Confocal 3D Research Articles

Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments

BackgroundThe thymus, responsible for T cell-mediated adaptive immune system, has a structural and functional complexity that is not yet fully understood. Until now, thymic anatomy has been studied using histological thin sections or confocal microscopy 3D reconstruction, necessarily for limited volumes.MethodsWe used Phase Contrast X-Ray Computed Tomography to address the lack of whole-organ volumetric information on the microarchitecture of its structural components. We scanned 15 human thymi (9 foetal and 6 postnatal) with synchrotron radiation, and repeated scans using a conventional laboratory x-ray system. We used histology, immunofluorescence and flow cytometry to validate the x-ray findings.ResultsApplication to human thymi at pre- and post-natal stages allowed reliable tracking and quantification of the evolution of parameters such as size and distribution of Hassall’s Bodies and medulla-to-cortex ratio, whose changes reflect adaptation of thymic activity. We show that Hassall’s bodies can occupy 25% of the medulla volume, indicating they should be considered a third thymic compartment with possible implications on their role. Moreover, we demonstrate compatible results can be obtained with standard laboratory-based x-ray equipment, making this research tool accessible to a wider community.ConclusionsOur study allows overcoming the resolution and/or volumetric limitations of existing approaches for the study of thymic disfunction in congenital and acquired disorders affecting the adaptive immune system.

Comparison of facial skin ageing in healthy Asian and Caucasian females quantified by in vivo line-field confocal optical coherence tomography 3D imaging.

Quantitative biomarkers of facial skin aging were investigated in 109 healthy Asian female volunteers, aged 20 to 70 years. In vivo 3D Line-field Confocal Optical Coherence Tomography (LC-OCT) imaging, enhanced by Artificial Intelligence (AI)-based quantification algorithms, was utilized to compute various metrics, including stratum corneum thickness (SC), viable epidermal (VE) thickness, and Dermal-Epidermal Junction (DEJ) undulation along with cellular metrics for the temple, cheekbone, and mandible. Comparison with data from a cohort of healthy Caucasian volunteers revealed similarities in the variations of stratum corneum and viable epidermis layers, as well as cellular shape and size with age in both ethnic groups. However, specific findings emerged, such as larger, more heterogeneous nuclei in both layers, demonstrated by an increase in nuclei volume and their standard deviation, and increased network atypia, all showing significant age-related variations. Caucasian females exhibited a flatter and more homogeneous epidermis, evidenced by a decreased standard deviation of the number of layers, and a less dense cellular network with fewer cells per layer, indicated by a decrease in cell surface density. Ethnicity-wise comparisons highlighted distinct biological features specific to each population. Asian individuals showed significantly higher DEJ undulation, higher compactness, and lower cell network atypia compared to their Caucasian counterparts across age groups. Differences in stratum corneum and viable epidermal thickness on the cheekbone were also significant. LC-OCT 3D imaging provides valuable insights into the aging process in different populations and underscores inherent biological differences between Caucasian and Asian female volunteers.

Internalisation of immunoglobulin light chains by cardiomyocytes in AL amyloidosis: what can biopsies tell us?

Background Cardiac involvement in systemic light chain amyloidosis (AL) leads to chronic heart failure and is a major prognosis factor. Severe cellular defects are provoked in cardiac cells by tissue-deposited amyloid fibrils of misfolded free immunoglobulin light chains (LCs) and their prefibrillar oligomeric precursors. Objective Understanding the molecular mechanisms behind cardiac cell cytotoxicity is necessary to progress in therapy and to improve patient management. One key question is how extracellularly deposited molecules exert their toxic action inside cardiac cells. Here we searched for direct evidence of amyloid LC uptake by cardiomyocytes in patient biopsies. Methods We immunolocalized LCs in cardiac biopsies from four AL cardiac amyloidosis patients and analysed histopathological images by high resolution confocal microscopy and 3D image reconstruction. Results We show, for the first time directly in patient tissue, the presence of LCs inside cardiomyocytes, and report their proximity to nuclei and to caveolin-3-rich areas. Our observations point to macropinocytosis as a probable mechanism of LC uptake. Conclusions Internalisation of LCs occurs in patient cardiomyocytes. This event could have important consequences for the pathogenesis of the cardiac disease by enabling interactions between amyloid molecules and cellular organelles inducing specific signalling pathways, and might bring new insight regarding treatment.

N2FXm, a method for joint nuclear and cytoplasmic volume measurements, unravels the osmo-mechanical regulation of nuclear volume in mammalian cells

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.

Enhancing the textural and rheological properties of fermentation-induced pea protein emulsion gels with transglutaminase.

The aim of this study was to assess how transglutaminase (TG) impacts the microstructure, texture, and rheological properties of fermentation-induced pea protein emulsion gels. Additionally, the study examined the influence of storage time on the functional properties of these gels. Fermentation-induced pea protein gels were produced in the presence or absence of TG and stored for 1, 4, 8, 12, and 16 weeks. Texture analysis, rheological measurements, moisture content and microstructure evaluation with confocal laser scanning microscopy (CLSM) and 3D image analysis were conducted to explore the effects of TG on the structural and rheological properties of the fermented samples. The porosity of the protein networks in the pea gels decreased in the presence of TG, the storage modulus increased and the textural characteristics were significantly improved, resulting in harder and more springy gels. The gel porosity increased in gels with and without TG after storage but the effect of storage on textural and rheological properties was limited, indicating limited structural rearrangement once the fermentation-induced pea protein emulsion gels are formed. Greater coalescence was observed for oil droplets within the gel matrix after 16 weeks of storage in the absence of TG, consistent with these protein structures being weaker than the more structurally stable TG-treated gels. This study shows that TG treatment is a powerful tool to enhance the textural and rheological properties of fermentation-induced pea protein emulsion gels.

SSL Depth: self-supervised learning enables 16× speedup in confocal microscopy-based 3D surface imaging [Invited

SSL Depth: self-supervised learning enables 16× speedup in confocal microscopy-based 3D surface imaging [Invited

Comparison of surface roughness parameters Ra/Sa and Rz/Sz with different measuring devices

ObjectivesThe aim of this in-vitro study was to investigate the influence of the measuring devices (mechanical and optical) on the surface values Ra/Sa and Rz/Sz of different materials and machined surfaces. Material and methodsMechanical (contact profilometry (CP): Perthometer S6P, Perthen Mahr, G) and optical (scanning electron microscope (SEM): Phenom, FEI, NL; confocal 3D laser scanning microscope (CLSM): VK-100, Keyence, J) measuring devices were used to determine the surface roughness Ra/Sa and Rz/Sz. Glass-ceramic (Empress, Ivoclar-Vivadent, FL), zirconia (Cercon HT, Dentsply, D), composite (Grandio, Voco, D), denture base material (Palapress, Kulzer, D) and titanium (grade 4) were investigated (n = 10 measurements) after surface finishing: sandblasting (Al2O3; 250 μm), diamond treatment (80 μm; wet), sandblasting (Al2O3; 50 μm), and polishing (grit of 4000; wet; Tegramin-25, Struers, D). Statistics: Shapiro-Wilk, ANOVA, Bonferroni post-hoc tests (α = 0.05). ResultsSurface characteristics Ra/Sa and Rz/Sz, which were detected with mechanical and optical measuring devices on different materials and surface finishing, showed significant (p ≤ 0.001, ANOVA) differences. Significant (p ≤ 0.045, Bonferroni) differences between CP, SEM and CLSM (line; area) were found, mainly for Ra/Sa. DiscussionThe surface roughness on different dental materials and differently machined surfaces were influenced by the individual mechanical and optical measuring devices. Optical methods measure a significantly higher roughness value for all materials and surface finishes.

Live Lymphoma and CLL Cells inside the Bone Marrow Fibroblasts: Implication in Residual Disease Persistence

Bruton tyrosine kinase inhibitors (BTKi) have generated remarkable clinical responses in patients with chronic lymphocytic leukemia (CLL) and other mature B-cell malignancies. However, deep, and durable response to BTK inhibition is uncommon. A significant number of patients with measurable residual disease relapse with resistance to therapies. Although mutational mechanisms responsible for resistance have been well described, it is not known what mechanism led to persistence of the minimal residual disease (MRD) in the first place. The tumor microenvironment (TME) contributes to drug resistance. Various roles for stroma participation have been described, however, none of these include the physical intracellular sheltering of tumor cells. Cell-in-cell (CIC) describes the microscopic observations of the embodiment of an intact cell by another whole cell ( Fig. 1A). Pathologists have reported this phenomenon sporadically in human fixed tissues for over a century. However, its biological, pathological, and clinical significance remains obscure. In the absence of systematic studies, understanding the significance of live CIC remains challenging. In this investigation, we studied primary human CLL samples using an ex vivo co-culture model system which mimics the CLL TME. In the TME system, CLL cells were cocultured with a primary bone marrow fibroblast line (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. Using the TME system, we have demonstrated that modeled drug response correlates well with patient clinical response to BTK inhibitor ibrutinib and BCL2 inhibitor venetoclax. Interestingly, with confocal microscopy and 3D reconstruction, we observed that CLL cells placed in the model system were actively internalized by BMF in all 41 CLL cases studied. CLL cells were live and mobile. Moreover, one CLL cell was captured halfway in ( Fig. 1B). The number of internalized CLL cells was quantified and correlated with patients' clinical and pathological features. No correlations were found between CIC and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that patients who were on treatment for CLL at the time of sample collection had higher levels of CIC than those not treated. We hypothesized that live CIC may be an important mechanism contributing to residual disease persistence and drug resistance. To test the hypothesis, we exposed CLL cells in the TME model system to covalent and noncovalent BTKi including ibrutinib, zanubrutinib, and pirtobrutinib at clinically achievable concentrations. Our results showed that drug exposure drove CLL cells into BMF. This happened specifically to the BTK inhibitors as BCL2 inhibitor venetoclax was not observed to cause such an increase in CIC. These laboratory findings are consistent with the clinical observations that the rate of MRD negativity is much higher with venetoclax treatment than with BTKi. Additionally, CIC was observed in both follicular lymphoma (FL) cell line and primary FL cells suggesting the phenomenon is not limited to CLL. Further, we found that CIC was stimulated by several B-cell growth factors. The chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, conditioned medium containing CXCL12 ligand promoted cell internalization, while plerixafor, a CXCR4 antagonist, largely prevented CIC from occurring. Furthermore, plerixafor blocks ibrutinib-driven CIC in cell lines as well as in primary CLL cells. Our study represents the first report implicating the CIC phenomenon in residual disease persistence using a cohort of patient-derived CLL cells assessed with 3D imaging and quantitative approach. These findings suggest that live CIC may be an important mechanism tumor cells use to avoid harmful drug insults, survive, persist, and later proliferate, eventually leading to clinical relapse. Targeting the CXCL12-CXCR4 axis may become a potential therapeutic strategy to minimize residual disease and future relapse. Fig.1. A. Images are reconstructed in 3D using Imaris v10.0. BMF cells are visualized as transparent red surfaces and CLL cells as green surfaces. B. One CLL cell was captured halfway in.

Miniaturization does not change conserved spider anatomy, a case study on spider Rayforstia (Araneae: Anapidae)

Miniaturization is an evolutionary trend observed in many animals. Some arachnid groups, such as spiders and mites, demonstrate a strong tendency toward miniaturization. Some of the most miniaturized spiders belong to the family Anapidae. In this study, using light and confocal microscopy and 3D modelling, we provide the first detailed description of the anatomy of a spider of the genus Rayforstia, which is only 900 µm long. In comparison with larger spiders, Rayforstia has no branching of the midgut in the prosoma and an increased relative brain volume. In contrast to many miniature insects and mites, the spider shows no reduction of whole organ systems, no allometry of the digestive and reproductive systems, and also no reduction of the set of muscles. Thus, miniature spider shows a more conserved anatomy than insects of a similar size. These findings expand our knowledge of miniaturization in terrestrial arthropods.

Impact of mucus modulation by N-acetylcysteine on nanoparticle toxicity

Human respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.

Design of Water-Soluble Rotaxane-Capped Superparamagnetic, Ultrasmall Fe3O4 Nanoparticles for Targeted NIR Fluorescence Imaging in Combination with Magnetic Resonance Imaging.

Integrating an NIR fluorescent probe with a magnetic resonance imaging (MRI) agent to harvest complementary imaging information is challenging. Here, we have designed water-soluble, biocompatible, noncytotoxic, bright-NIR-emitting, sugar-functionalized, mechanically interlocked molecules (MIMs)-capped superparamagnetic ultrasmall Fe3O4 NPs for targeted multimodal imaging. Dual-functional stoppers containing an unsymmetrical NIR squaraine dye interlocked within a macrocycle to construct multifunctional MIMs are developed with enhanced NIR fluorescence efficiency and durability. One of the stoppers of the axle is composed of a lipophilic cationic TPP+ functionality to target mitochondria, and the other stopper comprises a dopamine-containing catechol group to anchor at the surface of the synthesized Fe3O4 NPs. Fe3O4 NPs surface-coated with targeted NIR rotaxanes help to deliver ultrasmall magnetic NPs specifically inside the mitochondria. Two carbohydrate moieties are conjugated with the macrocycle of the rotaxane via click chemistry to improve the water solubility of MitoSQRot-(Carb-OH)2-DOPA-Fe3O4 NPs. Water-soluble, rotaxane-capped Fe3O4 NPs are used for live-cell mitochondria-targeted NIR fluorescence confocal imaging, 3D and multicolor imaging in combination with T2-weighted MRI on a 9.4 T MR scanner with a high relaxation rate (r2) of 180.7 mM-1 s-1. Biocompatible, noncytotoxic, ultrabright NIR rotaxane-capped superparamagnetic ultrasmall monodisperse Fe3O4 NPs could be a promising agent for targeted multimodal imaging applications.

Abstract P1027: Confocal And Mass Spectrometry-based Investigation Of REEP5 Depletion By AAV9 In The Mouse Heart

The sarco(endo)plasmic reticulum (SR/ER) is an essential regulator of many key cellular processes, especially those that play a role in the development and progression of cardiac disease. However, many aspects of its structural organization remain poorly defined. Receptor Expression Enhancing Protein 5 (REEP5) is a cardiac enriched SR/ER membrane protein, which regulates organization of the highly differentiated SR/ER network and responses to stress. In zebrafish models, genetic knock-out of reep5 results in cardiac functional defects and reduced heart rate. Within the cardiomyocyte, depletion of Reep5 in vitro results in decreased muscle cell contraction, disrupted Ca 2+ signaling and SR/ER luminal vacuolization. For these studies, in vivo cardiac knock-down of Reep5 in the mouse was achieved using recombinant adeno-associated virus serotype 9 (rAAV9)-mediated gene delivery. Cardiac tissues or isolated cardiomyocytes were harvested at 7 days through to 4 weeks following knock-down, for biochemical and functional assessments. We observed that the largest significant change in REEP5 expression occurred 4 weeks post-rAAV9 injection, correlating to a 78% knock-down, observed by immunoblotting (unpaired t-test, p<0.0001, n=6-8). 5 weeks following knock-down, mice developed lethal cardiac dysfunction. To assess the biochemical changes induced by Reep5 knock-down, we have established an organelle-specific cardiac proteomic profile of the microsome, mitochondria and cytosol, using subcellular fractionation and mass spectrometry (nLC-ESI-MS-HCD-MS). Coupled with high resolution confocal microscopy and 3D mapping techniques, we have examined localization and expression patterns of key SR/ER and mitochondrial proteins, that have altered expression following knock-down of Reep5 at the myocyte-level. These findings provide a detailed understanding of the role that REEP5 plays in maintaining ER homeostasis, SR/ER structure, and general organelle integrity. By identifying the mechanistic significance of REEP5 expression in the heart, we can work to delineate underappreciated pathways in cardiac muscle development.

Three-dimensional human germinal centers of different sizes in patients diagnosed with lymphadenitis show comparative constant relative volumes of B cells, T cells, follicular dendritic cells, and macrophages

Germinal centers (GCs) are some of the most important structures in the human immune system. As such, their cell types and functions have been thoroughly investigated. B cells, T cells, follicular dendritic cells (FDCs), and macrophages have widely been found to typically be aggregated in GCs. However, the amount of space occupied by each of these cell types has yet to be investigated. In this study, we conducted confocal laser-based 3D cell-volume quantification of typical GC cells under reactive conditions in lymphadenitis and investigated how volume proportions change during GC development. For this investigation, we used anti-CD3 (T cells), anti-CD20 and anti-Pax5 (B cells), anti-CD23 (FDCs), anti-CD68 (macrophages), and DAPI (nuclear staining). We detected average proportions of about 11% CD3, 9% CD20, 6% CD23, and 2% CD68 in the largest possible regions of interest within GCs. Interestingly, these values remained steady relatively independent of GC size. The remarkably low B cell proportion can be attributed to technical constraints given the use of the CD20 antibody in 3D. Applying the B cell marker Pax5, we found that about 44% of the volume was occupied by B cells after extrapolating the volume of B cell nuclei to that of whole B cells. We concluded that Pax5 is more suitable than anti-CD20 for 3D B cell quantification in GCs. The substantial unstained volume in GCs raises the question of whether other cell types fill these open spaces. Our 3D investigation enabled a unique morphological and volumetric evaluation of GC cells that balance their overall volumes in GCs.

Shelter in place: Live CLL cells inside the bone marrow fibroblasts and its implication in drug resistance

Introduction: Cell-in-cell (CIC) describes a century old observation where an intact cell can be seen inside another whole cell. It has been associated with human cancers, especially hematopoietic malignancies. However, most observations have been published as case reports. In the absence of systematic studies of case cohorts, understanding of the pathological significance of live CIC remains challenging. Methods: In this investigation, we studied primary CLL samples using an ex vivo co-culture model system which mimics the CLL tumor microenvironment (TME). In these TME models, CLL cells were cocultured with bone marrow fibroblasts (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4 and upregulate Ki67 expression. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. In this study, cells were observed and analyzed with confocal microscopy and 3D reconstruction. Results: We observed that CLL cells were actively internalized by BMF in 41 CLL cases studied. The internalized CLL cells were alive and mobile within the BMF’s cytoplasm. Moreover, the tumor cells were seen leaving their BMF host and re-entering into other BMFs. The number of internalized CLL cells were quantified and correlated with patients’ clinical and pathological parameters. No correlations were found between cell internalization and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that treated patients had higher level of CIC than those not treated. We hypothesized that live CIC may contribute to drug resistance and internalization may be an important mechanism for tumor cells to evade harmful drug insult and survive as residual disease. To test the hypothesis, we exposed CLL cells in the TME models to BTK inhibitors including ibrutinib and pirtobrutinib. Our results showed that the drug exposure drove CLL cells into BMF. Additionally, we observed that increased CIC was associated with higher cellular proliferation capacity. Importantly, chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, CXCL12 ligand promoted cell internalization, while a CXCR4 antagonist partially prevented CIC from occurring. The research was funded by: NCI Keywords: Chronic Lymphocytic Leukemia (CLL), Microenvironment, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

A high-precision multi-dimensional microspectroscopic technique for morphological and properties analysis of cancer cell

Raman and Brillouin scattering are sensitive approaches to detect chemical composition and mechanical elasticity pathology of cells in cancer development and their medical treatment researches. The application is, however, suffering from the lack of ability to synchronously acquire the scattering signals following three-dimensional (3D) cell morphology with reasonable spatial resolution and signal-to-noise ratio. Herein, we propose a divided-aperture laser differential confocal 3D Geometry-Raman-Brillouin microscopic detection technology, by which reflection, Raman, and Brillouin scattering signals are simultaneously in situ collected in real time with an axial focusing accuracy up to 1 nm, in the height range of 200 μm. The divided aperture improves the anti-noise capability of the system, and the noise influence depth of Raman detection reduces by 35.4%, and the Brillouin extinction ratio increases by 22 dB. A high-precision multichannel microspectroscopic system containing these functions is developed, which is utilized to study gastric cancer tissue. As a result, a 25% reduction of collagen concentration, 42% increase of DNA substances, 17% and 9% decrease in viscosity and elasticity are finely resolved from the 3D mappings. These findings indicate that our system can be a powerful tool to study cancer development new therapies at the sub-cell level.

A high temporal-spatial resolution three-dimensional imaging method using arrayed-focus structured illumination with differential axial enhancement

High precision and fast three-dimensional (3D) microscopic imaging technology plays an extremely important role in MEMS, micro optics, microelectronics, biomedicine and other fields. In this paper, a confocal microscopic 3D imaging method based on differential array focusing structured light illumination (DAF-SIM) is proposed. Theoretical analysis and experimental results show that under the 100x/NA 0.95 objective lens, the lateral resolution of this method reaches 173 nm, exceeding the optical diffraction limit, the axial sensitivity is better than 10 nm, and the time resolution is about 3 Hz@2.3 M Pixel, achieving the goal of high horizontal and axial resolution and time resolution. The high spatial and temporal resolution of DAF-SIM 3D micro imaging method provides a highly applicable, efficient and high-precision online micro terrain detection scheme for intelligent manufacturing and other fields.

3D atlas of cerebral neuropils with previously unknown demarcations in the honey bee brain.

Honey bees (Apis mellifera) express remarkable social interactions and cognitive capabilities that have been studied extensively. In many cases, behavioral studies were accompanied by neurophysiological and neuroanatomical investigations. While most studies have focused on primary sensory neuropils, such as the optic lobes or antennal lobes, and major integration centers, such as the mushroom bodies or the central complex, many regions of the cerebrum (the central brain without the optic lobes) of the honey bee are only poorly explored so far, both anatomically and physiologically. To promote studies of these brain regions, we used anti-synapsin immunolabeling and neuronal tract tracings followed by confocal imaging and 3D reconstructions to demarcate all neuropils in the honey bee cerebrum and close this gap at the anatomical level. We demarcated 35 neuropils and 25 fiber tracts in the honey bee cerebrum, most of which have counterparts in the fly (Drosophila melanogaster) and other insect species that have been investigated so far at this level of detail. We discuss the role of cerebral neuropils in multisensory integration in the insect brain, emphasize the importance of this brain atlas for comparative studies, and highlight specific architectural features of the honey bee cerebrum.

Evidence of invivo exogen protein uptake by red blood cells: aputative therapeutic concept.

For some molecular players in red blood cells (RBCs), the functional indications and molecular evidence are discrepant. One such protein is transient receptor potential channel of canonical subfamily, member 6 (TRPC6). Transcriptome analysis of reticulocytes revealed the presence of TRPC6 in mouse RBCs and its absence in human RBCs. We transfused TRPC6 knockout RBCs into wild-type mice and performed functional tests. We observed the "rescue" of TRPC6 within 10 days; however, the "rescue" was slower in splenectomized mice. The latter finding led us to mimic the mechanical challenge with the cantilever of an atomic force microscope and simultaneously carry out imaging by confocal (3D) microscopy. We observed the strong interaction of RBCs with the opposed surface at around 200 pN and the formation of tethers. The results of both the transfusion experiments and the atomic force spectroscopy suggest mechanically stimulated protein transfer to RBCs as a protein source in the absence of the translational machinery. This protein transfer mechanism has the potential to be utilized in therapeutic contexts, especially for hereditary diseases involving RBCs, such as hereditary xerocytosis or Gárdos channelopathy.

Study of Microbial Sulfur Metabolism in a Near Real-Time Pathway through Confocal Raman Quantitative 3D Imaging.

As microbial sulfur metabolism significantly contributes to the formation and cycling of deep-sea sulfur, studying their sulfur metabolism is important for understanding the deep-sea sulfur cycle. However, conventional methods are limited in near real-time studies of bacterial metabolism. Recently, Raman spectroscopy has been widely used in studies on biological metabolism due to its low-cost, rapid, label-free, and nondestructive features, providing us with new approaches to solve the above limitation. Here, we used the confocal Raman quantitative 3D imaging method to nondestructively detect the growth and metabolism of Erythrobacter flavus 21-3 in the long term and near real time, which possessed a pathway mediating the formation of elemental sulfur in the deep sea, but the dynamic process was unknown. In this study, its dynamic sulfur metabolism was visualized and quantitatively assessed in near real time using 3D imaging and related calculations. Based on 3D imaging, the growth and metabolism of microbial colonies growing under both hyperoxic and hypoxic conditions were quantified by volume calculation and ratio analysis. Additionally, unprecedented details of growth and metabolism were uncovered by this method. Due to this successful application, this method is potentially significant for analyzing the in situ biological processes of microorganisms in the future. IMPORTANCE Microorganisms contribute significantly to the formation of deep-sea elemental sulfur, so studies on their growth and dynamic sulfur metabolism are important to understand the deep-sea sulfur cycle. However, near real-time in situ nondestructive metabolic studies of microorganisms remain a great challenge due to the limitations of existing methods. We thus used an imaging-related workflow by confocal Raman microscopy. More detailed descriptions of the sulfur metabolism of E. flavus 21-3 were disclosed, which perfectly complemented previous research results. Therefore, this method is potentially significant for analyzing the in-situ biological processes of microorganisms in the future. To our knowledge, this is the first label-free and nondestructive in situ technique that can provide temporally persistent 3D visualization and quantitative information about bacteria.

Confocal Imaging and 3D Reconstruction to Determine How Genetic Perturbations Impact Presynaptic Morphology at the Mouse Calyx of Held.

Neurons communicate via synapses-specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit. Key features Confocal imaging and 3D reconstruction of presynaptic terminals. Used with a virus-mediated expression of mEGFP to achieve efficient, cell-type specific labeling of the presynaptic compartment. Protocol was developed with the calyx of Held but is suitable for pre- and postsynaptic compartments of various neurons across multiple mammalian and invertebrate species.