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.

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.

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.

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.

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.

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

The use of domestic pigs in clinical training and biomedical research is expanding rapidly, increasing the need for reliable, noninvasive indicators of health and welfare. Vocal analysis offers a non-invasive promising tool, yet the acoustic repertoire of adult domestic pigs remains poorly defined. However, the vocalization repertoire of adult domestic pigs has yet to be characterized. This study characterizes the vocal repertoire of adult pigs housed in a biomedical research laboratory. Twelve mixed-breed pigs (2-3 months old; 5 males, 7 females) were recorded during routine husbandry and experimental procedures. Vocal classification was conducted using perceptual and objective clustering techniques. First, aural- visual (AV) inspection of spectrograms was used to construct a hierarchical repertoire. Second, a two-step cluster analysis based on six acoustic parameters (5% frequency, first quartile frequency, center frequency, 90% bandwidth, interquartile range bandwidth, and 90% duration) provided an objective classification. Agreement between methods was evaluated using Cramers V. A total of 1,136 vocalizations from 69 recordings were analyzed. AV classification revealed five major vocal classes-- grunt, squeal, complex, scream, and bark--subdividing into 16 distinct call types. Standardized definitions integrating descriptive and quantitative criteria are provided. The two-step cluster analysis identified two clusters as the optimal statistical solution, with moderate agreement between methods (Cramers V = 0.67, p < 0.0001). Most AV-defined call types aligned with previously reported repertoires, although whines, yelps, and stable screams were unique to this study. While two-cluster solutions are commonly reported, our findings indicate that richer acoustic structure exists and that high gradation among pig calls may limit the resolution of statistical clustering. These results establish a detailed acoustic framework for adult pig vocalizations and provide essential groundwork for developing predictive models to enhance welfare assessment and support comparative research in laboratory-housed pigs.

Endocavity ultrasound transducers, particularly transvaginal ultrasound (TVUS) probes, contain intricate structures such as notches, grooves, lens surfaces, and handle edges that are highly susceptible to microbial contamination yet difficult to disinfect using conventional high-level disinfection (HLD) methods. This study evaluated the efficacy of a novel ultraviolet-C light-emitting diode (UV-C LED) HLD system in eliminating microbial contamination from these complex probe surfaces. Two TVUS probes were sampled from predefined high-risk regions before and after disinfection following clinical use. Probe A was sampled at the top and bottom notches and both sides of the handle, while Probe B was assessed at the lens, edges, and bent groove regions. Microbial contamination was quantified using swab sampling, culture on agar plates, and identification via MALDI-TOF. Environmental sampling of examination and disinfection rooms was also performed. To assess this system robustness, probe sites were repeatedly inoculated with Bacillus subtilis spores and evaluated following UV-C treatment. Before UV-C treatment, contamination rates ranged from 25% to 57% across sampled regions, with microbial loads reaching up to 3.9 log CFU. Identified organisms included Staphylococcus epidermidis, Pseudomonas koreensis, Bacillus cereus, and Propionibacterium spp. Probe sheaths were also predominantly contaminated with Staphylococcus epidermidis., with counts reaching up to 4.3 log CFU, Environmental sampling revealed diverse microbiota, with higher contamination levels in examination rooms compared to disinfection areas. Following 90 seconds of UV-C exposure, no microbial growth was detected on any sampled site, indicating 100% decontamination. Additionally, UV-C treatment achieved >6.7 log reduction of B. subtilis spores across all tested regions. These findings demonstrate that UV-C LED technology provides rapid, effective, and consistent high-level disinfection of complex TVUS probe surfaces, supporting its potential as a rapid and reliable disinfection modality in clinical setting.

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

BackgroundTranscranial Magnetic Stimulation (TMS) studies identify the Resting Motor Threshold (RMT) to calibrate stimulation intensity. However, this procedure is time-consuming and subject to variability. We developed an automated procedure to improve the efficiency and standardization of RMT determination. New methodWe developed an algorithm that measures MEP amplitudes and automatically adjusts stimulation intensity to determine the RMT. Experiment 1 compared this automated method with the manual procedure in terms of reliability and equivalence. Experiment 2 developed a "Fast" automated process, assessing it against both the manual and initial automated procedures. ResultsAcross both experiments the automated approach demonstrated excellent test-retest reliability and strong agreement with the manual method (Intraclass Correlation Coefficients [&ge;]0.95), giving estimates of RMT statistically equivalent to those of manual measurements within {+/-}3% MSO, with the majority of comparisons within {+/-}2% MSO. Experiment 2 optimized the procedure, allowing empirical determination of the RMT in an average of <3 minutes with only 33-34 pulses. Comparison with existing methods RMT-Finder provides a reliable and time-efficient alternative to manual approaches. To the best of our knowledge RMT-Finder presents the first closed-loop feedback approach to identify the RMT without manual intervention. This procedure can improve standardization and reproducibility in TMS studies. ConclusionsAutomating RMT assessment allows rapid and highly reproducible assessment of this standard TMS measurement, making it viable for inclusion in routine clinical applications that require standardized procedures.

Cell and tissue-specific transcriptomic profiling of Caenorhabditis elegans is commonly achieved by fluorescence tagging or staining of targeted cell populations, often followed by fluorescence-activated cell sorting (FACS) and RNA sequencing. However, these approaches typically require separate strains for each labeled population, increasing labor and experimental variability while limiting direct comparison of multiple tissues within the same genetic background. To address this limitation and establish proof of concept, we engineered CELeidoscope, a multicolored C. elegans strain that enables spectral sorting of multiple major cell types within a single strain population. Strain construction was carried out using a high-throughput screening method that reduces the labor and plastic costs associated with transgene integration and outcrossing. Four primary tissues (body muscle, neurons, intestinal, and pharyngeal muscle cells) were tagged with spectrally distinct fluorescent proteins, allowing compatibility with viability and nucleic acid dyes. Using spectral flow cytometry, dissociated CELeidoscope cell suspensions could be sorted based on their spectral profiles, with cell recovery rates approximating the expected cell counts in whole organisms. Transcriptomic analysis of the sorted cell populations further validated the identity of the sorted populations, with recovered cells exhibiting gene expression signatures consistent with their intended cell and tissue identities. Together, these results establish CELeidoscope as a versatile tool for multiplexed cell-type isolation in C. elegans, providing a framework for tissue-specific analyses from a common strain background.

BACKGROUND: Cranioplasty is an essential procedure to restore cranial integrity, protect neural structures, and improve cosmetic outcomes. However, commercially available implants are often costly, limiting their accessibility in public healthcare systems. Three dimensional (3D) printing offers a low cost alternative for producing patient-specific solutions. METHODS: A retrospective case series of eight patients undergoing cranioplasty using customized polymethylmethacrylate (PMMA) implants fabricated with 3D printed molds was conducted. Computed tomography (CT) scans were used for segmentation and digital modeling. Patient specific molds were designed and printed preoperatively. Variables analyzed included design time, printing time, intraoperative workflow, and clinical outcomes. RESULTS: Design time ranged from approximately 1 hour for small defects to 3 hours for larger defects. Printing time ranged from 2 3 hours for smaller defects and up to 8 10 hours for larger reconstructions. Satisfactory aesthetic outcomes were achieved in 7 of 8 patients (87.5%). No major implant related complications were observed. CONCLUSION: Low cost 3D printing for PMMA cranioplasty is a feasible, accessible, and effective technique for cranial reconstruction, particularly in resource limited settings. Keywords: Cranioplasty; 3D printing; Cranial defect reconstruction; Low cost surgery; Patient specific implants; Polymethylmethacrylate; Skull reconstruction

Particle bombardment systems are widely used for plant transformation, but commercial devices are expensive and rely on high-pressure helium gas. This study aimed to develop a cost-effective and helium gas-free alternative using an air duster gun connected to a commercial compressor. A nozzle (for DNA with transgenes), gold particles (as DNA carriers), nozzle-to-sample distance, and a method for coating gold particles with DNA were optimized to yield better transformation efficiency in targeting onion epidermal cells and rice calli. From the rice calli transformed with the newly developed system (a tool to shoot genes with massive air from a compressor: TSGMAC), stable transgenic plants could be obtained. TSGMAC offers a low-cost and helium gas-free solution for plant transformation and genome editing and can enhance accessibility to particle bombardment-based techniques.

Motion artifacts remain a barrier to in vivo calcium imaging in Drosophila melanogaster larvae. Here, we evaluate a multimodal immobilization approach that combines a Pluronic F-127 (PF-127) hydrogel with brief diethyl ether vapor exposure (5 minutes, 25{degrees}C) and compare it against hydrogel-only immobilization using custom MATLAB-based analysis software that performs NoRMCorre rigid motion correction. In wide-field GFP recordings at 1 Hz over approximately 60 minutes (N = 15 per group), the multimodal condition significantly reduced motion across all three core metrics after FDR correction (all q < 0.001), with large effect sizes for mean speed (Hedges g = -1.18) and median step size (g = -1.36). In a secondary analysis of the first 30 minutes, uniformly large effect sizes (|g| = 1.10-1.51) were observed, consistent with stronger initial chemical immobilization that partially wanes over the recording period. We implemented a dual-flag quality control system that distinguishes motion data reliability from ROI detection eligibility. Control calcium recordings (33.33 Hz, [~]5 minutes; N = 23) yielded 368 ROIs with a mean SNR 30.4 {+/-} 16.9 and an event rate of 0.228 {+/-} 0.113 Hz. Experimental recordings (N = 21) yielded 295 ROIs with SNR 18.0 {+/-} 10.6 and event rate 0.309 {+/-} 0.188 Hz. SNR was higher in controls (Cliffs{delta} = 0.50, p < 0.001), while event rate was modestly higher in the experimental group at the ROI level ({delta} = -0.22, p < 0.001), though this difference did not reach significance at the sample level, suggesting altered but not suppressed calcium dynamics. These results support a practical, accessible immobilization workflow for larval calcium imaging. HighlightsO_LIBrief ether + hydrogel approach reduces larval motion 85-91% vs. hydrogel alone C_LIO_LIDual-flag QC system separates motion reliability from calcium ROI eligibility C_LIO_LICalcium event rates not suppressed under multimodal immobilization C_LIO_LIComplete MATLAB pipeline for motion analysis and calcium imaging provided C_LIO_LIAccessible protocol requires only standard laboratory supplies C_LI

Quantitative histological analysis of skeletal muscle morphometry provides critical insights into muscle physiology but remains labor-intensive and technically demanding. While recent developments in machine-learning-based image segmentation techniques have facilitated large-scale tissue analysis, existing tools that automate muscle morphometry analysis are largely tailored to mammalian models, with limited applicability to teleosts. Moreover, there is a lack of effective tools for visualizing spatial organization and morphometric variability of teleost muscle fibers, a feature that is important for understanding hyperplastic muscle growth dynamics in teleosts. In this study, we show that cytoplasmic staining combined with deep learning-based cell segmentation offers a robust and accurate approach for automated muscle morphometry analysis in developing zebrafish. We also introduce a FIJI2 plugin, implemented in Jython, that streamlines both morphometric analysis and visualization. This tool accommodates shallow and deep learning-based segmentation techniques and incorporates novel quantification and visualization methods suited to teleost-specific muscle features, including mosaic hyperplasia dynamics. The plugin features an intuitive graphical user interface and is designed for flexibility, with minimal constraints regarding species, image quality, or staining protocol. Its modular architecture allows it to be used as a baseline for automated muscle morphometry analysis, while permitting integration with other tools and workflows.