Journal of Visualized Experiments

Optimized histological techniques are crucial for visualizing cellular morphology across zebrafish tissues. Here, we report a rapid and reliable hematoxylin and Oil Red O (H-ORO) staining protocol for frozen sections that can be completed in less than three minutes. Mayers hematoxylin is used for nuclear staining, followed by Oil Red O (ORO) to visualize lipid-rich structures such as the endomysium surrounding myofibers, white matter of the brain, and myelin layers of major axonal tracts. Importantly, our optimized H-ORO protocol preserves tissue integrity and minimizes artifacts such as myofiber shrinkage commonly observed with ethanol-based hematoxylin and eosin (H&E) staining in both frozen and paraffin sections.

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f059d9org.highwire.dtl.DTLVardef@9aeffforg.highwire.dtl.DTLVardef@27cd1org.highwire.dtl.DTLVardef@150b7d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

We describe a standardized and reproducible procedure to generate human organotypic lung cultures from surgical lung resection for the study of respiratory infections. The protocol details tissue harvesting, biopsy punching, mechanical slicing, culture at the air-liquid interface. This technique enables robust ex vivo infections of human lung tissue with respiratory viruses, including Influenza A and Nipah. The described system can be used to study host-pathogen interactions, analyze innate immune responses, and evaluate antiviral candidates in physiologically relevant human lung tissue. For complete details on the use and execution of this protocol, please refer to Cezard et al1.

IntroductionImproving the efficiency and accuracy of annotation and extraction of performance data from mouse behavioural tasks will improve both the throughput and scientific value of preclinical research. MethodsHere, we present and validate an automated pipeline for the annotation and quantification of performance in a mouse olfactory habituation-dishabituation task, using a single side-view camera, resulting in occluded body parts. We created a pipeline for task analysis, combining DeepLabCut, for pose-estimation, and SimBA, for behavioural classification to automatically quantify odour interaction (sniffing time) in a three-odour (water, familiar mouse social odour, novel mouse social odour) variant of the task. We used a subset of previously published, fully manually annotated datasets to train the models and unseen videos from the same study to validate the utility of our machine learning pipeline. Results and conclusionOur analysis pipeline estimated behavioural performance in the task with high accuracy, and the data produces similar technical and biological results to manual methods when analysed by linear mixed modelling. Thus, we validated the utility of our new pipeline for the automated scoring of this mouse sensory task.

Octopus vulgaris and other cephalopods are of increasing interest as neurobiological model organisms. This protocol describes a method to record calcium activity from individual cells in acute brain slices from Octopus vulgaris hatchlings during exogenous application of neurotransmitters. Using this protocol, we characterized single-cell responses to specific neurotransmitters in the optic lobes, which process visual information. The approach is readily adaptable to other cephalopods and small invertebrate species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/711860v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1564eaeorg.highwire.dtl.DTLVardef@147b682org.highwire.dtl.DTLVardef@11f3b85org.highwire.dtl.DTLVardef@17c9d70_HPS_FORMAT_FIGEXP M_FIG C_FIG

Investigating the neurophysiology of nociception is aided by electrophysiological recording from dorsal root ganglion (DRG) neurons. Because DRG neurons are heterogeneous with overlapping electrophysiological properties, methods to distinguish neuron subtypes are valuable for properly interpreting the measurements and drawing conclusions. Automated patch clamp recording offers an approach for conducting these experiments at higher throughput than conventional recording methods, but identification of neuron subtypes is challenging. We developed a method for recording from acutely isolated mouse DRG neurons using automated patch clamp recording coupled to optogenetic stimulation that was capable of discerning NaV1.8 and TRPV1 expressing neuron subpopulations. This approach can facilitate physiological and pharmacological studies of DRG neurons with potential value in developing and testing targeted analgesic agents.

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This protocol addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step methods specify reagents, equipment, acquisition parameters, and expected results. While recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, this protocol establishes a flexible approach to standardize organelle quantification to understand multiple organelles simultaneously in their cellular contexts. Basic Protocol 1: Mitochondrial Quantification Basic Protocol 2: Myofibril Quantification Basic Protocol 3: Golgi Apparatus Morphometry Basic Protocol 4: Endoplasmic Reticulum Network Analysis Alternate Protocol 1: Super-Resolution Imaging Protocol

BackgroundLong-term home-cage monitoring is essential to quantify spontaneous locomotor and social behaviors in group-housed mice, but analysis of high-density RFID tracking data remains a barrier to reproducibility. New methodsWe developed IntelliProfiler 2.0, a fully R-based pipeline tailored to the eeeHive 2D floor-mounted RFID array. The workflow performs data import from text logs, preprocessing, coordinate reconstruction, missing-value handling, feature extraction, statistical testing, and visualization in a single environment. Behavioral metrics include travel distance, close contact ratio (CCR), and a newly implemented inter-individual distance metric. ResultsIn four-day recordings of group-housed C57BL/6J mice (8 males and 8 females), IntelliProfiler 2.0 captured circadian phase-dependent locomotion and proximity patterns and reproduced sex-dependent differences consistent with prior analyses while incorporating updated hardware specifications. Radar-chart summaries enabled intuitive comparison of multidimensional behavioral profiles and inter-individual variability across light/dark phases. Comparison with existing methodsCompared with IntelliProfiler 1.0 and multi-tool workflows, IntelliProfiler 2.0 consolidates analysis into a single, script-based R pipeline, reducing operational complexity and improving reproducibility. The updated implementation supports recent manufacturer-driven changes, including antenna renumbering and multi-USB data export. ConclusionsIntelliProfiler 2.0 provides a reproducible, extensible framework for high-throughput behavioral phenotyping of group-housed mice and is scalable across hardware configurations, including simplified single-board recordings. HighlightsO_LIEnd-to-end R pipeline for eeeHive 2D floor-based RFID tracking analysis C_LIO_LIStandardized setup with comprehensive manuals and protocols C_LIO_LIInter-individual distance metric to quantify group spatial structure C_LIO_LICircadian- and sex-dependent behavioral profiling in group-housed mice C_LIO_LIRadar-charts summarize multidimensional behavioral profiles and variability C_LI

Precision-cut lung slices (PCLS) have emerged as a powerful tool for studying the biology of viable human lung tissue. However, the presence of agarose impurities compromises RNA yield and integrity during the extraction process. We tested whether using an alternative Plant kit RNA extraction method to wash agarose impurities or pre-dissolving agarose from PCLS implementing a dissolving buffer for routine RNA isolation in gel-electrophoresis would improve RNA quantity, quality, and integrity. Our results show that RNA quantity and integrity are highly compromised when using a conventional method of RNA extraction. The plant kit and dissolution of agarose increased the RNA quantity to 0.42{+/-}0.11 and 0.65{+/-}0.17 {micro}g/PCLS (measured by the Qubit) and integrity number to 6.60{+/-}0.59 and 9.13{+/-}0.39 (measured by the Bioanalyzer), respectively. The presence of impurities in conventional and Plant kit extractions misled to an overestimation of the RNA quantity and quality using the NanoDrop. The Plant kit and agarose dissolution showed a significant transcript integrity increase in GUSB (p<0.0001) and COL1A1 (p<0.05) expression, validating these methods over conventional extraction. We encourage laboratories applying PCLS experimentation to implement alternative methods to remove agarose impurities during RNA extraction, as well as to rely on sensitive quantitative techniques, such as the Qubit and Bioanalyzer, for RNA quantification and integrity measurements.

As human space exploration expands to the Moon, Mars, and beyond, there is a growing need to study the effects of altered gravity on the microbial systems that we will bring with us for life support. Because spaceflight experiment opportunities are rare and resource-intensive, most space biology experiments are conducted using ground-based simulators. The most common microgravity simulator for microbial experiments, the rotating wall vessel, can approximate the low-shear and low-turbulence conditions that characterize microgravity. However, current designs do not allow for real-time measurement of growth or metabolic activity during rotation: experiments require destructive sampling or disruption of the microgravity simulation conditions. Here, we describe the development of an in situ spectroscopy system compatible with the Cell Spinpod rotating wall vessel, which enables measurement of both optical absorbance and fluorescence with high temporal resolution, producing growth curves similar to those from an off-the-shelf plate reader. These results are validated using two common microbial hosts: Escherichia coli and Saccharomyces cerevisiae. The Spinpod Optical System has the potential to diversify the types of microbiology experiments possible in simulated microgravity, allowing the measurement of not only growth curve parameters but also metabolic activity, gene expression, or community dynamics. It thus has the potential to improve the quality of experiments seeking to characterize microbial responses to spaceflight conditions.

Longitudinal brain imaging is essential for understanding neural mechanisms. Here, we present a saline-free, chronic preparation for repeated neural recording in adult Drosophila over multiple days. We describe steps for mounting flies, performing manual surgery on the head cuticle without external saline, and resealing the opening to create a transparent optical window. We demonstrate the utility of this approach by tracking single-neuron spiking and neuronal calcium dynamics over 7-10 days. This protocol is potentially applicable to other insect species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/706199v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@abeb34org.highwire.dtl.DTLVardef@deaf93org.highwire.dtl.DTLVardef@1d8fc24org.highwire.dtl.DTLVardef@91a696_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundPhotobiomodulation (PBM) therapy has demonstrated therapeutic potential in promoting cellular repair, modulating inflammation, and enhancing mitochondrial function. Platelet-rich plasma (PRP) is widely used in regenerative medicine due to its concentration of growth factors and cytokines. Very small embryonic-like stem cells (VSELs), a rare population of pluripotent stem cells present in adult tissues, have emerged as a potential contributor to tissue regeneration. While PBM and PRP are used in combination, how VSELs or Multi-lineage stress enduring (MUSE) cells are at play, and the biological mechanisms underlying their synergistic effects remain incompletely characterized. ObjectiveThis exploratory pilot study aimed to evaluate whether application of the MD Biophysics laser to autologous PRP is associated with measurable changes in VSEL-related antibody marker expression, and to identify directional trends to inform future controlled studies. MethodsPRP samples were collected from participants across seven test dates (July 2024 to February 2025), yielding 18 participant-session datasets. Samples were analyzed before (Pre) and after (Post) laser application using flow cytometry conducted at a UCLA Flow Cytometry Laboratory. Four VSEL-associated antibody markers were assessed: CD45-CD34+, CXCR4+, CD133+, and SSEA-4+. Analyses were descriptive and focused on paired differences and directional trends due to the exploratory design and absence of a control group. ResultsThree of four VSEL-associated markers (CXCR4+, CD133+, and SSEA-4+) demonstrated a group-level increase in median paired differences following laser application. Directional increases were observed in 12/18 sessions for CXCR4+, 10/18 for CD133+, and 9/18 for SSEA-4+. CD45-CD34+ showed a near-equal distribution of increases and decreases. Ki-67 positivity indicated the presence of viable, proliferative cells. While no findings reached statistical significance due to limited sample size, consistent directional trends were observed across multiple markers. ConclusionApplication of PBM to autologous PRP was associated with directional increases in multiple VSEL-associated antibody markers, suggesting a potential role for stem cell activation or mobilization in the mechanism of action. Although preliminary and not statistically powered, these findings provide hypothesis-generating evidence supporting further investigation. The observed trends informed iterative protocol refinement and establish a foundation for future controlled, adequately powered studies to evaluate clinical efficacy and underlying biological mechanisms.

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Computational bioacoustics has seen significant advances in recent decades. However, the rate of insights from automated analysis of bioacoustic audio lags behind our rate of collecting the data - due to key capacity constraints in data annotation and bioacoustic algorithm development. Gaps in analysis methodology persist: not because they are intractable, but because of resource limitations in the bioacoustics community. To bridge these gaps, we advocate the open science method of data challenges, structured as public contests. We conducted a bioacoustics data challenge named BioDCASE, within the format of an existing event (DCASE). In this work we report on the procedures needed to select and then conduct useful bioacoustics data challenges. We consider aspects of task design such as dataset curation, annotation, and evaluation metrics. We report the three tasks included in BioDCASE 2025 and the resulting progress made. Based on this we make recommendations for open community initiatives in computational bioacoustics.

MOTIVATIONLongitudinal molecular studies of the mouse brain are limited by the need for terminal tissue collection. This prevents analysis of preexisting molecular states and their evolution within the same individual. We developed a stereotactic microbiopsy technique that enables minimally invasive sampling of defined brain regions in vivo. The method preserves survival while yielding material suitable for RNA and nuclei isolation. It provides a practical solution for linking baseline molecular states to subsequent behavioural, pharmacological, or disease-related outcomes. SUMMARYThis study presents a stereotactic microbiopsy technique for sampling defined brain regions in living mice, enabling transcriptomic and epigenomic analyses without sacrificing the animal. The method will allow pre-intervention tissue collection, making it possible to separate preexisting molecular differences from experience- or treatment-induced changes. We show that microbiopsies yield sufficient, high-quality RNA and chromatin for sequencing, with minimal tissue damage that largely resolves over time. The procedure uses standard stereotactic equipment and achieves reproducible spatial precision when the syringe is stabilised. This approach provides a practical framework for within-subject molecular comparisons, reducing animal use and enabling longitudinal profiling of the living mouse brain. It establishes a foundation for investigating how baseline molecular states influence later physiological or behavioural outcomes.

Dance is a core form of human-environment interaction and a powerful medium for emotional expression, yet dancers are routinely exposed to environmental affective cues that may shape their movement. We tested whether a negative emotional context induced immediately before improvisation alters dance biomechanics. Twenty professional dancers performed two 3-min improvised dances. Between dances, they viewed either Neutral or Negatively valenced pictures from the International Affective Picture System (IAPS; 2 min 40 s, 5 s per image). Eye tracking verified attention to the visual stream. Mood was assessed at four time points (PT1-PT4) using the Brazilian Mood Scale (BRAMS), and full-body, three-dimensional kinematics were captured at 300 Hz using a 9-camera optoelectronic system (Qualisys) and processed to measure global movement amplitude and expansion. Negative IAPS exposure increased tension, depression, fatigue, and decreased vigor from PT2 to PT3. Biomechanically, the Negative Stimulus dancers showed a significant reduction in global movement amplitude after negative IAPS exposure, with reduced movement amplitude of the body extremities. In contrast, global movement expansion remained unchanged; that is, the extremities were not positioned closer or farther from the pelvis. Neutral images produced no mood change and no measurable modulation of movement amplitude or expansion. Together, these results support the hypothesis that improvised dance carries biomechanical signatures of the dancers current affective state, beyond the intended expressive content, and provide an automated motion-capture workflow for studying emotion-movement coupling in spontaneous dance. HighlightsNegative visual context shifted dancers mood toward negative affect Negative images reduced movement amplitude in improvised dance Movement expansion remained stable despite mood induction Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/711707v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@aeaacdorg.highwire.dtl.DTLVardef@14f9bf5org.highwire.dtl.DTLVardef@18805fcorg.highwire.dtl.DTLVardef@1411256_HPS_FORMAT_FIGEXP M_FIG C_FIG

Currently, implantation of electroencephalogram (EEG) electrodes in laboratory animals is time-consuming and requires specialized equipment. We present a novel method for EEG recordings in mice that utilizes thin needle electrodes. These electrodes are inserted into the skull at predetermined locations by gently pressing them against the bone surface. To ensure stable fixation of the implant, hook-shaped needles are positioned along the lateral aspects of the skull. The electrodes are connected to a multipin connector and secured to the skull using dental composite, after which the animal is allowed to recover from anesthesia. Importantly, procedures such as skull drilling and screw placement are not required, allowing the entire surgery to be completed in less than 15 minutes. Consequently, this EEG implantation approach is rapid and minimally invasive. Results of our studies indicate that EEG recordings obtained with needle electrodes are not inferior to those obtained with screw electrodes. Overall, the method is designed to enhance the accuracy and efficiency of EEG recording studies while improving animal welfare. O_LISimplifies the placement of EEG electrodes. C_LIO_LIReduces the time required for electrode implantation. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/715731v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@e5608org.highwire.dtl.DTLVardef@1325ea4org.highwire.dtl.DTLVardef@1e37202org.highwire.dtl.DTLVardef@1521bb8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Space-based platforms currently represent the most accurate means to experimentally assess the influence of the space environment on biological systems. However, performing such experiments remains technically challenging and requires highly specialized instrumentation. This study describes the current development and hardware qualification of ExocubeBio, a miniaturized experimental platform for in-situ biological space exposure. This experiment is scheduled for installation on the exterior of the International Space Station in 2027, as part of Exobio, the European Space Agencys new generation exobiology exposure facility. ExocubeBio will expose live microbial samples to the low Earth orbit environment, and combine autonomous in-situ optical density and fluorescence measurements, with the capacity to return preserved samples to Earth. Achieving these experimental goals requires a specialized, robust and reliable hardware system. The ExocubeBio hardware testing described here includes assessment of material biocompatibility and durability, functional validation of the miniaturized fluidic system, and optimization of the integrated optical subsystem for optical density and fluorescence measurements. These results demonstrate that the ExocubeBio experimental hardware components can each execute their core functional and operational requirements; subsystems allow for sample exposure, in-situ measurements of microbial cultures, and the chemical preservation of samples for post-flight analysis. As ExocubeBio transitions from hardware development to mission readiness, the results presented here validate the overall design and engineering approaches utilized. By combining the strengths of in-situ monitoring and sample return, ExocubeBio represents a significant advancement in space-based experimentation, and will provide new insights into microbial responses to the space environment.

BackgroundFlow virometry (FV) - the application of flow cytometry to viruses - has historically been hindered by the inability of cytometers to detect particles below [~]300 nm in size. However, advances in optics and fluidics have enabled cytometers primarily designed for cells to detect viruses and extracellular vesicles (EVs) through light scatter alone. In 2024, the CytoFLEX nano was released, marketed for the detection of particles as small as 40 nm; however, its performance has yet to be compared to a conventional instrument for FV. MethodsFV was utilized to evaluate performance of the CytoFLEX nano and a conventional flow cytometer (CytoFLEX S). Instrument scatter sensitivity was assessed using NIST beads (40-400 nm), and virus stocks [human immunodeficiency virus (HIV), human coronaviruses (HCoV)-229E and HCoV-OC43]. For fluorescence analysis, HIV virions were stained with PE- and BV421-conjugated antibodies targeting virion incorporated proteins (CD38, CD44), individually and in combination. Finally, HIV stocks were labeled with antibodies against the envelope (Env) glycoprotein and tetraspanins (CD9, CD81) to assess EVs within virus preparations. ResultsCompared to the CytoFLEX S, the CytoFLEX nano exhibited substantially greater scatter sensitivity, reflected by up to 50-fold higher signal-to-noise ratio across NIST-traceable beads and virus samples. This enabled clearer resolution of smaller populations, including bead populations < 70 nm that were undetectable on the CytoFLEX S, as well as improved resolution across all viruses. While both instruments reliably detected stained proteins on HIV virions, the CytoFLEX nano revealed a distinct population of tetraspanin-positive EVs within HIV stocks that was undetected on the CytoFLEX S. Using GFP-tagged HIV, we identified Env+ particles lacking GFP, indicating the presence of Env on EVs. ConclusionsThe CytoFLEX nano exhibited markedly improved scatter sensitivity compared to the CytoFLEX S, improving detection of viruses and enabling detection of EV populations that were undetectable on the conventional instrument. While both platforms performed similarly for surface protein labeling, additional consideration of spectral overlap was needed with the CytoFLEX nano in multicolor experiments. These findings highlight that the complementary strengths of each platform can be utilized to more comprehensively characterize virus and EV populations, providing new opportunities to investigate nanoparticle heterogeneity.