networks in mitosis.

Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood1. Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.

Hypothesis-driven quantitative fluorescence microscopy - the importance of reverse-thinking in experimental design.

One of the challenges in modern fluorescence microscopy is to reconcile the conventional utilization of microscopes as exploratory instruments with their emerging and rapidly expanding role as a quantitative tools. The contribution of microscopy to observational biology will remain enormous owing to the improvements in acquisition speed, imaging depth, resolution and biocompatibility of modern imaging instruments. However, the use of fluorescence microscopy to facilitate the quantitative measurements necessary to challenge hypotheses is a relatively recent concept, made possible by advanced optics, functional imaging probes and rapidly increasing computational power. We argue here that to fully leverage the rapidly evolving application of microscopes in hypothesis-driven biology, we not only need to ensure that images are acquired quantitatively but must also re-evaluate how microscopy-based experiments are designed. In this Opinion, we present a reverse logic that guides the design of quantitative fluorescence microscopy experiments. This unique approach starts from identifying the results that would quantitatively inform the hypothesis and map the process backward to microscope selection. This ensures that the quantitative aspects of testing the hypothesis remain the central focus of the entire experimental design.

FMNL2 regulates dynamics of fascin in filopodia.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

Hydra image processor: 5-D GPU image analysis library with MATLAB and python wrappers.

SUMMARY: Light microscopes can now capture data in five dimensions at very high frame rates producing terabytes of data per experiment. Five-dimensional data has three spatial dimensions (x, y, z), multiple channels (λ) and time (t). Current tools are prohibitively time consuming and do not efficiently utilize available hardware. The hydra image processor (HIP) is a new library providing hardware-accelerated image processing accessible from interpreted languages including MATLAB and Python. HIP automatically distributes data/computation across system and video RAM allowing hardware-accelerated processing of arbitrarily large images. HIP also partitions compute tasks optimally across multiple GPUs. HIP includes a new kernel renormalization reducing boundary effects associated with widely used padding approaches. AVAILABILITY AND IMPLEMENTATION: HIP is free and open source software released under the BSD 3-Clause License. Source code and compiled binary files will be maintained on http://www.hydraimageprocessor.com. A comprehensive description of all MATLAB and Python interfaces and user documents are provided. HIP includes GPU-accelerated support for most common image processing operations in 2-D and 3-D and is easily extensible. HIP uses the NVIDIA CUDA interface to access the GPU. CUDA is well supported on Windows and Linux with macOS support in the future.

Practical Considerations in Particle and Object Tracking and Analysis.

The rapid advancement of live-cell imaging technologies has enabled biologists to generate high-dimensional data to follow biological movement at the microscopic level. Yet, the "perceived" ease of use of modern microscopes has led to challenges whereby sub-optimal data are commonly generated that cannot support quantitative tracking and analysis as a result of various ill-advised decisions made during image acquisition. Even optimally acquired images often require further optimization through digital processing before they can be analyzed. In writing this article, we presume our target audience to be biologists with a foundational understanding of digital image acquisition and processing, who are seeking to understand the essential steps for particle/object tracking experiments. It is with this targeted readership in mind that we review the basic principles of image-processing techniques as well as analysis strategies commonly used for tracking experiments. We conclude this technical survey with a discussion of how movement behavior can be mathematically modeled and described. © 2019 by John Wiley & Sons, Inc.

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.