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.

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO dyes should be used with caution on live samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/560001v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b8bab4org.highwire.dtl.DTLVardef@e9a84forg.highwire.dtl.DTLVardef@291058org.highwire.dtl.DTLVardef@1f0245d_HPS_FORMAT_FIGEXP M_FIG C_FIG

This protocol describes a versatile in vivo zebrafish embryo infection model with Candida albicans that enables precise microinjection and monitoring of defined infection readouts to compare experimental conditions (e.g., injection site and incubation temperature). The core workflow consists of quantitative microinjection of DiI-labeled C. albicans into the hindbrain ventricle of 30-48 hpf embryos, incubation at 23 deg C, and daily survival scoring to evaluate condition-dependent mortality. We calibrate the inoculum by hemocytometer counts and by plating the same injection suspension to determine colony-forming units (CFU) per pulse, allowing CFU per embryo to be reported and experiments to be compared across laboratories. Survival is analyzed with Kaplan-Meier curves and, when groups are compared, Cox models. DiI fluorescence verifies successful delivery, excludes mis-injections, and permits semi-quantitative burden comparisons between injection sites and temperatures at 24-48 hpi; beyond [~]48 hpi, signal becomes unreliable due to dye dilution and redistribution. The protocol is designed to be reproducible without transgenic reporters or confocal microscopy and includes practical guidance on randomization, blinding, sample-size planning, and reporting.

Tissues consist of various components and if these can be deleted or reserved, their location and proportion can be detected. Scanning acoustic microscopy (SAM) calculates the attenuation of sound (AOS) through tissue sections to obtain histological images without staining. AOS values are reduced as tissue components break down. Here, we digested specific components in tissues using enzymes and followed the process with AOS imaging over time. In addition, we applied specific dyes and antibodies to inhibit enzyme activity and maintain a specific component in the section. We used specific enzymes to degrade tissues that contain the enzymes substrate, such as collagenase for bone, elastase for skin and arteries, actinase for amyloid-positive cervical arteries and lymph nodes, amylase for the corpora amylacea (CA) of the brain and DNase and haematoxylin for adenocarcinomas. Collagenase digested bone and cartilage to clearly visualise the internal structure. The structural components had characteristic AOS values, which gradually decreased. Elastases break elastic fibres in the skin and arteries differently between young and old individuals. The dermis and tunica media of arteries in the elderly fracture more easily than those of younger individuals. Actinase digested the cervical artery except for amyloid deposits, which were preferentially detected by Congo red staining. Actinase-digested lymphoid cells remained horseradish peroxidase (HRP)-staining positive. Amylase digested some CAs, which became periodic acid-Schiff (PAS) staining negative and diminished in size by electron microscopy observation. Cell nuclei were digested and deleted by DNase except for those stained with HRP. Residual nuclear images of AOS matched those of light microscopy, and haematoxylin staining inhibited DNase digestion of the nucleus. Specific inhibition of enzymes preserved the target cells and materials. SAM observation can monitor the tissue breakdown process, which provides an advantage over light microscopy as no staining is required and exhibits higher sensitivity to detect fragile structures.

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.

BackgroundO_LIMany Drosophila species use acoustic communication during courtship and studies of these communication systems have provided insight into neurobiology, behavioral ecology, ethology, and evolution. C_LIO_LIRecording Drosophila courtship sounds and associated behavior is challenging, especially at high throughput, and previously designed devices are relatively expensive and complex to assemble. C_LI ResultsO_LIWe present construction plans for a modular system utilizing mostly off-the-shelf, relatively inexpensive components that provides simultaneous high-resolution audio and video recording of 96 isolated or paired Drosophila individuals. C_LIO_LIWe provide open-source control software to record audio and video. C_LIO_LIWe designed high intensity LED arrays that can be used to perform optogenetic activation and inactivation of labelled neurons. C_LIO_LIThe basic design can be modified to facilitate novel study designs or to record insects larger than Drosophila. C_LIO_LIFewer than 96 microphones can be used in the system if the full array is not required or to reduce costs. C_LI ImplicationsO_LIOur hardware design and software provide an improved platform for reliable and comparatively inexpensive high-throughput recording of Drosophila courtship acoustic and visual behavior and perhaps for recording acoustic signals of other small animals. C_LI

The fission yeast Schizosaccharomyces pombe is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar, from different suppliers, were toxic. Interestingly, the inhibitory effect was attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/597796v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@41d7c1org.highwire.dtl.DTLVardef@bbec24org.highwire.dtl.DTLVardef@18c6f76org.highwire.dtl.DTLVardef@e9b707_HPS_FORMAT_FIGEXP M_FIG C_FIG Take-awayO_LISporadically, batches of agar from different suppliers strongly inhibit the plating efficiency of S. pombe spores and vegetative cells on minimal media. C_LIO_LIQuality control checks that are not quantitative or that use only rich media can provide false assurances on the quality of the agar. C_LIO_LIVendors should conduct rigorous, thorough, organism-specific tests for potential toxicity of each lot of agar as a pre-condition for its sale. C_LI

BackgroundRodent sleep scoring in principally reliant on electroencephalogram (EEG) and electromyogram (EMG), but this approach is invasive, can be expensive, and requires expertise and specialized equipment. Affordable, simple to use, and noninvasive ways to accurately quantify rodent sleep are needed.\n\nNew methodWe developed and validated a new method for sleep-wake staging in mice using cost-effective, noninvasive electric field (EF) sensors that detect respiration and other movements. We validated recordings from EF sensors attached to the exterior of specialty chambers used to continuously capture sleep with EEG/EMG, then compared this to EF sensors attached to vivarium home-cages.\n\nResultsEF sensors quantified 3-state sleep architecture (wake, rapid eye movement - REM - sleep, and non-REM sleep) with high agreement (>93%) and comparable inter- and intra-scorer error as expert EEG/EMG scoring. Novices given an instruction document with examples were able to score sleep comparable to expert scorers (>91% agreement). Additionally, EF sensors were able to quantify 3-state sleep scoring in traditional mouse home cages.\n\nComparison with existing methodMost noninvasive sleep assessment technology requires animal contact, altered cage environments, and/or can only discern 2 states of arousal (wake or asleep). The EF sensors are able to discriminate REM from non-REM sleep accurately and from outside the animals home cage.\n\nConclusionsEF sensors provide a simple and reliable method to accurately score 3-state sleep architecture; (i) from outside the typical home cage, (ii) where noninvasive approaches are preferred, or (iii) which EEG/EMG is not possible.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC=\"FIGDIR/small/794552v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (43K):\norg.highwire.dtl.DTLVardef@1b36cf3org.highwire.dtl.DTLVardef@b5fdc9org.highwire.dtl.DTLVardef@28a6dforg.highwire.dtl.DTLVardef@e39a5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Studying adipogenesis and adipocyte biology requires the isolation of primary preadipocytes from adipose tissues. However, primary preadipocytes have a limited lifespan, can only undergo a finite number of divisions, and often lose their original biological characteristics before becoming senescent. The repeated isolation of fresh preadipocytes, particularly from young pups or aged animals, is costly and time-consuming. Immortalization of these cells offers a solution by overcoming cellular senescence and maintaining proliferative capacity, allowing for long-term studies without the continuous need to isolate new cells from animals. Immortalized cell lines thus provide a consistent and reproducible experimental model, significantly reducing variability across different animals. However, successfully establishing immortalized preadipocyte cell lines presents challenges, including selecting appropriate adipose tissue depots, isolating primary preadipocytes, and choosing an effective immortalization strategy. In this study, we present optimized protocols and share first-hand experiences establishing immortalized brown and white preadipocyte cell lines from young and aging mice. These protocols offer a valuable resource for researchers studying adipogenesis, metabolism, and adipocyte biology. Support Protocol 1: Retrovirus production Basic Protocol 1: Isolation and culture of primary brown and white preadipocytes from mouse interscapular brown adipose tissue (iBAT) and subcutaneous white adipose tissue (sWAT) in the same region Basic Protocol 2: Immortalization of mouse brown and white preadipocytes Basic Protocol 3: Selection of immortalized preadipocytes Basic Protocol 4: Selection of single-cell clones of immortalized preadipocytes Support Protocol 2: Cryopreservation of immortalized preadipocytes Support Protocol 3: Wake up and culture of immortalized preadipocytes Support Protocol 4: Subculture and expansion of immortalized preadipocytes Basic Protocol 5: Differentiation of immortalized mouse brown and white preadipocytes Support Protocol 5: Lipid droplet staining and nucleus counterstaining Support Protocol 6: Mitochondria staining and nucleus counterstaining

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350{euro} in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/543100v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@6dfcb7org.highwire.dtl.DTLVardef@ea7fbcorg.highwire.dtl.DTLVardef@1681e78org.highwire.dtl.DTLVardef@a88d51_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Fluid shear stress is a key modulator of cellular physiology in vitro and in vivo, but its effects are under-investigated due to requirements for complicated induction methods. Herein we report the validation of ShearFAST; a smartphone application that measures the rocking profile on a standard laboratory cell rocker and calculates the resulting shear stress arising in tissue culture plates. ShearFAST measured rocking profiles were validated against a graphical analysis and also against measures reported by an 8-camera motion tracking system. ShearFAST angle assessments correlated well with both analyses (r [≥]0.99, p [≤]0.001) with no significant differences in pitch detected across the range of rocking angles tested. Rocking frequency assessment by ShearFAST also correlated well when compared to the two independent validatory techniques (r [≥]0.99, p [≤]0.0001), with excellent reproducibility between ShearFAST and video analysis (mean frequency measurement difference of 0.006 {+/-} 0.005Hz) and motion capture analysis (mean frequency measurement difference of 0.008 {+/-} 0.012Hz) These data make the ShearFAST assisted cell rocker model make it an attractive approach for economical, high throughput fluid shear stress experiments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/929513v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1d8c1eborg.highwire.dtl.DTLVardef@1a346eborg.highwire.dtl.DTLVardef@8503a8org.highwire.dtl.DTLVardef@b31a3f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single-nucleus RNA sequencing enables high-resolution transcriptomic profiling of brain tissue, facilitating detailed analysis of cell identity in models of neurodegeneration and repair. Here, we describe a protocol for isolating nuclei from long-term human stem cell-derived grafts in the rat brain, incorporating vibratome sectioning, graft dissection, nuclear extraction, and fluorescence-activated sorting. This workflow supports analysis of human neurons embedded within host tissue or sensitive to dissociation, offering a powerful approach to assess graft composition, integration, and neuronal identity in living brain. For complete details on the use and execution of this protocol, please refer to Fiorenzano et al.1 Subject areasSingle nucleus RNA sequencing, stem cell biology, neuroscience, transplantation, cell replacement therapy Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/672369v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1ac3b90org.highwire.dtl.DTLVardef@7aab65org.highwire.dtl.DTLVardef@18a8fd4org.highwire.dtl.DTLVardef@1e8bfae_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIStep-by-step vibratome sectioning of xenografted rat brain tissue. C_LIO_LIPrecise dissection of human stem cell-derived grafts from host brain sections. C_LIO_LIExtraction of intact nuclei from fragile, grafted neurons. C_LIO_LIIsolation of single nuclei via fluorescence-activated nuclei sorting (FANS) for snRNA-seq sample preparation. C_LI

SUMMARYCurrent protocols for the differentiation of cardiomyocytes from human induced pluripotent stem cells (iPSCs) generally require prolonged time in culture and result in heterogeneous cellular populations. We present a method for the generation of beating cardiomyocytes expressing specific ventricular markers after just 14 days. Addition of the pan-retinoic acid receptor inverse agonist BMS 493 to human iPSCs for the first 8 days of differentiation resulted in increased protein expression of the ventricular isoform of myosin regulatory light chain (MLC2V) from 18.7% {+/-} 1.72% to 55.8% {+/-} 11.4% (p <0.0001) in cells co-expressing the cardiac muscle protein troponin T (TNNT2). Increased MLC2V expression was also accompanied by a slower beating rate (49.4 {+/-} 1.53 vs. 93.0 {+/-} 2.81 beats per minute, p <0.0001) and increased contraction amplitude (201% {+/-} 8.33% vs. 100% {+/-} 10.85%, p <0.0001) compared to untreated cells. Improved directed differentiation will improve in vitro cardiac modeling.

This Protocol Extension describes adapting an existing cryoinjury protocol to study heart regeneration in zebrafish1. We recently characterized a novel hepatic cryoinjury model that induces localized necrosis, inflammation, and fibrosis, which resolves as the liver regenerates. Here, we present a detailed description of the hepatic cryoinjury procedure. One of the key advantages of this model is that it allows direct comparison of healthy and injured liver parenchyma within individual animals. Our hepatic cryoinjury model provides a rapid and reproducible platform to investigate the molecular and cellular mechanisms underlying fibrosis and liver regeneration. This Protocol Extension aims to extend the reach of the cryoinjury approach for deployment in studying liver regeneration.

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex. Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

The embryonic zebrafish is an ideal system for lipid analyses with relevance to many areas of bioscience research, including biomarkers and therapeutics. Research in this area has been hampered by difficulties in extracting, identifying and quantifying lipids. We employed 1H-NMR at 700MHz to profile lipids in developing zebrafish embryos. The optimal method for lipidomics in embryonic zebrafish incorporated rapid lipid extraction using chloroform and an environment without oxygen depletion. Pools of 10 embryos gave the most acceptable signal-to-noise ratio, and the inclusion of chorions in the sample had no significant effect on lipid abundances. Embryos, bisected into cranial (head and yolk sac) and caudal (tail) regions, were compared by principal component analysis and analysis of variance. The lipid spectra (including lipid annotation) are available in the public repository MetaboLights (MTBLS2396).

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

BackgroundManual analysis of histopathological images is often not only time-consuming and painstaking but also prone to error from subjective evaluation criteria and human error. To address these issues, we created a fully automated workflow to enumerate jejunal crypts in a microcolony survival assay to quantify gastrointestinal damage from radiation. Methods and MaterialsAfter abdominal irradiation of mice, jejuna were obtained and prepared on histopathologic slides, and crypts were counted manually by trained individuals. The automated workflow (AW) involved obtaining images of jejunal slices from the irradiated mice, followed by cropping and normalizing the individual slice images for resolution and color; using deep learning-based semantic image segmentation to detect crypts on each slice; using a tailored algorithm to enumerate the crypts; and tabulating and saving the results. A graphical user interface (GUI) was developed to allow users to review and correct the automated results. ResultsCrypts counted manually exhibited a mean absolute percent deviation of (34 {+/-} 26)% between individuals vs the group mean across counters, which was reduced to (11 {+/-} 6)% across the 3 most-experienced counters. The AW processed a sample image dataset from 60 mice in a few hours and required only a few minutes of active user effort. AW counts deviated from experts mean counts by (10 {+/-} 8)%. The AW thereby allowed rapid, automated evaluation of the microcolony survival assay with accuracy comparable to that of trained experts and without subjective inter-observer variation. HighlightsO_LIWe fully automated the digital image analysis of a microcolony survival assay C_LIO_LIAnalyzing 540 images takes a few hours with only minutes of active user effort C_LIO_LIThe automated workflow (AW) is just as accurate as trained experts C_LIO_LIThe AW eliminates subjective inter-observer variation and human error C_LIO_LIHuman review possible with built-in graphical user interface C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/627578v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@4d178eorg.highwire.dtl.DTLVardef@1457d39org.highwire.dtl.DTLVardef@120644eorg.highwire.dtl.DTLVardef@1996992_HPS_FORMAT_FIGEXP M_FIG C_FIG