Serial Optical Sections Research Articles

Perspective Article: Space-time dynamics of genome replication studied with super-resolved microscopy.

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

Automated 3D scoring of fluorescence in situ hybridization (FISH) using a confocal whole slide imaging scanner

Fluorescence in situ hybridization (FISH) is a technique to visualize specific DNA/RNA sequences within the cell nuclei and provide the presence, location and structural integrity of genes on chromosomes. A confocal Whole Slide Imaging (WSI) scanner technology has superior depth resolution compared to wide-field fluorescence imaging. Confocal WSI has the ability to perform serial optical sections with specimen imaging, which is critical for 3D tissue reconstruction for volumetric spatial analysis. The standard clinical manual scoring for FISH is labor-intensive, time-consuming and subjective. Application of multi-gene FISH analysis alongside 3D imaging, significantly increase the level of complexity required for an accurate 3D analysis. Therefore, the purpose of this study is to establish automated 3D FISH scoring for z-stack images from confocal WSI scanner. The algorithm and the application we developed, SHIMARIS PAFQ, successfully employs 3D calculations for clear individual cell nuclei segmentation, gene signals detection and distribution of break-apart probes signal patterns, including standard break-apart, and variant patterns due to truncation, and deletion, etc. The analysis was accurate and precise when compared with ground truth clinical manual counting and scoring reported in ten lymphoma and solid tumors cases. The algorithm and the application we developed, SHIMARIS PAFQ, is objective and more efficient than the conventional procedure. It enables the automated counting of more nuclei, precisely detecting additional abnormal signal variations in nuclei patterns and analyzes gigabyte multi-layer stacking imaging data of tissue samples from patients. Currently, we are developing a deep learning algorithm for automated tumor area detection to be integrated with SHIMARIS PAFQ.

Automatic three-dimensional imaging for blastomere identification in early-stage embryos based on brightfield microscopy

As the first step in manual or automated preimplantation genetic diagnosis (PGD), accurate locating of the blastomere is essential. In order to further improve success rates of the PGD process, it is necessary to obtain three-dimensional information of the blastomeres. In this study, a practical and effective pipeline that allows conventional brightfield microscopy systems to provide 3D imaging feedback for individual blastomeres inside embryos is proposed. The technique consists of two main phases, namely the blastomere identification and the 3D modeling of individual blastomeres. In the first phase, after a series of optical sections are obtained along the z-axis (z-stack), the embryo is located in the z-stack and the blastomere outline is then identified inside the embryo using a coarse-to-fine strategy; In the second phase, a probabilistic model combined with the machine learning approach is designed for the boundary estimation between individual blastomeres, and the 3D coordinate and model of each blastomere are obtained through further relocalization and reconstruction in the z-stack. Experimental results demonstrate that the proposed technique is valid and accurate compared with human perception. Specifically, the proposed technique achieves 90.32% precision rate, and the bias of blastomere position between the proposed method and human segmentation is 1.028 μm.

Quantitative Research and Characterization of the Loess Microstructure in the Bai Lu Tableland, Shaanxi Province, China

Loess is a special geotechnical material with strong structural properties, and the microstructural characteristics of loess significantly influence its macroscopic physical features, mechanical properties, and catastrophic behavior. In this paper, serial samples were extracted from the continuous loess and paleosol strata of the Bai Lu tableland; with these samples, the optical microscopy‐based serial sectioning method was adopted to study the quantitative characterization and variation in the loess microstructure. Three‐dimensional characteristics and quantitative parameters of the particles, pores, and throats of the loess were obtained. The results indicate that the volume, Eq‐Radius, and major‐minor axis ratio of the loess particles satisfy third‐order, third‐order, and second‐order Gaussian distributions, respectively. The Eq‐Radius of the loess pores and throats satisfies a first‐order Gaussian distribution, and the throat channel length satisfies a gamma distribution. With increasing stratum depth, the particles become more flattened, the throat radii become larger and the pore channels become slenderer. The variation in fitting parameters and the correlations between the macrophysical and mechanical properties of loess were then explored. The study of the microstructure of loess contributes to a better understanding of the catastrophic behavior of loess and the physical mechanism of geologic hazards in this area.

A 3D molecular atlas of the chick embryonic heart

We present a detailed analysis of gene expression in the 2-day (HH12) embryonic chick heart. RNA-seq of 13 micro-dissected regions reveals regionalised expression of 15,570 genes. Of these, 132 were studied by in situ hybridisation and a subset (38 genes) was mapped by Optical Projection Tomography or serial sectioning to build a detailed 3-dimensional atlas of expression. We display this with a novel interactive 3-D viewer and as stacks of sections, revealing the boundaries of expression domains and regions of overlap. Analysis of the expression domains also defines some sub-regions distinct from those normally recognised by anatomical criteria at this stage of development, such as a previously undescribed subdivision of the atria into two orthogonal sets of domains (dorsoventral and left-right). We also include a detailed comparison of expression in the chick with the mouse and other species.

3-D Image Reconstruction of Biological Organelles With a Robot-Aided Microscopy System for Intracellular Surgery

Intracellular surgery suffers from a low success rate due to the lack of three-dimensional (3-D) position feedback of selected organelles. In this study, we developed a novel robot-aided microscopy system and 3-D reconstruction algorithm to conduct intracellular surgery with 3-D information. A series of optical sections along the vertical direction was obtained by microscopy lens movement. A 3-D reconstruction model of the specimen was realized after several deconvolution, segmentation, and reconstruction processes. Simulations and experiments were performed to verify the accuracy of the proposed algorithm. Furthermore, 3-D reconstruction position feedback was applied to extract mitochondria via a robot-aided wide-field fluorescence system. The proposed approach facilitates intracellular surgeries, such as organelle biopsy and cell injection.

Evaluation of Dental Composite Resin Interface and Adhesion Using Laser Scanning Confocal Microscopy

The aim of the study was to evaluate and to compare the interface and marginal leakage of dental composite resins using confocal laser scanning biological microscopy (CM). Ten class II cavities were prepared on the proximal surfaces of 10 extracted teeth, previously kept in 10% formalin to preserve the bonding properties of the tooth structures. Cavities were restored with dental composite resin using the �open sandwich� technique and then assessed using CM. Open marginal adaptations at the interfaces inside the composite resins were identified. The results obtained with CM helped to eliminate and reduce the background information collected from a distance from the focal plane and allowed for the acquisition of serial optical sections from thick tooth specimens. In vitro assessments were thus made using CM on the teeth structure, regarding the occurrence of potential micro-leakages at the restoration interface.

Analysis of 3D Cellular Organization of Fixed Plant Tissues Using a User-guided Platform for Image Segmentation

The advent of non-invasive, high-resolution microscopy imaging techniques and computational pipelines for high-throughput image processing has contributed to gain insights in plant organ morphogenesis at the cellular level. Confocal scanning laser microscopy (CSLM) allows the generation of three dimensional images constituted of serial optical sections reporting on stained subcellular structures. Fluorescent labels of cell walls or cell membranes, either chemically or through reporter proteins, are particularly useful for the analyses of tissue organization and cellular shapes in 3D. Image segmentation based on cell boundary signals is used as an input to generate 3D-segments representing cells. These digitalized, 3D objects provide quantitative data on cell shape, size, geometry, position or on (intercellular) intensity signals if additional reporters are used. Herein, we report a detailed, annotated workflow for image segmentation using microscopic data. We used it in the context of a study of tissue patterning during ovule primordium development in Arabidopsis thaliana. Whole carpels are stained for cell boundaries using a modified pseudo-Schiff propidium iodide (mPS-PI) protocol, 3D images are acquired at high resolution by CSLM, segmented and annotated for individual cell types using ImarisCell. This allows for quantitative analyses of cell shape and cell number that are relevant for tissue morphodynamic studies.

Whole-brain neural network analysis (connectomics) using cell lineage-based neuron-labeling method.

The brain is a computing machine that receives input signals from sensory neurons, calculates best responses to changing environments, and sends output signals to motor muscles. How such computation is materialized remains largely unknown. Understanding the entire wiring network of neural connections in the brain, which is recently called the connectomics (connection + omics), should provide indispensable insights on this problem.To resolve the circuit diagram from the tangled thickets of neural fibers, only a small subset of neurons should be visualized at one time. Previous studies visualized such selective cells by injecting dyes or by detecting specific molecules or gene expression patterns using antibodies and expression driver strains. These approaches were unfortunately not efficient enough for identifying all the brain cells in a comprehensive and systematic manner.Neurons are generated by neural stem cells. The entire neural population can therefore be divided into a finite number of families - or clones - of the cells that are the progeny of each single stem cell. The central brain of the fruit fly Drosophila melanogaster consists of about 15,000 neurons per side and is made by utmost 100 stem cells. By genetically labeling one of such stem cells and tracing the projection patterns of its progeny in the adult brain, we were able to identify the neural projections of almost all the clonal cell groups.To visualize these neural projections, we made serial optical sections of the fly brain using laser confocal microscopy. Because of its relatively small size (0.6-mm wide and less than 0.3-mm thick), the entire fly brain can be imaged using high-resolution objectives with n.a. 1.2. Neuronal fibers are visualized by ectopically expressed cytoplasmic and membrane-bound fluorescent proteins, and the output synaptic sites are visualized with ectopically expressed tag proteins that are fused with the proteins associated with synaptic vesicles. In addition, density of all the synapses was visualized using an antibody against synaptic proteins. Signal distributions of the latter data were put into the template using 3D non-linear elastic morphing. The data of other channels are morphed with the same formula. By doing so, neural projection data obtained from different brain samples can directly be compared in the 3D space.Neurons generated by each stem cell turned out to form lineage-specific sets of projections that arborize in distinct regions of the brain, arguably serving specific aspects of brain functions. We named such clonally associated projection units the clonal units. Different parts of the brain were formed by distinct sets of overlapping clonal units. By tracing the projections made by each clonal unit, we were able to establish a comprehensive connection diagram between different brain regions. Microscopic analysis of neuronal fibers thus leads to the understanding of the entire brain neural network.

Organization of columnar inputs in the third optic ganglion of a highly visual crab

Motion information provides essential cues for a wide variety of animal behaviors such as mate, prey, or predator detection. In decapod crustaceans and pterygote insects, visual codification of object motion is associated with visual processing in the third optic neuropile, the lobula. In this neuropile, tangential neurons collect motion information from small field columnar neurons and relay it to the midbrain where behavioral responses would be finally shaped. In highly ordered structures, detailed knowledge of the neuroanatomy can give insight into their function. In spite of the relevance of the lobula in processing motion information, studies on the neuroarchitecture of this neuropile are scant. Here, by applying dextran-conjugated dyes in the second optic neuropile (the medulla) of the crab Neohelice, we mass stained the columnar neurons that convey visual information into the lobula. We found that the arborizations of these afferent columnar neurons lie at four main lobula depths. A detailed examination of serial optical sections of the lobula revealed that these input strata are composed of different number of substrata and that the strata are thicker in the centre of the neuropile. Finally, by staining the different lobula layers composed of tangential processes we combined the present characterization of lobula input strata with the previous characterization of the neuroarchitecture of the crab’s lobula based on reduced-silver preparations. We found that the third lobula input stratum overlaps with the dendrites of lobula giant tangential neurons. This suggests that columnar neurons projecting from the medulla can directly provide visual input to the crab’s lobula giant neurons.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Cochleovestibular nerve development is integrated with migratory neural crest cells

The cochleovestibular (CV) nerve, which connects the inner ear to the brain, is the nerve that enables the senses of hearing and balance. The aim of this study was to document the morphological development of the mouse CV nerve with respect to the two embryonic cells types that produce it, specifically, the otic vesicle-derived progenitors that give rise to neurons, and the neural crest cell (NCC) progenitors that give rise to glia. Otic tissues of mouse embryos carrying NCC lineage reporter transgenes were whole mount immunostained to identify neurons and NCC. Serial optical sections were collected by confocal microscopy and were compiled to render the three dimensional (3D) structure of the developing CV nerve. Spatial organization of the NCC and developing neurons suggest that neuronal and glial populations of the CV nerve develop in tandem from early stages of nerve formation. NCC form a sheath surrounding the CV ganglia and central axons. NCC are also closely associated with neurites projecting peripherally during formation of the vestibular and cochlear nerves. Physical ablation of NCC in chick embryos demonstrates that survival or regeneration of even a few individual NCC from ectopic positions in the hindbrain results in central projection of axons precisely following ectopic pathways made by regenerating NCC.

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Abstract 248: Stearoyl-CoA desaturase 1 is a key factor for lung cancer stem cell survival and expansion.

Abstract In recent years studies of cancer development and recurrence have been significantly influenced by the Cancer Stem Cell (CSC) hypothesis. According to this, cancer is sustained by highly-positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after treatment with debulking agents. Growth of cancer cells as non-adherent spheroids in non differentiating conditions is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from Malignant Pleural Effusions (MPE) of patients affected by adenocarcinoma of the lung are able to form spheroids in non-adherent conditions supplemented with selected growth factors, that these spheroids upregulate markers associated with stemness, can be serially propagated in vitro through several passages and, finally, give rise efficiently to tumors, which reproduce the same histopathological features of the original human tumor when implanted in immunodeficient mice. By expression profiling we also identified a set of genes whose expression is significantly upregulated in lung tumor spheroids vs adherent cultures. One of the most strongly upregulated gene was Stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study we show both by RNA interference or through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in primary tumor cultures. SCD1 inhibition causes a dramatic impairment of survival of cells which express ALDH and other markers of stemness in vitro. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem like properties. Furthermore, SCD1-inhibited lung cancer spheroids were strongly impaired in their in vivo tumorigenicity. These results suggest that SCD1 is a critical target in lung cancer tumor initiating cells. Citation Format: Gennaro Ciliberto, Alessia Noto, Claudia De Vitis, Roberta Barzellotti, Giuseppe Roscilli, Luigi Aurisicchio, Salvatore Raffa, Alberto Ricci, Salvatore Mariotta, Maria Rosaria Torrisi, Maria Rosaria Giovagnoli, Rita Mancini. Stearoyl-CoA desaturase 1 is a key factor for lung cancer stem cell survival and expansion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 248. doi:10.1158/1538-7445.AM2013-248

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo-4, Propidium iodide, and di-8-Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin-eosin, Masson's tri-chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure.

Statistical organelle dissection of Arabidopsis guard cells using image database LIPS

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

2D and 3D mapping of microindentations in hydrated and dehydrated cortical bones using confocal laser scanning microscopy

We report on responses of hydrated and dehydrated cortical bone tissues to mechanical loading applied by a Vickers indenter. The Vickers indentations were imaged in two- and three-dimensions (2D and 3D) using confocal laser scanning microscopy (CLSM) to understand mechanical behavior of bone tissues. Serial optical sections of indentation patterns of dry and wet bones were collected using CLSM. The indention surface structures were mapped using topographical CLSM imaging. The observation of CLSM shows the fundamental indentation responses for both the hydrated and dehydrated bone tissues were plastic deformation. No visible fracture was observed in the Vickers indentation patterns in the wet bone tissue, while non-propagating lamellar microcracks occurred in the dry bone tissue. This indicates that drying resulted in increased brittleness of the bone tissue. The Vickers hardness values of dry bone tissue were significantly higher than those of wet bone tissue at any applied loads (analysis of variation, ANOVA, p < 0.05). The resolution limits of confocal microscopy were also discussed for bone tissue scanning.

3-D STUDY OF VESSELS IN PERIPHERAL PLACENTAL VILLI

The microvascular bed of the human term placenta is located in the mature intermediate and terminal villi. Despite repeated studies, a commonly accepted model of its spatial organization is not yet available. In the present study, the 3-D arrangement of vessels in mature intermediate and terminal placental villi was examined using the combination of confocal microscopy and image processing. Fixed and paraffin embedded placental samples were cut into 120 μm thick sections. Serial optical sections were captured by a confocal microscope and used as an initial data for 3-D reconstruction. The reconstructed segments of the villous tree show parallel vessels of mature intermediate villi giving rise to the capillaries of terminal villi. The signs of longitudinal and sprouting capillary growth as well as growth of new villi were observed. The presented method allowed us to visualize large parts of peripheral placental villi, which is useful for elucidating the arrangement of their vascular bed.

Diagnostic morphometric applicability of confocal laser scanning microscopy in Osteoarchaeology

AbstractThe confocal laser scanning microscope (CLSM) has become an essential tool for a wide range of biological and clinical studies and imaging applications. The major imaging modes of the CLSM include single and serial optical sections from thick specimens (100 µm), multiple wavelength images and three‐dimensional reconstruction. Such images provide measurement of length, surface or volume of objects. Surprisingly, this advanced microscopic method has been very rarely used in anthropological research, although numerous clinical and basic science research projects showed its value to study, bone growth, bone micro‐architecture and 3D bone morphometry. We present the basic principles and advantages of CLSM and outline practical aspects of specimen preparation, image collection and digital image processing specifically appropriate for historical bone and hair material. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Three-dimensional reconstruction of living mouse liver tissues using cryotechniques with confocal laser scanning microscopy

Soluble proteins and glycogen particles are well preserved in paraffin-embedded sections prepared by in vivo cryotechnique (IVCT) and cryobiopsy followed by freeze substitution fixation. We performed confocal laser scanning microscopic analyses on the distributions of glycogen with periodic acid-Schiff (PAS) staining and serum proteins with immunostaining for mouse liver tissues. Livers of fully fed mice showed a strong fluorescence signal of PAS staining in all hepatocytes and immunofluorescence of immunoglobulin kappa light chain (Igκ) in blood vessels and bile canaliculi. However, some hepatocytes in mechanically damaged livers were PAS-negative and Igκ-immunopositive, showing extraction of glycogen particles and infiltration of serum proteins in hepatocytes. By three-dimensional (3D) reconstruction of serial optical sections, interconnecting hepatic sinusoids and bile canaliculi were detected with Igκ immunostaining between trabecular hepatocytes that were PAS stained. In PAS-stained samples under fasting conditions, interstitial structures along sinusoids were clarified in vivo by 3D reconstruction because of the lower PAS staining intensity of hepatocytes. In addition, 100-μm-thick eosin-stained slices provided 3D structural images more than 30 μm in thickness away from tissue surfaces, showing blood vessels with flowing erythrocytes and networks of bile ducts and canaliculi. IVCT and cryobiopsy with histochemical analyses enabled us to visualize native hepatocytic glycogen and 3D structures, such as vascular networks, reflecting their living states by confocal laser scanning microscopy.