Mitochondrial sulfide promotes life span and health span through distinct mechanisms in developing versus adult treated Caenorhabditis elegans.

Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic.

Capillary imbibition of confined monodisperse emulsions in microfluidic channels.

We study imbibition of a monodisperse emulsion into high-aspect ratio microfluidic channels with the height h comparable to the droplet diameter d. Two distinct regimes are identified in the imbibition dynamics. In a strongly confined system (the confinement ratio d/h = 1.2 in our experiments), the droplets are flattened between the channel walls and move more slowly compared to the average suspension velocity. As a result, a droplet-free region forms behind the meniscus (separated from the suspension region by a sharp concentration front) and the suspension exhibits strong droplet-density and velocity fluctuations. In a weaker confinement, d/h = 0.65, approximately spherical droplets move faster than the average suspension flow, causing development of a dynamically unstable high-concentration region near the meniscus. This instability results in the formation of dense droplet clusters, which migrate downstream relative to the average suspension flow, thus affecting the entire suspension dynamics. We explain the observed phenomena using linear transport equations for the particle-phase and suspension fluxes driven by the local pressure gradient. We also use a dipolar particle interaction model to numerically simulate the imbibition dynamics. The observed large velocity fluctuations in strongly confined systems are elucidated in terms of migration of self-assembled particle chains with highly anisotropic mobility.

Mitochondrial hydrogen sulfide supplementation improves health in the C. elegans Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin gene. The symptoms of DMD share similarities with those of accelerated aging. Recently, hydrogen sulfide (H2S) supplementation has been suggested to modulate the effects of age-related decline in muscle function, and metabolic H2S deficiencies have been implicated in affecting muscle mass in conditions such as phenylketonuria. We therefore evaluated the use of sodium GYY4137 (NaGYY), a H2S-releasing molecule, as a possible approach for DMD treatment. Using the dys-1(eg33) Caenorhabditis elegans DMD model, we found that NaGYY treatment (100 µM) improved movement, strength, gait, and muscle mitochondrial structure, similar to the gold-standard therapeutic treatment, prednisone (370 µM). The health improvements of either treatment required the action of the kinase JNK-1, the transcription factor SKN-1, and the NAD-dependent deacetylase SIR-2.1. The transcription factor DAF-16 was required for the health benefits of NaGYY treatment, but not prednisone treatment. AP39 (100 pM), a mitochondria-targeted H2S compound, also improved movement and strength in the dys-1(eg33) model, further implying that these improvements are mitochondria-based. Additionally, we found a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice. Overall, our results suggest that H2S deficit may contribute to DMD pathology, and rectifying/overcoming the deficit with H2S delivery compounds has potential as a therapeutic approach to DMD treatment.

Microfluidic tapered aspirators for mechanical characterization of microgel beads.

In this study, we report a microfluidic approach for the measurement of mechanical properties of spherical microgel beads. This technique is analogous to tapered micropipette aspiration, while harnessing the benefits of microfluidics. We fabricate alginate-based microbeads and determine their mechanical properties using the microfluidic tapered aspirators. Individual microgel beads are aspirated and trapped in tapered channels, the deformed equilibrium shape is measured, and a stress balance is used to determine the Young's modulus. We investigate the effect of surface coating, taper angle, and bead diameter and find that the measured modulus is largely insensitive to these parameters. We show that the bead modulus increases with alginate concentration and follows a trend similar to that of the modulus measured using standard uniaxial compression. The critical pressure to squeeze out the beads from the tapered aspirators was found to depend on both the modulus and the bead diameter. Finally, we demonstrate how temporal changes in bead moduli due to enzymatic degradation of the hydrogel could be quantitatively determined. The results from this study highlight that the microfluidic tapered aspirators are a useful tool to measure hydrogel bead mechanics and have the potential to characterize dynamic changes in mechanical properties.

Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration.

Regular physical exercise is the most efficient and accessible intervention known to promote healthy aging in humans. The molecular and cellular mechanisms that mediate system-wide exercise benefits, however, remain poorly understood, especially as applies to tissues that do not participate directly in training activity. The establishment of exercise protocols for short-lived genetic models will be critical for deciphering fundamental mechanisms of transtissue exercise benefits to healthy aging. Here we document optimization of a long-term swim exercise protocol for Caenorhabditis elegans and we demonstrate its benefits to diverse aging tissues, even if exercise occurs only during a restricted phase of adulthood. We found that multiple daily swim sessions are essential for exercise adaptation, leading to body wall muscle improvements in structural gene expression, locomotory performance, and mitochondrial morphology. Swim exercise training enhances whole-animal health parameters, such as mitochondrial respiration and midlife survival, increases functional healthspan of the pharynx and intestine, and enhances nervous system health by increasing learning ability and protecting against neurodegeneration in models of tauopathy, Alzheimer's disease, and Huntington's disease. Remarkably, swim training only during early adulthood induces long-lasting systemic benefits that in several cases are still detectable well into midlife. Our data reveal the broad impact of swim exercise in promoting extended healthspan of multiple C. elegans tissues, underscore the potency of early exercise experience to influence long-term health, and establish the foundation for exploiting the powerful advantages of this genetic model for the dissection of the exercise-dependent molecular circuitry that confers system-wide health benefits to aging adults.

Roll maneuvers are essential for active reorientation of Caenorhabditis elegans in 3D media.

Locomotion of the nematode Caenorhabditis elegans is a key observable used in investigations ranging from behavior to neuroscience to aging. However, while the natural environment of this model organism is 3D, quantitative investigations of its locomotion have been mostly limited to 2D motion. Here, we present a quantitative analysis of how the nematode reorients itself in 3D media. We identify a unique behavioral state of C. elegans-a roll maneuver-which is an essential component of 3D locomotion in burrowing and swimming. The rolls, associated with nonzero torsion of the nematode body, result in rotation of the plane of dorsoventral body undulations about the symmetry axis of the trajectory. When combined with planar turns in a new undulation plane, the rolls allow the nematode to reorient its body in any direction, thus enabling complete exploration of 3D space. The rolls observed in swimming are much faster than the ones in burrowing; we show that this difference stems from a purely hydrodynamic enhancement mechanism and not from a gait change or an increase in the body torsion. This result demonstrates that hydrodynamic viscous forces can enhance 3D reorientation in undulatory locomotion, in contrast to known hydrodynamic hindrance of both forward motion and planar turns.

Catastrophic thermal destabilization of two-dimensional close-packed emulsions due to synchronous coalescence initiation.

The mechanisms for phase separation in highly concentrated emulsions when subjected to a thermal phase transition remain to be elucidated. Here, we create a hexagonally close-packed monodisperse emulsion in 2D and show that during a cool-heat cycle, the emulsion fully destabilizes akin to phase separation. The mechanism for this catastrophic destabilization is found to be spontaneous coalescence initiation that synchronously occurs between every solidified droplet and its neighbors. This synchronous coalescence initiation establishes system-wide network connectivity in the emulsion causing large-scale destabilization. This system-wide coalescence initiation is found to be insensitive to droplet size and tested surfactants, but dependent on network connectivity and crystal content of individual droplets.

The integrin-adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans.

The integrin-adhesome network, which contains >150 proteins, is mechano-transducing and located at discreet positions along the cell-cell and cell-extracellular matrix interface. A small subset of the integrin-adhesome is known to maintain normal muscle morphology. However, the importance of the entire adhesome for muscle structure and function is unknown. We used RNA interference to knock down 113 putative Caenorhabditis elegans homologs constituting most of the mammalian adhesome and 48 proteins known to localize to attachment sites in C. elegans muscle. In both cases, we found >90% of components were required for normal muscle mitochondrial structure and/or proteostasis vs. empty vector controls. Approximately half of these, mainly proteins that physically interact with each other, were also required for normal sarcomere and/or adhesome structure. Next we confirmed that the dystrophy observed in adhesome mutants associates with impaired maximal mitochondrial ATP production (P < 0.01), as well as reduced probability distribution of muscle movement forces compared with wild-type animals. Our results show that the integrin-adhesome network as a whole is required for maintaining both muscle structure and function and extend the current understanding of the full complexities of the functional adhesome in vivo.

Collective dynamics of non-coalescing and coalescing droplets in microfluidic parking networks.

We study the complex collective dynamics mediated by flow resistance interactions when trains of non-coalescing and coalescing confined drops are introduced into a microfluidic parking network (MPN). The MPN consists of serially connected loops capable of parking arrays of drops. We define parking modes based on whether drops park without breakage or drop fragments are parked subsequent to breakage or drops park after coalescence. With both non-coalescing and coalescing drops, we map the occurrence of these parking modes in MPNs as a function of system parameters including drop volume, drop spacing and capillary number. We find that the non-coalescing drops can either park or break in the network, producing highly polydisperse arrays. We further show that parking due to collision induced droplet break-up is the main cause of polydispersity. We discover that collisions occur due to a crowding instability, which is a natural outcome of the network topology. In striking contrast, with coalescing drops we show that the ability of drops to coalesce rectifies the volume of parked polydisperse drops, despite drops breaking in the network. We find that several parking modes act in concert during this hydrodynamic self-rectification mechanism, producing highly monodisperse drop arrays over a wide operating parameter space. We demonstrate that the rectification mechanism can be harnessed to produce two-dimensional arrays of microfluidic drops with highly tunable surface-to-volume ratios, paving the way for fundamental investigations of interfacial phenomena in emulsions.

FHOD-1/profilin-mediated actin assembly protects sarcomeres against contraction-induced deformation in C. elegans.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from C. elegans results in thin body-wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that actin polymerization by FHOD-1 is required for its function in muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and profilins PFN-2 and PFN-3 to promote body-wall muscle growth. We further demonstrate dense bodies in fhod-1 and pfn-3 mutants are less stable than in wild type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin depolymerization factor/cofilin homolog UNC-60B in body-wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, and may account for the abnormally slow growth and reduced strength of body-wall muscle in fhod-1 mutants. Overall, these results show the importance of FHOD protein-mediated actin assembly to forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

Generation of chemical concentration gradients in mobile droplet arrays via fragmentation of long immiscible diluting plugs.

We report a one-step passive microfluidic technique to generate arrays of moving droplets containing variation of chemical concentration between individual drops. We find that a concentration gradient can be established in a long diluting plug by on-chip dilution and extraction of samples via orthogonal coalescence of the plug with a static array of sample drops. The diluting plug containing the gradient is subsequently fragmented by a droplet generator. We show that the technique is flexible, as the dilution range can be tuned by a variety of control parameters including the carrier fluid flow rate, volume of diluting plugs, and stationary drops. We also find that the concentration gradients have a fine resolution and are reproducible to within 2% relative standard deviation. As one demonstrative application, we show the suitability of the technique for generating a dose-response curve for an enzyme inhibition assay. Because of the ability to inject multiple plugs, our technique has the potential for unlimited as well as sequential dilution of a series of substrates. Thus, our method could be valuable as a high-throughput and high-resolution screening tool for assays that require interrogation of the response of one or more target species to numerous distinct chemical concentrations.

Microfluidic production of spherical and nonspherical fat particles by thermal quenching of crystallizable oils.

We report the microfluidic production of spherical and nonspherical fat particles from crystallizable oils. The method is based on microfluidic generation of oil droplets at a cross-junction followed by thermal solidification downstream in a microcapillary. We vary the drop production conditions and the device temperature and demonstrate that the size, shape, and crystallinity can be controlled. By measuring thermal gradients in the microcapillary, we show that crystalline fat particles are best produced when the device temperature is below the onset temperature of bulk fat crystallization. To produce monodisperse nonspherical fat particles, we find that the carrier fluid flow rate needs to be sufficiently high to provide strong hydrodynamic forces to transport the confined rod-like particles. We identify the scaling relationship between geometric confinement and particle elasticity necessary to maintain the nonspherical shape. Thus, our study provides guidelines for the production of spherical and nonspherical fat particles that can be potentially used for controlling microstructure, rheology, and drug encapsulation in foods, cosmetics, and pharmaceutical creams that employ crystallizable oils.

A Compact Imaging Platform for Conducting C. elegans Phenotypic Assays on Earth and in Spaceflight.

The model organism Caenorhabditis elegans is used in a variety of applications ranging from fundamental biological studies, to drug screening, to disease modeling, and to space-biology investigations. These applications rely on conducting whole-organism phenotypic assays involving animal behavior and locomotion. In this study, we report a 3D printed compact imaging platform (CIP) that is integrated with a smart-device camera for the whole-organism phenotyping of C. elegans. The CIP has no external optical elements and does not require mechanical focusing, simplifying the optical configuration. The small footprint of the system powered with a standard USB provides capabilities ranging from plug-and-play, to parallel operation, and to housing it in incubators for temperature control. We demonstrate on Earth the compatibility of the CIP with different C. elegans substrates, including agar plates, liquid droplets on glass slides and microfluidic chips. We validate the system with behavioral and thrashing assays and show that the phenotypic readouts are in good agreement with the literature data. We conduct a pilot study with mutants and show that the phenotypic data collected from the CIP distinguishes these mutants. Finally, we discuss how the simplicity and versatility offered by CIP makes it amenable to future C. elegans investigations on the International Space Station, where science experiments are constrained by system size, payload weight and crew time. Overall, the compactness, portability and ease-of-use makes the CIP desirable for research and educational outreach applications on Earth and in space.

Deep learning assisted holography microscopy for in-flow enumeration of tumor cells in blood.

Currently, detection of circulating tumor cells (CTCs) in cancer patient blood samples relies on immunostaining, which does not provide access to live CTCs, limiting the breadth of CTC-based applications. Here, we take the first steps to address this limitation, by demonstrating staining-free enumeration of tumor cells spiked into lysed blood samples using digital holographic microscopy (DHM), microfluidics and machine learning (ML). A 3D-printed module for laser assembly was developed to simplify the optical set up for holographic imaging of cells flowing through a sheath-based microfluidic device. Computational reconstruction of the holograms was performed to localize the cells in 3D and obtain the plane of best focus images to train deep learning models. We developed a custom-designed light-weight shallow Network dubbed s-Net and compared its performance against off-the-shelf CNN models including ResNet-50. The accuracy, sensitivity and specificity of the s-Net model was found to be higher than the off-the-shelf ML models. By applying an optimized decision threshold to mixed samples prepared in silico, the false positive rate was reduced from 1 × 10-2 to 2.77 × 10-4. Finally, the developed DHM-ML framework was successfully applied to enumerate spiked MCF-7 breast cancer cells and SkOV3 ovarian cancer cells from lysed blood samples containing white blood cells (WBCs) at concentrations typical of label-free enrichment techniques. We conclude by discussing the advances that need to be made to translate the DHM-ML approach to staining-free enumeration of actual CTCs in cancer patient blood samples.

Comprehensive Profiling of Cancer-Associated Cells in the Blood of Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy to Predict Pathological Complete Response.

Neoadjuvant chemotherapy (NAC) can affect pathological complete response (pCR) in breast cancers; the resection that follows identifies patients with residual disease who are then offered second-line therapies. Circulating tumor cells (CTCs) and cancer-associated macrophage-like cells (CAMLs) in the blood can be used as potential biomarkers for predicting pCR before resection. CTCs are of epithelial origin that undergo epithelial-to-mesenchymal transition to become more motile and invasive, thereby leading to invasive mesenchymal cells that seed in distant organs, causing metastasis. Additionally, CAMLs in the blood of cancer patients are reported to either engulf or aid the transport of cancer cells to distant organs. To study these rare cancer-associated cells, we conducted a preliminary study where we collected blood from patients treated with NAC after obtaining their written and informed consent. Blood was collected before, during, and after NAC, and Labyrinth microfluidic technology was used to isolate CTCs and CAMLs. Demographic, tumor marker, and treatment response data were collected. Non-parametric tests were used to compare pCR and non-pCR groups. Univariate and multivariate models were used where CTCs and CAMLs were analyzed for predicting pCR. Sixty-three samples from 21 patients were analyzed. The median(IQR) pre-NAC total and mesenchymal CTC count/5 mL was lower in the pCR vs. non-pCR group [1(3.5) vs. 5(5.75); p = 0.096], [0 vs. 2.5(7.5); p = 0.084], respectively. The median(IQR) post-NAC CAML count/5 mL was higher in the pCR vs. non-pCR group [15(6) vs. 6(4.5); p = 0.004]. The pCR group was more likely to have >10 CAMLs post-NAC vs. non-pCR group [7(100%) vs. 3(21.4%); p = 0.001]. In a multivariate logistic regression model predicting pCR, CAML count was positively associated with the log-odds of pCR [OR = 1.49(1.01, 2.18); p = 0.041], while CTCs showed a negative trend [Odds Ratio (OR) = 0.44(0.18, 1.06); p = 0.068]. In conclusion, increased CAMLs in circulation after treatment combined with lowered CTCs was associated with pCR.

Single-Cell Proliferation Microfluidic Device for High Throughput Investigation of Replicative Potential and Drug Resistance of Cancer Cells.

Introduction: Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes. Methods: We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin. Results: The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01 µM, 0.001 µM, 0.0001 µM), we observed that 50-80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension. Conclusions: The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00773-z.

Spaceflight Induces Strength Decline in Caenorhabditis elegans.

Background: Understanding and countering the well-established negative health consequences of spaceflight remains a primary challenge preventing safe deep space exploration. Targeted/personalized therapeutics are at the forefront of space medicine strategies, and cross-species molecular signatures now define the 'typical' spaceflight response. However, a lack of direct genotype-phenotype associations currently limits the robustness and, therefore, the therapeutic utility of putative mechanisms underpinning pathological changes in flight. Methods: We employed the worm Caenorhabditis elegans as a validated model of space biology, combined with 'NemaFlex-S' microfluidic devices for assessing animal strength production as one of the most reproducible physiological responses to spaceflight. Wild-type and dys-1 (BZ33) strains (a Duchenne muscular dystrophy (DMD) model for comparing predisposed muscle weak animals) were cultured on the International Space Station in chemically defined media before loading second-generation gravid adults into NemaFlex-S devices to assess individual animal strength. These same cultures were then frozen on orbit before returning to Earth for next-generation sequencing transcriptomic analysis. Results: Neuromuscular strength was lower in flight versus ground controls (16.6% decline, p < 0.05), with dys-1 significantly more (23% less strength, p < 0.01) affected than wild types. The transcriptional gene ontology signatures characterizing both strains of weaker animals in flight strongly corroborate previous results across species, enriched for upregulated stress response pathways and downregulated mitochondrial and cytoskeletal processes. Functional gene cluster analysis extended this to implicate decreased neuronal function, including abnormal calcium handling and acetylcholine signaling, in space-induced strength declines under the predicted control of UNC-89 and DAF-19 transcription factors. Finally, gene modules specifically altered in dys-1 animals in flight again cluster to neuronal/neuromuscular pathways, suggesting strength loss in DMD comprises a strong neuronal component that predisposes these animals to exacerbated strength loss in space. Conclusions: Highly reproducible gene signatures are strongly associated with space-induced neuromuscular strength loss across species and neuronal changes in calcium/acetylcholine signaling require further study. These results promote targeted medical efforts towards and provide an in vivo model for safely sending animals and people into deep space in the near future.

Growth kinetics of microalgae in microfluidic static droplet arrays.

We investigated growth kinetics of microalgae, Chlorella vulgaris, in immobilized arrays of nanoliter-scale microfluidic drops. These static drop arrays enabled simultaneous monitoring of growth of single as well as multiple cells encapsulated in individual droplets. To monitor the growth, individual drop volumes were kept nearly intact for more than a month by controlling the permeation of water in and out of the microfluidic device. The kinetic growth parameters were quantified by counting the increase in the number of cells in each drop over time. In addition to determining the kinetic parameters, the cell-size distribution of the microalgae was correlated with different stages of the growth. The single-cell growth kinetics of C. vulgaris showed significant heterogeneity. The specific growth rate ranged from 0.55 to 1.52 day(-1) for different single cells grown in the same microfluidic device. In comparison, the specific growth rate in bulk-scale experiment was 1.12 day(-1). It was found that the average cell size changes significantly at different stages of the cell growth. The mean cell-size increased from 5.99 ± 1.08 to 7.33 ± 1.3 µm from exponential to stationary growth phase. In particular, when multiple cells are grown in individual drops, we find that in the stationary growth phase, the cell size increases with the age of cell suggesting enhanced accumulation of fatty acids in older cells.

Inertial focusing of particles and cells in the microfluidic labyrinth device: Role of sharp turns.

Inertial, size-based focusing was investigated in the microfluidic labyrinth device consisting of several U-shaped turns along with circular loops. Turns are associated with tight curvature and, therefore, induce strong Dean forces for separating particles; however, systematic studies exploring this possibility do not exist. We characterized the focusing dynamics of different-sized rigid particles, cancer cells, and white blood cells over a range of fluid Reynolds numbers R e f . Streak widths of the focused particle streams at all the turns showed intermittent fluctuations that were substantial for smaller particles and at higher R e f . In contrast, cell streaks were less prone to fluctuations. Computational fluid dynamics simulations revealed the existence of strong turn-induced Dean vortices, which help explain the intermittent fluctuations seen in particle focusing. Next, we developed a measure of pairwise separability to evaluate the quality of separation between focused streams of two different particle sizes. Using this, we assessed the impact of a single sharp turn on separation. In general, the separability was found to vary significantly as particles traversed the tight-curvature U-turn. Comparing the separability at the entry and exit sections, we found that turns either improved or reduced separation between different-sized particles depending on R e f . Finally, we evaluated the separability at the downstream expansion section to quantify the performance of the labyrinth device in terms of achieving size-based enrichment of particles and cells. Overall, our results show that turns are better for cell focusing and separation given that they are more immune to curvature-driven fluctuations in comparison to rigid particles.

Detection of live breast cancer cells in bright-field microscopy images containing white blood cells by image analysis and deep learning.

SIGNIFICANCE: Circulating tumor cells (CTCs) are important biomarkers for cancer management. Isolated CTCs from blood are stained to detect and enumerate CTCs. However, the staining process is laborious and moreover makes CTCs unsuitable for drug testing and molecular characterization. AIM: The goal is to develop and test deep learning (DL) approaches to detect unstained breast cancer cells in bright-field microscopy images that contain white blood cells (WBCs). APPROACH: We tested two convolutional neural network (CNN) approaches. The first approach allows investigation of the prominent features extracted by CNN to discriminate in vitro cancer cells from WBCs. The second approach is based on faster region-based convolutional neural network (Faster R-CNN). RESULTS: Both approaches detected cancer cells with higher than 95% sensitivity and 99% specificity with the Faster R-CNN being more efficient and suitable for deployment presenting an improvement of 4% in sensitivity. The distinctive feature that CNN uses for discrimination is cell size, however, in the absence of size difference, the CNN was found to be capable of learning other features. The Faster R-CNN was found to be robust with respect to intensity and contrast image transformations. CONCLUSIONS: CNN-based DL approaches could be potentially applied to detect patient-derived CTCs from images of blood samples.