支气管扩张症的X线平片、CT和HRCT对照分析

目的:对支气管扩张的X线平片、CT和HRCT表现做一对比分析,以提高诊疗水平。方法:取病理证实支扩20例,分别摄有胸正侧位片,常规CT扫描和HRCT扫描。HRCT选薄层,大矩阵和高分辨率算法。结果:显示X光平片漏诊率高(8/20),特异性低(3/20)。CT较平片显示肺“盲区”效果好,有粘液嵌塞者,常规CT像上有时不易与血管性病变鉴别,HRCT可显示部分含气的支气管腔存在。结论:HRCT扫描诊断支扩特异性高,可与支气管造影相媲美,它不仅显示支扩范围、程度和部位。还能显示小叶中央性改变。常规CT较平片显示率高。     

Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography

We provide a catalog of 3D cryo soft X-ray tomography (cryo-SXT) images obtained from ～6 to 12μm thick mouse adenocarcinoma cells. Included are multiple representative images of nuclei, nucleoli, nuclear membrane, nuclear membrane channels, mitochondria, lysosomes, endoplasmic reticulum, filaments and plasma membrane, plus three structures not previously described by cryo-SXT, namely Golgi, microvilli and nuclear-membrane blebs. Sections from the 3D cryo-SXT tomograms for all the preceding structures closely resemble those seen by thin-section transmission electron microscopy (TEM). Some structures such as nuclear-membrane channels and nuclear-membrane blebs are more easily detected by cryo-SXT than TEM most likely due to their better contrast and cellular preservation in cryo-SXT combined with the ability to rapidly locate these structures within a full 3D image. We identify and discuss two current limitations in cryo-SXT: variability in image quality and difficulties in detecting weaker contrast structures such as chromatin and various nuclear bodies. Progress on these points is likely to come from the solution of several technical problems in image acquisition, plus the implementation of advanced cryo soft X-ray microscopy approaches such as phase contrast or optical sectioning.     

Correction of distortion of histologic sections of arteries

Histologic sections of arteries can be used to generate three-dimensional (3D) geometric models and identify structural constituents. However, geometric distortions are introduced by fixation, embedding and sectioning; distortions which can, for example, lead to errors in stresses predicted by finite element models. We developed a method to measure and correct for distortions caused by acrylic processing and applied it to intact, healthy porcine coronary arteries. Micro-computed tomography was used to image arteries in the fresh and embedded states. Tissue blocks were sectioned, stained and imaged using a light microscope. Each section contained four registration marks used to determine strains introduced by sectioning and staining. Using these three image sets, 3D geometric models were generated and distortions were measured. Fixation, processing, and embedding resulted in shrinkage of 6.4+/-2.3% axially and 35.4+/-5.0% in mean cross-sectional area (n=5). Shrinkage in a cross section was well characterized by a uniform, equibiaxial strain. Sectioning and staining resulted in additional compressive strains in the sectioning direction of 0.067+/-0.011 and, in the direction perpendicular to sectioning, of 0.023+/-0.005 (n=5). These strains are assumed uniform and form the basis for correcting section geometry. Reconstructions using corrections for sectioning and shrinkage-related distortions had errors of 1.6+/-0.5% (n=5) and 4.0+/-1.7% (n=5), respectively.     

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.     

High-resolution CT scoring system-based grading scale predicts the clinical outcomes in patients with idiopathic pulmonary fibrosis

Background

The 2011 idiopathic pulmonary fibrosis (IPF) guidelines are based on the diagnosis of IPF using only high-resolution computed tomography (HRCT). However, few studies have thus far reviewed the usefulness of the HRCT scoring system based on the grading scale provided in the guidelines. We retrospectively studied 98 patients with respect to assess the prognostic value of changes in HRCT findings using a new HRCT scoring system based on the grading scale published in the guidelines.

Methods

Consecutive patients with IPF who were diagnosed using HRCT alone between January 2008 and January 2012 were evaluated. HRCT examinations and pulmonary function tests were performed at six-month intervals for the first year after diagnosis. The HRCT findings were evaluated using the new HRCT scoring system (HRCT fibrosis score) over time. The findings and survival rates were analyzed using a Kaplan-Meier analysis.

Results

The HRCT fibrosis scores at six and 12 months after diagnosis were significantly increased compared to those observed at the initial diagnosis (p < 0.001). The patients with an elevated HRCT fibrosis score at six months based on a receiver operating characteristic (ROC) curves analysis had a poor prognosis (log-rank, hazard ratio [HR] 2.435, 95% CI 1.196-4.962; p = 0.0142). Furthermore, among the patients without marked changes in %FVC, those with an elevated score above the cut-off value had a poor prognosis (HR 2.192, 95% CI 1.003-4.791; p = 0.0491).

Conclusions

Our data demonstrate that the HRCT scoring system based on the grading scale is useful for predicting the clinical outcomes of IPF and identifying patients with an adverse prognosis when used in combination with spirometry.     

Micro-computed tomography: an alternative method for shark ageing

Micro-computed tomography (microCT) produced 3D reconstructions of shark Carcharhinus brevipinna vertebrae that could be virtually sectioned along any desired plane, and upon which growth bands were readily visible. When compared to manual sectioning, it proved to be a valid and repeatable means of ageing and offers several distinct advantages over other ageing methods.     

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.     

Development and validation of a generic 3D model of the distal femur

The development and validation of a virtual generic 3D model of the distal femur using computer graphical methods is presented. The synthesis of the generic model requires the following steps: acquisition of bony 3D morphology using standard computed tomography (CT) imaging; alignment of 3D models reconstructed from CT images with a common coordinate system; computer graphical sectioning of the models; extraction of bone contours from the image sections; combining and averaging of extracted contours; and 3D reconstruction of the averaged contours. The generic models reconstructed from the averaged contours of six cadaver femora were validated by comparing their surface geometry on a point to point basis with that of the CT reconstructed reference models. The mean errors ranged from 0.99 to 2.5 mm and were in agreement with the qualitative assessment of the models.     

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.Download video file.^{(204M, mov)}     

Development and validation of a generic 3D model of the distal femur

The development and validation of a virtual generic 3D model of the distal femur using computer graphical methods is presented. The synthesis of the generic model requires the following steps: acquisition of bony 3D morphology using standard computed tomography (CT) imaging; alignment of 3D models reconstructed from CT images with a common coordinate system; computer graphical sectioning of the models; extraction of bone contours from the image sections; combining and averaging of extracted contours; and 3D reconstruction of the averaged contours.

The generic models reconstructed from the averaged contours of six cadaver femora were validated by comparing their surface geometry on a point to point basis with that of the CT reconstructed reference models. The mean errors ranged from 0.99 to 2.5 mm and were in agreement with the qualitative assessment of the models.     

Karyology of a Marine Non-Motile Dinoflagellate, Pyrocystis lunula

Dinoflagellates have a unique and interesting intracellular architecture such as permanently condensed chromosomes throughout the cell cycle. However the study of dinoflagellate chromosomes is not amendable because of the unusually higher number of chromosomes and problems in sample preparation. The species of Pyrocystis spend most of their life cycle as vegetative cyst forms and have been used as experimental organisms for bioluminescence and circadian rhythms. Here, we documented the content of DNA in different life stages and the chromosome karyology in a marine non-motile dinoflagellate Pyrocystis lunula, through light and fluorescent microscopy, serial ultra-thin sectioning, and three dimension (3D) modeling. The DNA content doubles during DNA synthesis and in the end of the cell division two separate daughter cells have the approximately same fluorescent values for the mother cells. Using serial ultra-thin sectioning and 3D modeling, we report the first ultrastructural karyogram. The cells chosen were at the end of karyokinesis. A total of 98 chromosomes were counted and assigned to 49 pairs. In this species, DNA synthesis appears to occur before, or during asexual division and P. lunula lives a diplontic life cycle.     

Stereology meets electron tomography: towards quantitative 3D electron microscopy

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

中耳胆脂瘤的影像诊断:高分辨率计算机断层成像与磁共振成像的比较研究

目的:评估和比较高分辨率计算机断层成像(high resolution computer tomography,HRCT)与磁共振成像(magnetic resonance imaging,MRI)对中耳胆脂瘤的诊断价值。方法:回顾性分析临床初步诊断为中耳胆脂瘤患者55人(63耳)在术前5天内进行的16排HRCT和1.5T MRI诊断结果,以病理诊断为基础对HRCT和MRI的诊断结果进行对比研究,统计分析HRCT和MRI的灵敏度、特异度、阳性预期值、阴性预期值,以及Kappa检验两者之间的内在观察一致性。结果:63耳中,病理确诊为中耳胆脂瘤40耳。HRCT正确诊断30例,假阳性11例,假阴性10例,灵敏度、特异度、阳性预期值以及阴性预期值分别为75%、52.17%、73.17%、54.55%。MRI正确诊断37例,假阳性和假阴性各为3例,灵敏度、特异度、阳性预期值以及阴性预期值分别为92.5%、86.96%、75%和86.96%。HRCT与MRI诊断结果之间的内在观察一致性Kappa=0.274(P0.05)。剔除既往有手术史的10耳,只针对无手术史的53耳,HRCT与MRI诊断结果之间的内在观察一致性增高,吻合度一般,Kappa=0.462(P0.05)。结论:MRI的总体阳性检出率高于HRCT,尤其是对中耳胆脂瘤手术后复发的诊断;HRCT与MRI两者相结合对于中耳胆脂瘤的明确诊断具有互补作用。     

The grid sectioning technique: a study of catalase platelets.

The grid sectioning technique has been used to obtain the two missing principal axis projections of orthorhombic catalase platelets and to measure directly the unit cell c-value. The negatively stained platelets have a unit cell c-dimension of half that proposed by Unwin (1975) from powder X-ray diffraction. The precision of the grid sectioning technique in positioning sections along a specimen axis shows that the growth fault lines usually observed on negatively stained catalase platelets are rows of missing molecules filled with stain. From these sections conclusions are drawn concerning the action of negative stain on a specimen, the microtomy process, and the specimen/supporting film interaction. Finally the value of microtomy for detailed structural analysis of biological objects is emphasized.     

Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-''light-sheet'', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-''light-sheet'' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.     

Three-dimensional X-ray observation of atmospheric biological samples by linear-array scanning-electron generation X-ray microscope system

Recently, we developed a soft X-ray microscope called the scanning-electron generation X-ray microscope (SGXM), which consists of a simple X-ray detection system that detects X-rays emitted from the interaction between a scanning electron beam (EB) and the thin film of the sample mount. We present herein a three-dimensional (3D) X-ray detection system that is based on the SGXM technology and designed for studying atmospheric biological samples. This 3D X-ray detection system contains a linear X-ray photodiode (PD) array. The specimens are placed under a CuZn-coated Si₃N₄ thin film, which is attached to an atmospheric sample holder. Multiple tilt X-ray images of the samples are detected simultaneously by the linear array of X-ray PDs, and the 3D structure is calculated by a new 3D reconstruction method that uses a simulated-annealing algorithm. The resulting 3D models clearly reveal the inner structure of the bacterium. In addition, the proposed method can easily be used for diverse samples in a broad range of scientific fields.     

Highlights of HRCT imaging in IPF

High-resolution computed tomography (HRCT) imaging has a central role in the diagnosis of interstitial lung diseases, particularly in the evaluation of patients with suspected idiopathic pulmonary fibrosis (IPF). In approximately half of cases, HRCT scans are sufficient to allow a confident IPF diagnosis. Advances in HRCT scanning and interpretation have facilitated improved accuracy for use in diagnosing IPF, eliminating the need for a surgical biopsy in many patients. HRCT may also have a role to play in predicting the prognosis of the disease;. The role of routine follow-up with HRCT to monitor patients with IPF remains unclear due to lack of sufficient evidence, although, sometimes follow-up HRCT might be necessary to rule out progressive disease in patients with undetermined diagnosis. Advances in the field of HRCT imaging are discussed, along with insights into the clinical utility of this procedure in the diagnosis and management of IPF.     

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists.This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method.Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。