The application and development of electron microscopy for three-dimensional reconstruction in life science: a review.

Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.

[Effects of upstream laboratory processes on the digitization of histological slides]. / Auswirkungen vorgeschalteter Laborprozesse auf die Digitalisierung histologischer Schnittpräparate.

BACKGROUND: Several factors in glass slide (GS) preparation affect the quality and data volume of a digitized histological slide. In particular, reducing contamination and selecting the appropriate coverslip have the potential to significantly reduce scan time and data volume. GOALS: To objectify observations from our institute's digitization process to determine the impact of laboratory processes on the quality of digital histology slides. MATERIALS AND METHODS: Experiment 1: Scanning the GS before and after installation of a central console in the microtomy area to reduce dirt and statistical analysis of the determined parameters. Experiment 2: Re-coverslipping the GS (post diagnostics) with glass and film. Scanning the GS and statistical analysis of the collected parameters. CONCLUSION: The targeted restructuring in the laboratory process leads to a reduction of GS contamination. This causes a significant reduction in the amount of data generated and scanning time required for the digitized sections. Film as a coverslip material minimizes processing errors in contrast to glass. According to our estimation, all the above-mentioned points lead to considerable cost savings.

Combining array tomography with electron tomography provides insights into leakiness of the blood-brain barrier in mouse cortex.

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

Micro-plastination: technique for obtaining slices below 250 µm for the visualization of microanatomy in morphological and pathological morphology protocols / Micro-plastinación: técnica para la obtención de cortes inferiores a 250 µm para la visualización de la microanatomía en protocolos experimentales de morfología y patología

Plastination has revolutionized the study and research of anatomy, thanks to the biosecurity and indefinite preservation of human and animal bodies and organs. This paper presents the concept of Micro-Plastination, an ultra-thin sheet plastination technique, to obtain ultra-thin slices, of a thickness of less than 250 µm, for the identification and visualization of the microanatomy of any anatomical region in morphological and pathological experimental protocols.

La plastinación ha revolucionado el estudio y la investigación de la anatomía, gracias a la conservación biosegura y por tiempo indefinido de cadáveres y órganos humanos y animales. En este trabajo se presenta el concepto de Micro-Plastinación, técnica de plastinación de cortes ultrafinos para la obtención de cortes ultradelgados, de un grosor inferior a los 250 µm, para la identificación y visualización de la microanatomía de cualquier región anatómica en protocolos de morfología experimental.

Repair of Inaccessible Ventral Dural Defect in Thoracic Spine: Double Layered Duraplasty

We propose a double layered (intradural and epidural patch) duraplasty that utilizes Lyoplant and Duraseal. We examined a 47-year-old woman after decompression for thoracic ossification of posterior longitudinal ligament was performed in another hospital. On postoperative day 7, she complained of weakness in both legs. Postoperative magnetic resonance imaging (MRI) showed cerebrospinal fluid (CSF) collection with cord compression. In the operative field, we found 2 large dural defects on the ventral dura mater. We performed a conventional fat graft with fibrin glue. However, the patient exhibited neurologic deterioration, and a postoperative MRI again showed CSF collection. We performed dorsal midline durotomy and inserted a intradural and epidural Lyoplant patch. She immediately experienced diminishing back pain postoperatively. Her visual analog scale and motor power improved markedly. Postoperative MRIs performed at 2 and 16 months showed no spinal cord compression or CSF leakage to the epidural space. We describe a new technique for double layered duraplasty. Although we do not recommend this technique for all dural repairs, double-layered duraplasty may be useful for repairing large inaccessible dural tears in cases of persistent CSF leakage refractory to conventional management.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall / 华中科技大学学报（医学）（英德文版）

Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

Melanocortin-4 receptor expression in the cuneiform nucleus is involved in modulation of opioidergic signaling / 华中科技大学学报（医学）（英德文版）

Substantial evidence has suggested that deep brain stimulation of the cuneiform nucleus has become a remarkable treatment option for intractable pain, but the possible mechanism is poorly understood. Using a melanocortin-4 receptor (MC4R)-green fluorescent protein (GFP) reporter knockin mouse, we showed that a large number of MC4R-GFP-positive neurons were expressed in the cuneiform nucleus. Immunofluorescence revealed that approximately 40%-50% of MC4R-GFP-positive neurons expressed mu opioid receptors, indicating that they were opioidergic signaling. Our findings support the hypothesis that MC4R expression in the cuneiform nucleus is involved in the modulation of opioidergic signaling.

Establishment of localization ultrathin section for cytopathic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a localization ultrathin section method through which target cytopathic cells could be sectioned in situ.</p><p><b>METHODS</b>Lab-Tek Chamber slide system (177402) was selected as resin embedding mould. Cells infected with Human adenovirus type 5 (Ad5) or A/HN/SWL3/ 2009 (H1N1) influenza virus were embedded in situ as models. Target cytopathic cells were exposed by trimming, sectioned and observed under transmission electron microscope (TEM).</p><p><b>RESULTS</b>Target cells could be sectioned in situ and virus particles could be found easily on sections.</p><p><b>CONCLUSION</b>A localization ultrathin sectioning method was established and this technique could be applied in virus detection in cytopathic cells to improve TEM detection efficiency.</p>

Histomorfometria do corno uterino de gatas (Felis catus) submetidas à ovariosalpingohisterectomia / Histomorphometry of the uterine horn of queens (Felis catus) uterus submitted to ovaryhisterectomy

Dados histomorfométricos foram obtidos de cornos uterinos de gatas nulíparas (n=6), primíparas (n= 6), multíparalterações mais marcantes nas gatas tratadas; as variações morfológicas menos marcantes foram relativas ao diâmetro e epitélio glandulares; a presença de glândulas endometriais dilatadas foi encontrada somente nas gatas tratadas.

Histomorphometric data were obtained from cats uterine horns: either nulliparous (n = 6), primiparous (n = 6), multiparous (n = 6) and treated with contraceptive (n = 6). The material was collected after surgery, fixed in paraformaldehyde and embedded in paraplast® resin to be sliced in a microtome. The obtained sections were stained with hematoxylin -eosin and measured under a light microscope: uterine wall total thickness (μm), endometrium total height, endometrial glands diameter and glandular epithelia height, total myometrium, internal and external myometrium and vascular layer thickness. It was concluded that: 1 -contraceptive use and number of pregnancies altered the uterine structure, 2 -one pregnancy does not appear to affect the uterine lining structures as occurs in multiparous cats, 3 – there was no variation on the evaluated structures between nulliparous and primiparous cats except for inner myometrium, 4 - the total myometrium hight and the endometrium hight showed similar variations except for the contraceptive treated cats group, 5 - the outer myometrium showed marked changes in the treated cats, 6 – the less marked morphological variations were for the endometrial glands diameter and glandular epithelium hight, 7 - the presence of dilated endometrial glands was found only in treated cats.

Role of dilute alcohol in the removal of fine wrinkles from paraffin sections, a histo-technical study / Rol del alcohol diluido en la eliminación de arrugas finas desde secciones de parafina, un estudio histotécnico

Histotechnology is concerned with processing and preparing of the body tissue in such a manner as to enable a satisfactory study of it. Section cutting is an integral part of histology and histopathology. It is an art by itself requiring skill and knowledge on the part of technician or the person who needs to do section cutting. In the routine method of preparing paraffin sections, it is often encountered by the presence of artefacts like fine wrinkles or folds. Attempts have been made to remove the wrinkles by floating the sections in the warm water bath. However this method has not been able to remove all the wrinkles from the sections. We have designed a simple and reliable method, in which the paraffin sections were floated over the ethyl alcohol diluted with water (1:15) before they were placed in the water bath. Through this method, we have been able to get the wrinkle free sections of superior quality. The advantage of our method is that, it is easy to prepare the dilute alcohol and is cost effective. This method can be used by the histology and pathology technicians and the researchers.

Histotecnología se refiere a la elaboración y preparación de los tejidos del cuerpo de tal forma que permitan un estudio satisfactorio de éste. El corte seccionado es una parte integral de la histología y la histopatología. Es un arte por sí mismo que requiere habilidad y conocimiento por parte del técnico o la persona que tiene que hacer el corte de la sección. En el método de rutina de la preparación de las secciones de parafina se encuentra a menudo la presencia de artefactos como arrugas finas o pliegues. Se han hecho intentos para eliminar las arrugas por flotación de las secciones en el baño de agua tibia. Sin embargo, este método no ha sido capaz de eliminar todas las arrugas de las secciones. Hemos diseñado un método sencillo y fiable, en el que las secciones de parafina fueron colocadas por flotación sobre alcohol etílico diluido con agua (1:15) antes de ser colocado en el baño de agua. A través de este método, hemos sido capaces de obtener secciones sin arrugas de calidad superior. La ventaja de nuestro método es que, es fácil de preparar el alcohol diluido y es rentable. Este método puede ser utilizado por los técnicos de histología, patología e investigadores.

Computerized three-dimensional reconstruction of cochlear microstructures / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore an useful and practical method for three-dimensional reconstruction of cochlear microstructure.@*METHOD@#A digital image dataset of magnified forty times of human cochlear tissue can be get from the serial sections of transverse cedukol images of male cadavers temporal bone. Photoshop 6.0 and Amira 3.0 software were used to deal with the digital image dataset, such as image-connecting and image inner-location and three-dimensional reconstruction of cochlear and scala media.@*RESULT@#The result of three-dimensional reconstruction of the cedukol images represents the anatomy of human cochlear and scala media transparently and objectively.@*CONCLUSION@#The three-dimensional reconstruction technique used in the study is practical for three-dimensional of inner ear microstructure.

Avaliação in vitro, com microscopia óptica, da incidência de istmos em molares inferiores / In vitro evaluation with optical microscopy of isthmus incidence in mandibular molars

A proposição desse estudo foi verificar a incidência da fusão dos canais, tanto nas raízes mesiais quanto nas distais, em molares inferiores. Esse estudo foi aprovado pelo Comitê de Ética da Universidade de Fortaleza sob o número 181.388, de 20 de dezembro de 2012. Para a pesquisa foram selecionados 150 molares inferiores, sendo 50 primeiros molares (Grupo I), 50 segundos molares (Grupo II) e 50 terceiros molares (Grupo III). As raízes de todos os dentes foram seccionadas com disco de carborundum. Para melhor visualização da área de istmo, foi utilizada magnificação visual com microscopia óptica e limas manuais. Com a metodologia empregada, verificou-se que: no Grupo I, 76% dos canais mesiais e 53,7% dos distais possuíam comunicação; no Grupo II, 86% dos canais mesiais e 40,6% dos distais apresentaram área de istmo entre os canais radiculares; por fim, no Grupo III, 91% dos canais mesiais e 13,5% dos distais se comunicavam por istmos. Assim, por meio da metodologia empregada, foi possível verificar que a incidência de istmos conectando os canais radiculares presentes nas raízes dos molares inferiores foi expressiva nos três grupos.

Simulation and data analysis of stereological modeling based on virtual slices / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a computer-assisted stereological model for simulating the process of slice section and evaluate the relationship between section surface and estimated three-dimensional structure.</p><p><b>METHODS</b>The model was designed by mathematic method as a win32 software based on the MFC using Microsoft visual studio as IDE for simulating the infinite process of sections and analysis of the data derived from the model. The linearity of the fitting of the model was evaluated by comparison with the traditional formula.</p><p><b>RESULTS</b>The win32 software based on this algorithm allowed random sectioning of the particles distributed randomly in an ideal virtual cube. The stereological parameters showed very high throughput (>94.5% and 92%) in homogeneity and independence tests. The data of density, shape and size of the section were tested to conform to normal distribution. The output of the model and that from the image analysis system showed statistical correlation and consistency.</p><p><b>CONCLUSION</b>The algorithm we described can be used for evaluating the stereologic parameters of the structure of tissue slices.</p>

Technique of rat cochlea slicing and study of rat spiral ganglion neurons by infrared visual slice patch clamp method / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To establishing the cochlea slice technique and infrared visual slice patch clamp method in order to observe the electrophysiological characteristics of rat spiral ganglion neurons (SGN) METHODS: SD rats were divided into three groups according to postnatal days old (0-2 d, 3-6 d and 7-14 d). Making slice of SD rat cochlear quickly, using infrared differential interference contrast (IR-DIC) technique, together with slice patch clamp, the electrophysiological characteristics of rat spiral ganglion neurons were observed, and factors which affected the quality of cochlear slice and recording of patch clamp were analyzed.</p><p><b>RESULTS</b>The success rate of 3-6 days SD was the highest, and 2-4 pieces of slice could be made from each cochlea. Cochlea connecting with partial skull and integrity of cochlear hull were the key for making slice, and the angle of modiolus axis should be adjusted to be parallel to the knife and the preparing time should be shorter. The SGN cell of good condition could be easily found and the seal test became easier with the help of infrared visual slice patch clamp method. The rest membrane potential was (-45.6 +/- 5.3) mV (x +/- s, n=52) and the current of Na+ and K+ could be activated.</p><p><b>CONCLUSIONS</b>Cochlear slice technique can retain structural integrity, cell viability and their association in cochlea, which suggest that this technique provides carrier for electrophysiological study of rat spiral ganglion neurons, and patch clamp with infrared videomicroscopy method can be used to make direct real-time observation in electrophysiological experiments of SGN, which can provide important technique support and reference for deep study of electrophysiological characteristics of SGN and auditory neurotransmission in cochlea.</p>

Study of methods of decalcification for making united slices of tooth and affiliated periodontic tissues / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the methods of decalcification for making united slices of tooth and affiliated periodontic tissues.</p><p><b>METHODS</b>Twenty-one samples containing dog molars and affiliated periodontic tissues were divided into seven mean groups. The pH value of solution, time of decalcification, weight and volume of samples, and content of decalcified calcium were detected. The slices were observed by HE, specific, and immunohistochemical stain.</p><p><b>RESULTS</b>The velocity of decalcification increased with decrease of solution pH. The weight of samples lightened by 37.61%, the volume reduced by 25.97% on average, and calcium decalcified was 174.49 mg per gram humid samples. The EDTA decalcification was slowest, but it was best. Decalcification was fast in Plank-Rycho solution while the section was worst, and faster in the formyl solution containing aluminium chloride than in EDTA, and the section was better.</p><p><b>CONCLUSIONS</b>The 50% formyl solution containing aluminium chloride is an ideal decalcifying solution.</p>

Study with large slice technique and tissue microarray on lymph node involvement and micro-metastasis of rectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study lymph node involvement and micro-metastasis of rectal cancer with large slice technique and tissue microarray.</p><p><b>METHODS</b>Large slice technique, combined with tissue microarray,was used in pathologic study of 31 patients after total mesorectal excision (TME) for rectal cancer.</p><p><b>RESULTS</b>Nine hundred and ninety- two lymph nodes were harvested and 148 were positive. More than 40% of positive lymph nodes were located in the outer layer of the mesorectum and in the same side of the mesorectum as the primary tumor was. Circumferential margin involvement was observed in 12 cases and correlated with the numbers of metastatic lymph nodes (Beta =1.166, P=0.041). Micrometastasis was found in 9 cases with negative pathological lymph nodes, but not correlated with tumor differentiation and stage (P> 0.05).</p><p><b>CONCLUSION</b>Large slice technique combined with tissue microarray facilitates the detection of lymph node involvement and micrometastasis. There is a predominance of lymph node metastasis in the outer layer and the same side of the mesorectum. Micrometastasis can be discovered in different stages of rectal cancer.</p>

Crescimento do coraçäo no período embrionário humano: estudo relativo ao segundo mês gestacional / The growth of the heart in human embryonic period: study related to the 2nd gestacional month

O crescimento cardíaco foi estudado em 27 embriöes humanos estadiados no período pós-somítico (estádios 15 a 23 da Carnegie Institution of Washington). O volume cardíaco foi determinado multiplicando-se a área de imagens cardíacas (planimetria por contagem de pontos em cortes histológicos seriados) pela espessura da microtomia. O aumento do volume do coraçäo foi relacionado à idade gestacional, ao tamanho vertex-coccyx e ao peso corporal total do embriäo. Os dados, transformados em logaritmos, foram analisados em relaçöes bivariadas pelo método alométrico (log y = k log x + log b). O ajuste das regressöes foi feito por três técnicas: mínimos quadrados, eixo maior (principal), e eixo maior reduzido. As inclinaçöes das retas indicam alometria positiva do volume cardíaco em relaçäo à idade embrionária e ao tamanho vertex-coccyx e isometria em relaçäo ao peso do embriäo

Diferentes localizações dos paragânglios interatriais no coração humano adulto / Different localizations of interatrial paraganglia in the adult human heart

O estudo de cortes seriados do terço superior do septo interatrial de 20 coraçöes humanos adultos mostram três variantes de paragânglios, tomando-se por base o tecido que os envolve diretamente: a - paragânglios imersos na gordura interatrial, encontrados em 90% dos casos; b-paragânglios intraganglionares, encontrados em 25% dos casos, localizando-se excentricamente na intimidade de gânglios nervosos encapsulados do septo interatrial; c - paragânglios intramiocárdicos, observados em 10% dos coraçöes. Foi realizada reaçäo cromafim em um paragânglio intraganglionar e em dois paragânglios imersos na gordura, sendo positiva em todos. Säo discutidos os papéis funcionais destes paragânglios, particularmente sua possível influência na modulaçäo da atividade ganglionar e atuaçäo como quimio ou mecanorreceptores

Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

The effects of methylmercury (MeHg) on histochemical demonstration of the NADPH-diaphorase (NADPH-d) activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 µg MeHg and 100 g fish containing 0.03 µg MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 µg/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1) and the other animals were perfused 6 months later (group 2). After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80). Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg) in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P<0.05). These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

Registration and 3D rendering of serial tissue section images / 生物医学工程学杂志

It is an important morphological research method to reconstruct the 3D imaging from serial section tissue images. Registration of serial images is a key step to 3D reconstruction. Firstly, an introduction to the segmentation-counting registration algorithm is presented, which is based on the joint histogram. After thresholding of the two images to be registered, the criterion function is defined as counting in a specific region of the joint histogram, which greatly speeds up the alignment process. Then, the method is used to conduct the serial tissue image matching task, and lies a solid foundation for 3D rendering. Finally, preliminary surface rendering results are presented.