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bioRxiv2024

CellSexID: Sex-Based Computational Tracking of Cellular Origins in Chimeric Models

Cell tracking in chimeric models is essential yet challenging, particularly in developmental biology, regenerative medicine, and transplantation research. Existing methods such as fluorescent labeling and genetic barcoding are technically demanding, costly, and often impractical for dynamic or heterogeneous tissues. Here, we introduce CellSexID, a computational framework that leverages sex as a surrogate marker for cell origin inference. Using a machine learning model trained on single-cell transcriptomic data, CellSexID accurately predicts the sex of individual cells, enabling in silico distinction between donor and recipient cells in sex-mismatched settings. The model identifies minimal sex-linked gene sets through ensemble feature selection and has been validated using both public datasets and experimental flow sorting, confirming the biological relevance of predicted populations. We further demonstrate CellSexIDs applicability beyond chimeric models, including organ transplantation and multiplexed sample demultiplexing. As a scalable and cost-effective alternative to physical labeling, CellSexID facilitates precise cell tracking and supports diverse biomedical applications involving mixed cellular origins.

bioinformatics↗

bioRxiv2024

Mechanical stress orients stomata division to form tissue scale alignments.

The last stomatal division aligns with the leafs main axis in many species [1]. Understanding how cellular events such as these are coordinated across organ scales remains a challenge in developmental biology. In Arabidopsis, polarised proteins guide the asymmetric divisions in the early stomatal lineage. These proteins show organ scale alignment and may be sensitive to mechanical stress [2]. In contrast, what determines the orientation and alignment of the critical final division is unknown [3]. Here we use an artificial system where every cell adopts the fate of a stomata pore [4] making it easy to visualise their alignment. Combining this system with simultaneous time-lapse imaging on both sides of the cotyledon we are able to compare the stomatal orientation relative to the organ axis, the cell major axis, and the principal directions of growth. Using finite element modelling on a realistic template enabled us to identify differential growth-derived stress patterns as a factor coordinating stomata division at the organ scale. Mechanical perturbation confirmed the influence of tensile stress on stomata division orientation. Through this study, we have identified a mechanism that can explain this nearly century-old observation.

plant biology↗

bioRxiv2024

Effects of transmembrane phenylalanine residues on γ-secretase-mediated Notch-1 proteolysis

{gamma}-Secretase is a presenilin-containing intramembrane aspartyl protease complex that cleaves within the transmembrane domain (TMD) of nearly 150 substrates, with the amyloid precursor protein (APP) being the most well studied. APP cleavage by {gamma}-secretase generates amyloid {beta}-peptides (A{beta}) that pathologically deposit in Alzheimers disease. APP TMD substrate undergoes initial endoproteolysis ({varepsilon}-cleavage) followed by processive carboxypeptidase trimming of long A{beta} intermediates in [~]tripeptide intervals. Although {gamma}-secretase cleavage of Notch1 is essential in developmental biology and altered in many cancers, the processing of this cell-surface receptor is relatively understudied. Only one sequence specificity rule is known for {gamma}-secretase substrate processing: Aromatic residues such as phenylalanine are not tolerated in the P2 position with respect to any processing event on the APP TMD. Here we show using biochemical and mass spectrometry (MS) techniques that this specificity rule holds for Notch1 as well. Analysis of products from the reactions of purified enzyme complex and Notch1 TMD substrate variants revealed that P2 Phe relative to {varepsilon}-site cleavage reduced proteolysis and shifted initial cleavage N-terminally by one residue. Double Phe mutation near the {varepsilon} site resulted in reduced proteolysis with shifting to two major initial cleavage sites, one N-terminally and one C-terminally, both of which avoid Phe in the P2 position. Additionally, three natural Phe residues were mutated to corresponding residues in the APP TMD, which led to increased {varepsilon} proteolysis. Thus, Phe residues can affect the enzyme reaction rate as well as cleavage site specificity in the Notch1 TMD.

biochemistry↗

bioRxiv2024

Cell-cell communication controls the timing of gastruloid symmetry-breaking

How cell fate decisions coordinate with tissue-scale morphogenesis remains a major challenge in developmental biology. Gastruloids, three-dimensional aggregates of pluripotent stem cells that self-organise and break symmetry via polarised Brachyury/T expression, provide an ideal system to address this question. By generating gastruloids with defined initial proportions of T-expressing cells we show that fate decisions occur collectively, with cell-fate proportions influencing the transition rates. Mechanical measurements reveal differences in surface tension between T-positive and T-negative tissues, consistent with radial cell sorting. Finally, incorporating fate dynamics and mechanics into a computational model recapitulates the sequential symmetry-breaking events observed in vitro. Our findings identify a mechanochemical mechanism underlying axis formation, and demonstrate how multicellular systems can robustly self-organise without external signalling cues.

biophysics↗

bioRxiv2024

Spatial Transcriptomics Reveals Expression Gradients in Developing Wheat Inflorescences at Cellular Resolution

The diversity of plant inflorescence architectures is specified by gene expression patterns. In wheat (Triticum aestivum), the lanceolate-shaped inflorescence (spike) is defined by rudimentary spikelets at the base which initiate first but subsequently lag in development compared with central spikelets. While previous studies identified gene expression differences between central and basal inflorescence sections, the spatio-temporal dynamics and gradients along the apical-basal axis remain poorly resolved due to bulk tissue-level techniques. Here, using spatial transcriptomics, we profiled 200 genes across four stages of wheat inflorescence development to cellular resolution. Cell segmentation and unsupervised clustering identified 18 expression domains and their enriched genes, revealing dynamic spatio-temporal organisation along the apical-basal axis of the inflorescence. Along this axis, we uncovered distinct and spatially coordinated gene expression gradients patterning meristems prior to the visible delay in basal spikelet development. This study demonstrates the potential for spatial transcriptomics time-series to advance plant developmental biology.

plant biology↗

bioRxiv2024

An epigenetic clock for Xenopus tropicalis reveals age-associated sites enriched in H4K20me3-marked heterochromatin

DNA methylation clocks have been widely used for accurate age prediction, but most studies have been carried out on mammals. Here we present a clock for the aquatic frog Xenopus tropicalis, a widely used model organism in developmental biology and genomics. The clock, trained on targeted bisulfite sequencing data, achieved a cross-validation median absolute error of 0.70 years. To construct the clock, we collected DNA methylation data from 192 frogs at genomic regions containing CpG sites previously shown to have age-associated methylation in Xenopus. We found that CpG sites with methylation levels significantly increasing or decreasing with age are enriched in heterochromatic regions marked with H4K20me3 and H3K9me3. CpG sites with positively age-associated methylation levels are enriched in bivalent chromatin and gene bodies with H3K36me3, and tend to be proximal to lowly expressed genes. These epigenetic features of aging are similar to those found in mammals, suggesting evolutionary conservation of epigenetic aging mechanisms. Our clock enables future aging biology experiments that leverage the unique properties of amphibians.

genomics↗

bioRxiv2025

Epigenome-wide analysis reveals novel DNA methylation signatures significantly associated with the infant pupillary light reflex, a candidate intermediate phenotype for autism.

Autism is a highly heterogeneous neurodevelopmental condition, currently diagnosed based on behavioural characteristics. Candidate early intermediate phenotypes, such as the Pupillary Light Reflex (PLR), a reflexive constriction of the pupil in response to increased optical luminance, may provide insights into etiological factors and potential biomarkers, such as DNA methylation (DNAm), involved in the emergence of autism. We conducted epigenome-wide DNAm association analyses of 9-, 14-, 24-month PLR onset latency and constriction amplitude in a sample of 51 infants enriched for autism family history, using buccal DNA collected at 9-months. Our epigenome-wide analysis (EWAS) identified four stringently significant differentially methylated probes (p < 2.4 x 10-7) associated with cross-section PLR latency measurements at 14- and 24-months, and with 14-to 24-month PLR latency developmental change. Differentially methylated probes associated with PLR amplitude were identified, but at a less stringent discovery threshold (p < 5 x 10-5). Our region analyses identified several significant differentially methylated regions associated with both PLR latency and amplitude Downstream exploratory pathway analysis identified enrichment for multiple developmental biological processes, as well as several susceptibility genes to autism and related neurodevelopmental conditions including NR4A2, HNRNPU and NAV2. Our findings provide novel insight into the role of DNAm in PLR development and illuminate biological mechanisms underpinning altered PLR in infancy in emerging autism.

neuroscience↗

bioRxiv2025

Multiple neural network approaches, including use of topological data analysis, enhances classification of human induced pluripotent stem cell colonies by treatment condition

Understanding how stem cells organize to form early tissue layers remains an important open question in developmental biology. Helpful in understanding this process are biomarkers or features that signal when a significant transition or decision occurs. We show such features from the spatial layout of the cells in a colony are sufficient to train neural networks to classify stem cell colonies according to differentiation protocol treatments each colony has received. We use topological data analysis to derive input information about the cells positions to a four-layer feedforward neural network. We find that despite the simplicity of this approach, such a network has performance similar to the traditional image classifier ResNet. We also find that network performance may reveal the time window during which differentiation occurs across multiple conditions. Author summaryOur understanding of how stem cells determine what specialized cells to differentiate into is still incomplete. One aspect of understanding this process involves identifying when key decisions about a cells fate occur. We explored whether by looking at the layout of the cells of the colony, we can infer knowledge regarding the eventual phenotypes the cells are differentiating towards. We train an algorithm to recognize cell type using spatial information by taking as input the number and size of holes that appear among the colonys cells. We find this method succeeds in its classification, similar to an industry-grade image classifier.

systems biology↗

bioRxiv2025

Selection-free CRISPR-Cas9 editing protocol for distant Dictyostelid species

Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Most research focusses on Dictyostelium discoideum, which - due to its ease of culturing and genetic tractability - has been adopted as a model species in the fields of developmental biology, cell biology and microbiology. Over decades, genome editing methods in D. discoideum have steadily improved but remain relatively time-consuming and limited in scope, effective in a few species only. Here, we introduce a CRISPR-Cas9 editing protocol that is cloning-free, selection-free, highly-efficient, and effective across Dictyostelid species. After optimizing our protocol in D. discoideum, we obtained knock-out efficiencies of [~]80% and knock-in efficiencies of [~]30% without antibiotic selection. Efficiencies depend on template concentrations, insertion sizes, homology arms and target sites. Since our protocol is selection-free, we can isolate mutants as soon as one day post-transfection, vastly expediting the generation of knock-outs, fusion proteins and expression reporters. Our protocol also makes it possible to generate several knock-in mutations simultaneously in the same cells. Boosted by cell-sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing protocol to phylogenetically distant Dictyostelid species, which diverged hundreds of millions of years ago and have never been genome edited before. Our protocol therefore opens the door to performing broad-scale genetic interrogations across Dicyostelids.

genetics↗

bioRxiv2025

scContrast: A contrastive learning based approach for encoding single-cell gene expression data

Single-cell RNA sequencing (scRNA-seq) captures gene expression at a individual cell resolution, which reveals critical insights into cellular diversity, disease processes, and developmental biology. However, a key challenge in scRNA-seq analysis is clustering similar cells across multiple batches, particularly when distinct sequencing protocols are used. In this work, we present scContrast, a semi-supervised contrastive learning method tailored for embedding scRNA-seq data from both plate- and droplet-based protocols into a universal representation space. By leveraging five simple augmentations, scContrast extracts biologically relevant signals from gene expression data while filtering out batch effects and technical artifacts. We trained scContrast on a subset of Tabula Muris tissues and evaluated its zero-shot performance on unseen tissues. Our results demonstrate that scContrast generalizes effectively to new tissues and outperforms the leading UCE approach in integrating scRNA-seq data from droplet- and plate-based sequencing protocols.

genomics↗

bioRxiv2025

Exploring non-invasive sexing of early chick embryos in intact eggs using Laser Speckle Contrast Imaging (LSCI) and Deep Neural Network (DNN)

The ability to image blood flow in early-stage avian embryos has significant applications in developmental biology, drug and vaccine testing, as well as determining sex differentiation. In this project, we used our recently developed laser speckle contrast imaging (LSCI) system to non-invasively image extraembryonic blood vessels and used these images to attempt early sex identification of chick embryos. Specifically, we captured images of blood vessels from 1,251 living chicken embryos between day three and day four of incubation. We then applied deep neural network (DNN) models to evaluate whether it is possible to differentiate sex based on vascular patterns. Using ResNetBiT and YOLOv5 models, our results indicate that sex differentiation from extraembryonic blood vessel images was not achievable with sufficiently high accuracy or statistical significance for practical use. Specifically, ResNetBiT had a five-fold cross-validated average accuracy of 59%{+/-}5% (fold-wise p-value, p [≤] 0.3) at day 3 and 61%{+/-}3% (fold-wise, p [≤] 0.04) at day 4. YOLOv5 had a five-fold cross-validated average accuracy of 55%{+/-}3% (fold-wise, p [≤] 0.3) at day 3 and 53%{+/-}3% (fold-wise, p [≤] 0.5) at day 4. Our findings suggest that using vascular pattern imaging alone is inconclusive for reliable early sex identification in chicken embryos.

bioengineering↗

bioRxiv2025

Single-Cell RNA Sequencing Defines Developmental Progression and Reproductive Transitions of Pneumocystis carinii

Pneumocystis species are host-obligate fungal pathogens that cause severe pneumonia in immunocompromised individuals. Despite their clinical importance, their life cycle remains poorly understood, in part because Pneumocystis depends on the host environment for most nutrients and requires sexual reproduction for survival, which occurs exclusively in vivo. This study presents the first single-cell RNA sequencing (scRNA-seq) atlas of Pneumocystis carinii, generated from isolated organisms recovered from the bronchoalveolar lavage fluid of infected rats to map the life cycle of P. carinii. Transcriptomes from 87,716 cells were analyzed using the 10x Genomics platform, revealing 13 transcriptionally distinct clusters representing key developmental stages, including biosynthetically active trophic forms, mating-competent intermediates, and asci undergoing sporulation. These states were characterized by expression of MAPK signaling components, {beta}-glucan-modifying enzymes, and spore-associated genes, respectively. The scRNA-seq data supports previous evidence that these host-obligate fungi undergo sexual reproduction and provide new insights into the gene expression patterns associated with different life cycle phases. Biomarkers associated with ascus formation identified by scRNA-seq were validated by RT-qPCR, showing decreased expression levels in ascus-depleted populations treated with anidulafungin, a drug that halts ascus formation. More broadly, this approach provides a strategy for studying the full life cycles of fungal pathogens that cannot be continuously cultured. ImportancePneumocystis species (spp.) are clinically significant fungal pathogens that cannot be sustainably cultured in vitro due to their host obligate nature. This longstanding limitation has impeded progress in understanding their life cycle and identifying therapeutic vulnerabilities. Here, we apply scRNA-seq to P. carinii isolated directly from infected rat lungs, generating the first transcriptional map of its developmental progression. Our results define discrete gene expression states associated with trophic growth, mating activation, and ascus formation, and provide transcriptional evidence for a structured life cycle clarifying key developmental transitions and identifying potential regulatory targets for therapeutic intervention. Importantly, this study demonstrates that scRNA-seq can resolve the developmental biology of host-restricted fungal pathogens that cannot be cultured in vitro. This approach offers a generalizable framework for investigating other unculturable or obligate microbial pathogens directly within their native host environments, where traditional experimental tools are limited.

microbiology↗

bioRxiv2025

Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

Cerebral organoids (COs) are multicellular, self-organized, in vitro, 3D brain-like tissues used for developmental biology, disease modelling and drug screening. However, their lack of vascularity renders them less physiologically accurate. Vascularization of COs remains challenging due to the different requirements between COs and vascular cells, limited vascular network penetration within the organoid, and the absence of luminal perfusion. Here, we devised an encapsulation approach in which human brain microvascular endothelial cells (HBMVECs) were delivered to developing COs from progressively degrading extracellular matrix (ECM)-based hydrogel droplets. By tuning this hydrogel concentration and media composition, we observed enhanced vascular-like network formation that expanded within the organoid tissue. Using pathway inhibitors, we showed that a subset of the endothelial cells (ECs) originated from the CO itself, promoting network integration. Endothelial networks displayed blood-brain barrier (BBB) features, including astrocytic end-footlike interactions, pericyte wrapping, and collagen-laminin basal lamina. Vascularized COs exhibited greater media internalization and up to three-fold lower apoptosis than non-vascularized COs. This comprehensive 3D neurovascular model is a promising platform for cerebrovascular research and drug testing applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/650161v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@175edb7org.highwire.dtl.DTLVardef@1499b91org.highwire.dtl.DTLVardef@184567borg.highwire.dtl.DTLVardef@149c8e4_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIAddition and angiogenic stimulation of human brain microvascular endothelial cells (HBMVECs) co-cultured with cerebral organoids (COs) generate multicellular vascular-like networks C_LIO_LIVascularization induces changes in organoid morphology but not on tissue stiffness C_LIO_LIEndothelial networks morphological features are correlated with the concentration of the supporting matrix C_LIO_LIA number of the endothelial cells (ECs) in the networks originate from the organoid itself C_LIO_LIEndothelial networks integrate within the organoid tissue interacting with astrocytes and pericyte-like cells, and are surrounded by basement membrane-like depositions C_LIO_LIVascularized COs exhibit higher media diffusion, and a reduced necrotic core compared to non-vascularized COs C_LI

bioengineering↗

bioRxiv2025

The zoo of the gene networks capable of pattern formation by extracellular signaling

A fundamental question of developmental biology is pattern formation, or how cells with specific gene expression end up in specific locations in the body to form tissues, organs and, overall, functional anatomy. Pattern formation involves communication through extracellular signals and complex intracellular gene networks integrating these signals to determine cell responses (e.g., further signaling, cell division, cell differentiation, etc.). In this article we address two question: 1) Are there any logical or mathematical principles determining which gene network topologies can lead to pattern formation by cell signaling over space in multicellular systems? 2) Can gene network topologies be classified into a small number of classes that entail similar dynamics and pattern transformation capacities? We combine logical arguments and mathematical proofs to show that, despite the large amount of formally possible gene network topologies, all gene network topologies capable of pattern formation fall into only three fundamental classes and their combinations. Additionally, we show that gene networks within each class share the same logic on how they lead to pattern formation and hence, lead to similar patterns. We characterize the main features of each class. This zoo includes the complete gene networks that, to the best of our knowledge, have been experimentally reported to lead to pattern formation as well as other gene networks that have not yet been found experimentally.

systems biology↗

bioRxiv2025

A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

The derivation of authentic embryonic stem cells (ESCs) from diverse mammalian species offers valuable opportunities for advancing regenerative medicine, studying developmental biology, and enabling species conservation. Here, we report the development of a robust, serum-free culture system, termed 6iL/E4 that enables the derivation and long-term self-renewal of ESCs from multiple mammalian species, including mouse, rat, bovine, rabbit, and human. Using systematic signaling pathway analysis, we identified key regulators--including GSK3, STAT3, PDGFR, BRAF, and LATS--critical for ESC maintenance across species. Additionally, inducible expression of KLF2 and NANOG enhances the naive pluripotency and chimeric potential of bovine ESCs. The E4 medium also supports stable ESC growth while minimizing lineage bias. These findings reveal conserved principles underlying ESC self-renewal across divergent mammalian species and provide a universal platform for cross-species stem cell research, disease modeling, and biotechnology applications. In BriefWang et al. developed 6iL/E4, a serum-free system sustaining ESCs from mouse, rat, bovine, rabbit, and human. These findings reveal conserved fundamental mechanisms governing ESC self-renewal across diverse mammalian species. HighlightsO_LIDeveloped 6iL/E4 system for ESC derivation across five mammalian species. C_LIO_LIPDGFR signaling inhibition as critical for ESC derivation across species. C_LIO_LIE4 medium improves ESC maintenance and avoids neural bias of traditional N2B27. C_LIO_LIInducible KLF2/NANOG enhances naive pluripotency and chimera formation in bovine. C_LI

cell biology↗

bioRxiv2025

Transient chromatin decompaction by histone deacetylation inhibition preferentially radiosensitizes cancerous breast epithelial cells at lower radiation doses

Radiation therapy plays a prominent role in breast cancer treatment, but the high doses of radiation damage both healthy and cancerous cells. Therefore, additional research is needed into combination therapies that could preferentially radiosensitize cancer cells compared to surrounding healthy tissue without causing deleterious side effects. Histone deacetylase inhibitor drugs (HDACis) have been tested as radiosensitizers in both basic research and clinical trials, but the long exposure time typically used in these treatments and the lack of matched healthy cell controls often leave aspects of their mechanism of action unclear. Here, we show that transient (2 hour) trichostatin A (TSA) treatment of cancerous and non-tumorigenic breast epithelial cell lines increases immediate DNA damage and decreases long term cell viability in both cell types at high radiation doses. Transient TSA treatment also causes an increase in DNA damage signals after 5 Gy in other cancer and healthy cell types: A375 melanoma cells and BJ5-ta fibroblasts. This suggests that chromatin decompaction acts to increase cellular vulnerability to initial DNA damage from high doses of radiation DNA damage in a cell type independent manner that does not rely on changes to DNA repair pathways caused by longer TSA treatment. However, responses to lower doses of radiation and long term survival are more cell type specific: only MCF7 cells experience an effect of TSA on DNA damage after 1 Gy radiation while MCF10a cells experience somewhat more evident cell viability effects of combined TSA and radiation treatment long term. Scope statementThis manuscript covers several topics that are core to the mission of Frontiers in Cell and Developmental Biology, including cancer cell biology as compared to non-cancerous cell function, cell death vs. survival, and epigenetics and chromosome structure. While some of the implications of this work touch on cancer therapeutics, the study itself focuses on the basics of the interplay between genome architecture and DNA damage and cellular survival / response. We noted several recent articles on related topics in this journal, including the effects of combination treatments on cancer cells (doi 10.3389/fcell.2025.1636288), radiotherapy effects on cells (doi 10.3389/fcell.2025.1568634), and DNA damage (doi 10.3389/fcell.2025.1575483). Our work is well suited for a Brief Research Report as it provides key but focused data on comparisons of radiosensitivity in response to transient chromatin decompaction in cancer vs. healthy cells.

cancer biology↗

bioRxiv2025

Title: KymoTip: High-throughput Characterization of Tip-growth Dynamics in Plant Cells

Live imaging data analysis often requires an objective, local, and accurate way of quantification of cell dynamics. In the research field of polarized tip-growth, the cell fluctuations and/or fluctuations in tip position and growth direction hampers automated analyses of huge amounts of imaging sequences. The fluctuated nature in data makes it unclear how cell shape and growth are linked to intracellular events that could be the actual driving force of cell growth. To overcome these difficulties, we developed a powerful and user-friendly tool called KymoTip with an available format. In this software, novel functions such as coordinate normalization, tip-bottom detection, and signal kymograph were implemented. We confirmed that not only plasma membrane-labeled fluorescent images, but also images such as bright-field and cortical microtubule markers --so long as the cell contours can be identified-- are amenable to KymoTip. Furthermore, by combining markers for cell contours with those that visualize intracellular structures, it becomes possible to quantitatively analyze various intracellular events, such as nuclear migration and calcium wave, in conjunction with cellular growth dynamics. Since KymoTip can be handled by non-specialist, it is expected to promote understanding of what happens at the sub- and cellular level with high throughput outcomes. Significance statementFaced with fluctuations in cell coordinates and cell tip positions, position correction of live imaging data and accurate detection of tip position are key challenges in plant developmental biology. We solved them with a powerful and user-friendly tool, KymoTip, that can realize cell position correction, cell tip detection with cell centerline, and quantification of intracellular events.

bioinformatics↗

bioRxiv2025

Low Angle Ring Illumination Stereomicroscopy (LARIS): A high-resolution imaging method of compound eyes in Drosophila and other insects.

The compound eyes of Drosophila are widely used to gain valuable insights into genetics, developmental biology, cell biology, disease biology, and gene regulation. Various parameters, such as eye size, pigmentation loss, formation of necrotic patches, and disorientation, fusion or disruption of ommatidial arrays are commonly assessed to evaluate eye development and degeneration. In this study, we developed an improved optical alignment imaging technique named "Low Angle Ring Illumination Stereomicroscopy" (LARIS), which provides high-contrast images of the Drosophila compound eye. By capturing images of the same eye using different optical alignments of the stereomicroscope, we achieved the highest resolution with minimal reflection through the LARIS method. The images captured using LARIS clearly show ommatidial fusion, disorientation, and pigmentation loss in the Drosophila eye compared to those obtained with conventional imaging method. We believe that LARIS will open new avenues for improved imaging of the compound eyes of Drosophila and other insects.

cell biology↗