3D Reconstruction of the Mitochondrial Network within the Neuronal Soma from SBF-SEM Volume Data.

Neurons contain three compartments, the soma, long axon, and dendrites, which have distinct energetic and biochemical requirements. Mitochondria feature in all compartments and regulate neuronal activity and survival, including energy generation and calcium buffering alongside other roles including proapoptotic signaling and steroid synthesis. Their dynamicity allows them to undergo constant fusion and fission events in response to the changing energy and biochemical requirements. These events, termed mitochondrial dynamics, impact their morphology and a variety of three-dimensional (3D) morphologies exist within the neuronal mitochondrial network. Distortions in the morphological profile alongside mitochondrial dysfunction may begin in the neuronal soma in ageing and common neurodegenerative disorders. However, 3D morphology cannot be comprehensively examined in flat, two-dimensional (2D) images. This highlights a need to segment mitochondria within volume data to provide a representative snapshot of the processes underpinning mitochondrial dynamics and mitophagy within healthy and diseased neurons. The advent of automated high-resolution volumetric imaging methods such as Serial Block Face Scanning Electron Microscopy (SBF-SEM) as well as the range of image software packages allow this to be performed.We describe and evaluate a method for randomly sampling mitochondria and manually segmenting their whole morphologies within randomly generated regions of interest of the neuronal soma from SBF-SEM image stacks. These 3D reconstructions can then be used to generate quantitative data about mitochondrial and cellular morphologies. We further describe the use of a macro that automatically dissects the soma and localizes 3D mitochondria into the subregions created.

Infection of Alfalfa Cotyledons by an Incompatible but Not a Compatible Species of Colletotrichum Induces Formation of Paramural Bodies and Secretion of EVs.

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase to invade host epidermal cells followed by a necrotrophic phase to spread through neighboring mesophyll and epidermal cells. We used serial block face-scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 h following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microscopy (FIB-SEM) revealed the formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60 h postinoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared with compatible fungus, which in turn was more than produced by noninfected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

HeLa cell segmentation using digital image processing.

Computational cell segmentation is a vital area of research, particularly in the analysis of images of cancer cells. The use of cell lines, such as the widely utilized HeLa cell line, is crucial for studying cancer. While deep learning algorithms have been commonly employed for cell segmentation, their resource and data requirements can be impractical for many laboratories. In contrast, image processing algorithms provide a promising alternative due to their effectiveness and minimal resource demands. This article presents the development of an algorithm utilizing digital image processing to segment the nucleus and shape of HeLa cells. The research aims to segment the cell shape in the image center and accurately identify the nucleus. The study uses and processes 300 images obtained from Serial Block-Face Scanning Electron Microscopy (SBF-SEM). For cell segmentation, the morphological operation of erosion was used to separate the cells, and through distance calculation, the cell located at the center of the image was selected. Subsequently, the eroded shape was employed to restore the original cell shape. The nucleus segmentation uses parameters such as distances and sizes, along with the implementation of verification stages to ensure accurate detection. The accuracy of the algorithm is demonstrated by comparing it with another algorithm meeting the same conditions, using four segmentation similarity metrics. The evaluation results rank the proposed algorithm as the superior choice, highlighting significant outcomes. The algorithm developed represents a crucial initial step towards more accurate disease analysis. In addition, it enables the measurement of shapes and the identification of morphological alterations, damages, and changes in organelles within the cell, which can be vital for diagnostic purposes.

Modeling the 3-dimensional structure of the silkworm's spinning apparatus in silk production.

The silk-spinning process of the silkworms transforms the liquid silk solution to a solid state under mild conditions, making it an attractive model for bioinspiration However, the precise mechanism behind silk expulsion remains largely unknown. Here we selected the silkworms as representative models to investigate the silk-spinning mechanism. We used serial block-face scanning electron microscopy (SBF-SEM) to reconstruct the three-dimensional structures of the spinnerets in silkworms at various stages and with different gene backgrounds. By comparing the musculature and duct deformation of these spinneret models during the spinning process, we were able to simulate the morphological changes of the spinneret. Based on the results, we proposed three essential factors for silkworm spinning: the pressure generated by the silk gland, the opening duct, and the pulling force generated by head movement. Understanding the silkworm spinning process provides insights into clarify the fluid-ejecting mechanism of a group of animals. Moreover, these findings are helpful to the development of biomimetic spinning device that mimics the push-and-pull dual-force system in silkworms. STATEMENT OF SIGNIFICANCE: The silkworms' spinning system produces fibers under mild conditions, making it an ideal candidate for bioinspiration. However, the mechanism of silk expulsion is unknown, and the three-dimensional structure of the spinneret is still uncertain. In this study, we reconstructed a detailed 3-dimensional model of the spinneret at near-nanometer resolution, and for the first time, we observed the changes that occur before and during the silk-spinning process. Our reconstructed models suggested that silkworms have the ability to control the spinning process by opening or closing the spinning duct. During the continuously spinning period, both the pressure generated by the silk gland and the pulling force resulting from head movement work in tandem to expel the silk solution. We believe that gaining a full understanding of the spinning process steps can advance our ability to spin synthetic fibers with properties comparable to those of native fibers by mimicking the natural spinning process.

Ultrastructure and 3D reconstruction of a diplonemid protist (Diplonemea) and its novel membranous organelle.

IMPORTANCE: The knowledge of cell biology of a eukaryotic group is essential for correct interpretation of ecological and molecular data. Although diplonemid protists are one of the most species-rich lineages of marine eukaryotes, only very fragmentary information is available about the cellular architecture of this taxonomically diverse group. Here, a large serial block-face scanning electron microscopy data set complemented with light and fluorescence microscopy allowed the first detailed three-dimensional reconstruction of a diplonemid species. We describe numerous previously unknown peculiarities of the cellular architecture and cell division characteristic for diplonemid flagellates, and illustrate the obtained results with multiple three-dimensional models, comprehensible for non-specialists in protist ultrastructure.

Automated large volume sample preparation for vEM.

New developments in electron microscopy technology, improved efficiency of detectors, and artificial intelligence applications for data analysis over the past decade have increased the use of volume electron microscopy (vEM) in the life sciences field. Moreover, sample preparation methods are continuously being modified by investigators to improve final sample quality, increase electron density, combine imaging technologies, and minimize the introduction of artifacts into specimens under study. There are a variety of conventional bench protocols that a researcher can utilize, though most of these protocols require several days. In this work, we describe the utilization of an automated specimen processor, the mPrep™ ASP-2000™, to prepare samples for vEM that are compatible with focused ion beam scanning electron microscopy (FIB-SEM), serial block face scanning electron microscopy (SBF-SEM), and array tomography (AT). The protocols described here aimed for methods that are completed in a much shorter period of time while minimizing the exposure of the operator to hazardous and toxic chemicals and improving the reproducibility of the specimens' heavy metal staining, all without compromising the quality of the data acquired using backscattered electrons during SEM imaging. As a control, we have included a widely used sample bench protocol and have utilized it as a comparator for image quality analysis, both qualitatively and using image quality analysis metrics.

Mitochondria morphometry in 3D datasets obtained from mouse brains with serial block-face scanning electron microscopy.

The dysfunction of mitochondria is linked with many diseases. In the nervous system, evidence of their implication in neurodegenerative disease is growing. Mitochondria health is assessed by their impact on cellular metabolism but alterations in their morphologies and locations in the cells can also be markers of dysfunctions. Light microscopy techniques allow us to look at mitochondria in vivo in cells or tissue. But in the case of the nervous system, in order to assess the precise location of mitochondria in different cell types and neuronal compartments (cell bodies, dendrites or axons), electron microscopy is required. While the percentage of volume occupied by mitochondria can be assessed on 2D images, alterations in length, branching, and interactions with other organelles require three-dimensional (3D) segmentation of mitochondria in volumes imaged at ultrastructural level. Nowadays three-dimensional volume electron microscopy (vEM) imaging techniques such as serial block face scanning electron microscopy (SBF-SEM) enable us to image 3D volumes of tissue at ultrastructural level and can be done routinely. Segmentation of all the neuropil is also successfully achieved at a large scale in the nervous system. Here, we show a workflow based on open access resources, which allows us to image, segment, and analyze mitochondria in 3D volumes of regions of interest in the mouse brain. Taking advantage of recent developments, e.g., pre-trained models for mitochondria, we speed up the reconstruction and analysis. We also critically assess the impact on the results of the different reconstruction methods chosen and the level of manual corrections invested.

Serial block-face scanning electron microscopy of Schmidtea mediterranea.

The flatworm planarian, Schmidtea mediterranea (Smed) is a master at regenerating and rebuilding whole animals from fragments. A full understanding of Smed's regenerative capabilities requires a high-resolution characterization of organs, tissues, and the adult stem cells necessary for regeneration in their native environment. Here, we describe a serial block face scanning electron microscopy (SBF-SEM) protocol, optimized for Smed specifically, for visualizing the ultrastructure of membranes and condensed chromosomes in this model organism.

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

Resin comparison for serial block face scanning volume electron microscopy.

Serial Block Face Scanning Electron Microscopy (SBF-SEM) is one of several volume electron microscopy (vEM) techniques whose purpose is to reveal the nanostructure of cells and tissues in three dimensions. As one of the earliest, and possibly most widely adopted of the disruptive vEM techniques there have been hundreds of publications using the method, although very few comparative studies of specimen preparation parameters. While some studies have focused on staining and specimen acquisition no comparison of resin embedding has yet been conducted. To this end we have surveyed the SBF-SEM literature to determine which resins are commonly used and compared them in both cellular and fixed tissue samples in an attempt to optimize sample preparation for: effectiveness of resin infiltration, resistance to charging and beam damage and clarity of image in the resulting data set. Here we present the results and discuss the various factors that go into optimizing specimen preparation for SBF-SEM.

From the beginnings to multidimensional light and electron microscopy of virus morphogenesis.

Individual functional viral morphogenesis events are often dynamic, short, and infrequent and might be obscured by other pathways and dead-end products. Volumetric live cell imaging has become an essential tool for studying viral morphogenesis events. It allows following entire dynamic processes while providing functional evidence that the imaged process is involved in viral production. Moreover, it allows to capture many individual events and allows quantitative analysis. Finally, the correlation of volumetric live-cell data with volumetric electron microscopy (EM) can provide crucial insights into the ultrastructure and mechanisms of viral morphogenesis events. Here, we provide an overview and discussion of suitable imaging methods for volumetric correlative imaging of viral morphogenesis and frame them in a historical summary of their development.

Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

Direct association with the vascular basement membrane is a frequent feature of myelinating oligodendrocytes in the neocortex.

BACKGROUND: Oligodendrocyte lineage cells interact with the vasculature in the gray matter. Physical and functional interactions between blood vessels and oligodendrocyte precursor cells play an essential role in both the developing and adult brain. Oligodendrocyte precursor cells have been shown to migrate along the vasculature and subsequently detach from it during their differentiation to oligodendrocytes. However, the association of mature oligodendrocytes with blood vessels has been noted since the discovery of this glial cell type almost a century ago, but this interaction remains poorly explored. RESULTS: Here, we systematically investigated the extent of mature oligodendrocyte interaction with the vasculature in mouse brain. We found that ~ 17% of oligodendrocytes were in contact with blood vessels in the neocortex, the hippocampal CA1 region and the cerebellar cortex. Contacts were made mainly with capillaries and sparsely with larger arterioles or venules. By combining light and serial electron microscopy, we demonstrated that oligodendrocytes are in direct contact with the vascular basement membrane, raising the possibility of direct signaling pathways and metabolite exchange with endothelial cells. During experimental remyelination in the adult, oligodendrocytes were regenerated and associated with blood vessels in the same proportion compared to control cortex, suggesting a homeostatic regulation of the vasculature-associated oligodendrocyte population. CONCLUSIONS: Based on their frequent and close association with blood vessels, we propose that vasculature-associated oligodendrocytes should be considered as an integral part of the brain vasculature microenvironment. This particular location could underlie specific functions of vasculature-associated oligodendrocytes, while contributing to the vulnerability of mature oligodendrocytes in neurological diseases.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Correlative light and volume electron microscopy to study brain development.

Recent advances in volume electron microscopy (EM) have been driving our thorough understanding of the brain architecture. Volume EM becomes increasingly powerful when cells and their subcellular structures that are imaged in light microscopy are correlated to those in ultramicrographs obtained with EM. This correlative approach, called correlative light and volume electron microscopy (vCLEM), is used to link three-dimensional ultrastructural information with physiological data such as intracellular Ca2+ dynamics. Genetic tools to express fluorescent proteins and/or an engineered form of a soybean ascorbate peroxidase allow us to perform vCLEM using natural landmarks including blood vessels without immunohistochemical staining. This immunostaining-free vCLEM has been successfully employed in two-photon Ca2+ imaging in vivo as well as in studying complex synaptic connections in thalamic neurons that receive a variety of specialized inputs from the cerebral cortex. In this mini-review, we overview how volume EM and vCLEM have contributed to studying the developmental processes of the brain. We also discuss potential applications of genetic manipulation of target cells using clustered regularly interspaced short palindromic repeats-associated protein 9 and subsequent volume EM to the analysis of protein localization as well as to loss-of-function studies of genes regulating brain development. We give examples for the combinatorial usage of genetic tools with vCLEM that will further enhance our understanding of regulatory mechanisms underlying brain development.

Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

Diversity in new flagellum tip attachment in bloodstream form African trypanosomes.

The closely related parasites Trypanosoma brucei, T. congolense, and T. vivax cause neglected tropical diseases collectively known as African Trypanosomiasis. A characteristic feature of bloodstream form T. brucei is the flagellum that is laterally attached to the side of the cell body. During the cell cycle, the new flagellum is formed alongside the old flagellum, with the new flagellum tip embedded within a mobile transmembrane junction called the groove. The molecular composition of the groove is currently unknown, which limits the analysis of this junction and assessment of its conservation in related trypanosomatids. Here, we identified 13 proteins that localize to the flagellar groove through a small-scale tagging screen. Functional analysis of a subset of these proteins by RNAi and gene deletion revealed three proteins, FCP4/TbKin15, FCP7, and FAZ45, that are involved in new flagellum tip attachment to the groove. Despite possessing orthologues of all 13 groove proteins, T. congolense and T. vivax did not assemble a canonical groove around the new flagellum tip according to 3D electron microscopy. This diversity in new flagellum tip attachment points to the rapid evolution of membrane-cytoskeleton structures that can occur without large changes in gene complement and likely reflects the niche specialization of each species.

Whole-cell observation of ZIO-stained Golgi apparatus in rat hepatocytes with serial block-face scanning electron microscope, SBF-SEM.

The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with serial block-face scanning electron microscope (SBF-SEM), a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae, and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of zinc, iodine and osmium (ZIO) staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: (i) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; (ii) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.

Connectome of the lamina reveals the circuit for early color processing in the visual pathway of a butterfly.

Connectomics has become a standard neuroscience methodology in a few model animals,1 with the visual system being a popular target of study.2-5 Combining connectomics with circuit and behavioral physiology, recent studies on the color vision of the fruit fly Drosophila melanogaster have focused on the mechanisms underlying early wavelength processing in the optic ganglia.6-8 However, the color vision capabilities of D. melanogaster are limited,9 compared with many flower-visiting insects.10,11 For example, a butterfly Papilio xuthus has six spectral classes of photoreceptors. Each ommatidium contains nine photoreceptors in one of three fixed combinations, making the eye an array of three spectrally distinct ommatidia types.12 Behaviorally, P. xuthus can detect 1 nm differences in light wavelength across the spectrum from ultraviolet to red, outperforming humans.13 What is the neuronal basis of such precise color vision? How does such a system evolve? Addressing these questions requires comparative studies at the circuit level. Here, we performed a connectome analysis in the first optic ganglion, the lamina, of P. xuthus. The lamina comprises cartridges, each typically containing nine photoreceptor axons from a single ommatidium and four second-order neurons. We found abundant inter-photoreceptor connections, which are absent in the lamina of D. melanogaster. We also identified connections between neighboring cartridges, particularly those receiving inputs from spectrally distinct ommatidia. The linear summation of synaptic connections well explains the spectral sensitivity of photoreceptors and second-order neurons in the lamina.