医学实验室的光学显微镜图像记录技术-显微照相术

结合技术进展和工作实践讨论光学显微镜照相的基本方法及其应用。     

液基细胞学检测在宫颈癌早期筛查中的价值

目的：探讨液基细胞技术在宫颈癌早期筛查中的应用价值。方法：对625例患者行TCT,用TBS诊断方法进行分析,对76例细胞学阳性及临床上高度可疑的50例共计126例患者行阴道镜检查,以评价TCT在临床上的应用价值,同时调查他们的疾病构成及年龄分布特征。结果：TBS诊断ASCUS40例,AGUS4例,ASC-H3例,LSIL18例,HSIL7例,SCC3例,AC1例。细胞学与阴道镜下活组织病理诊断符合率分别为LSIL72.22%,HSIL85.71%,SCC100%,76例患者中宫颈病变占4.64%,31~40岁的妇女为宫颈病变的最高发年龄段,占46.05%。结论：采用TCT技术对早期发现与治疗宫颈病变及宫颈癌有积极的临床作用。     

彩色多普勒对糖尿病视网膜病变球后动脉血流动力学变化的研究

目的研究2型糖尿病患者视网膜中央动脉（CRA）、睫状后动脉（PCA）的血流动力学改变以及CRA、PCA血流参数与糖化血红蛋白（GHb）及伴随疾病高血压间的相关性。方法收集58例2型糖尿病病人的GHb及并发症等相关危险因素资料,彩色多谱勒检查病人及30例健康对照者的视网膜中央动脉（CRA）、睫状后动脉（PCA）的收缩期血流峰值速度（PSV）、舒张末期血流速度（EDV）、阻力指数（RI）,比较分析各因素与糖尿病眼底动脉病变的相关性。结果随患者视网膜病变程度的加重,CRA、PCA的PSV、EDV呈进行性下降趋势,RI呈进行性升高趋势。与健康对照组相比,糖尿病无视网膜病变组CRA的血流参数已出现改变,且组间差异有统计学意义,而PCA的PSV的组间差异也有统计学意义。相关性分析显示：CRA、PCA的EDV与GHb呈显著负相关,RI与GHb呈显著正相关;CRA的PSV、EDV以及PCA的EDV与收缩压显著负相关,PCA的RI值与收缩压显著正相关。结论糖尿病患者球后动脉血流动力学改变早于眼底检出确诊视网膜病变的出现。CRA、PCA的血流动力学参数与患者GHb及高血压伴随疾病相关。彩色多普勒超声检测球后动脉血流动力学改变是评价糖尿病视网膜及脉络膜早期病变的有效方法。     

儿童髓鞘少突胶质细胞糖蛋白抗体相关炎性脱髓鞘疾病临床分析

目的 探讨儿童髓鞘少突胶质细胞糖蛋白(MOG)抗体相关炎性脱髓鞘疾病(IDD)患儿的临床表现、影像学特征、治疗方案及临床预后.方法 选择2014年6月1日至2020年9月30日,在中山大学孙逸仙纪念医院儿科确诊的7例MOG抗体相关IDD患儿为研究对象.其中,女性患儿为2例,男性为5例;平均年龄为8.3岁.回顾性分析该7...     

SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering–two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

With the rapid advances in both genotyping and phenotyping of single cells, bridging genotype and phenotype at the single-cell level is becoming a new frontier of science (1). Methods have been developed to shed light on the genotype–metabolism relationship of individual cells in a complex environment (2, 3), which is especially relevant for an in-depth understanding of complex microbial communities in the environment and host-associated microbiomes. For functional analyses of microbial communities, single-cell isotope probing is often performed in combination with nanoscale secondary ion mass spectrometry (NanoSIMS) (4–7), microautoradiography (MAR) (8, 9), or spontaneous Raman microspectroscopy (10–12) to visualize and quantify the incorporation of isotopes from labeled substrates. These methods can be combined with fluorescence in situ hybridization (FISH) using ribosomal ribonucleic acid (rRNA)-targeted probes (13), enabling a direct link between metabolism and identity of the organisms. In addition, Raman-activated cell sorting has been recently developed using either optical tweezers or cell ejection for downstream sequencing of the sorted cells (14–16). While these approaches have expanded the possibilities for functional analyses of microbiome members (17), all of the aforementioned methods suffer from extremely limited throughput. Consequently, only relatively few samples and cells per sample are typically analyzed in single-cell stable isotope probing studies, hampering a comprehensive understanding of the function of microbes in their natural environment.To overcome the limited throughput of Raman spectroscopy, coherent Raman scattering microscopy based on coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS) has been developed (18, 19). Compared with CARS, the SRS signal is free of the electronic resonance response (20) and is linear to molecular concentration, thus permitting quantitative mapping of biomolecules (21, 22). Both CARS and SRS microscopy have successfully been applied for studying single-cell metabolism in eukaryotes (23–26). In a label-free manner, SRS imaging has led to the discovery of an aberrant cholesteryl ester storage in aggressive cancers (27, 28), lipid-rich protrusions in cancer cells under starvation (29), and fatty acid unsaturation in ovarian cancer stem cells (30) and more recently, in melanoma (31, 32). CARS and SRS have also been harnessed to explore lipid metabolism in live Caenorhabditis elegans (33–36). Combined with stable isotope probing, SRS microscopy has allowed the tracing of glucose metabolism in eukaryotic cells (37, 38) and the visualization of metabolic dynamics in living animals (25). Recently, SRS was successfully applied to infer antibiotic resistance patterns of bacterial pure cultures and heavy water (D2O) metabolism (39). Yet, SRS microscopy has not been adapted for studying functional properties of members of microbiomes as SRS itself lacks the capability of identifying cells in a complex community.Here, we present an integrative platform that exploits the advantages of SRS for single-cell stable isotope probing together with two-photon FISH for the identification of cells in a high-throughput manner. To deal with the challenges in detecting low concentrations of metabolites inside small cells with diameters around 1 µm, we have developed a protocol that maximizes the isotope label content in cells and exploits the intense SRS signal from the Raman band used for isotope detection.Conventionally, FISH is performed separately by one-photon excited fluorescence microscopy (40). To enhance efficiency, we developed a system that implements highly sensitive SRS metabolic imaging with two-photon FISH using the same laser source. These efforts collectively led to a high-throughput platform that enables correlative imaging of cell identity and metabolism at a speed of 10 to 100 ms per cell. In comparison, it takes about 20 s to record a Raman spectrum from a single cell in a conventional spontaneous Raman FISH experiment (41, 42).Our technology enabled high-throughput analysis of single-cell metabolism in the human gut microbiome. In the human body, microbes have been shown to modulate the host’s health (43, 44). Analytical techniques looking into their activities and specific physiologies (i.e., phenotype) as a result of both genotype and the environment provide key information on how microbes function, interact with, and shape their host. As a proof of principle, we used stimulated Raman scattering–two-photon fluorescence in situ hybridization (SRS-FISH) to track the incorporation of deuterium (D) from D2O into a mixture of two distinct gut microbiota taxa. Incorporation of D from D2O into newly synthesized cellular components of active cells, such as lipids and proteins, occurs analogously to incorporation of hydrogen from water during the reductive steps of biosynthesis of various cellular molecules (10, 45, 46). Importantly, D incorporation from D2O has been shown to be reliable to track metabolic activity of individual cells within complex microbial communities in response to the addition of external substrates (10, 17, 47). When microbial communities are incubated in the presence of D2O under nutrient-limiting conditions, individual cells display only minimal activity and only minor D incorporation (11, 17, 47). In contrary, when cells are stimulated by the addition of an external nutrient, cells that can metabolize this compound become active and incorporate D into macromolecules, which lead to the presence of C-D bonds into the cell’s biomass. Consequently, D incorporation from D2O can be combined with techniques able to detect C-D signals, such as Raman-based approaches, and to track metabolic activity at the single-cell level in response to a variety of compounds. Here, we show that SRS-FISH enables fast and sensitive determination of the D content of individual cells while simultaneously unveiling their phylogenetic identity. We applied this technique to complex microbial communities by tracking in situ the metabolic responses of two major phylogenetic groups of microbes in the human gut (Bacteroidales and Clostridia spp.) and of a particular species within each group to supplemented host-derived nutrients. Our study revealed that 1) Clostridia spp. can actually outperform Bacteroidales spp. at foraging on the mucosal sugar fucose and shows 2) a significant interindividual variability of responses of these major microbiome taxa toward mucosal sugars. Together, our results demonstrate the capability of SRS-FISH to unveil the metabolism of particular microbes in complex communities at a throughput that is two to three orders of magnitude higher than other metabolism identity bridging tools, therefore providing a valuable multimodal platform to the field of single-cell analysis.     

急性脑梗死OCSP分型与影像学分型的关系

目的探讨急性脑梗死牛津郡社区卒中项目(OCSP)分型与影像学分型的对应关系。方法回顾性分析236例急性脑梗死患者早期OCSP分型情况及其影像学特征。结果按OCSP分型:完全性前循环梗死(TAC I)28例(11.9%),部分性前循环梗死(PAC I)71例(30.1%),腔隙性梗死(LAC I)94例(39.8%),后循环梗死(POC I)43例(18.2%)。临床分型与影像学分型一致者共有171例,分型总准确率72.5%(171/236);各亚型分型准确率分别为TAC I 76%(25/33),PAC I 81%(34/42),LAC I71%(81/114),POC I 66%(31/47)。结论OCSP分型能正确预测梗阻部位和大小,与影像学检查结果有良好的对应关系。     

血管瘤与脉管畸形的鉴别诊断研究进展

血管瘤和脉管畸形是婴幼儿比较常见的血管疾病,过去由于对两者的分类和诊断比较混乱,给临床治疗带来诸多不便,也给患者增加了不必要的痛苦。1982年,Mulliken和Glowacki提出了脉管性疾病的生物学新分类,将血管瘤和脉管畸形归类为2种性质完全不同的病变。血管瘤是多发于婴幼儿的良性肿瘤,大多数可以自行消退;而脉管畸形是血管结构的发育异常,不会自行消退,随患者的生长发育持续增长。因此,对于确诊的脉管畸形,应尽早采取干预措施。这就要求临床医师能及时、准确、有效地鉴别诊断血管瘤和脉管畸形。本文就目前关于血管瘤和脉管畸形的鉴别诊断进展作一综述。     

3.0T高分辨率磁共振成像技术评价颅内动脉粥样硬化的临床价值研究

目的 分析3.0T高分辨率磁共振成像(High-resolution magnetic resonance imaging, 3.0T HR-MRI)评价颅内动脉粥样硬化价值。方法 回顾2019年3月至2021年3月笔者医院63例疑似颅内中动脉粥样硬化性狭窄患者资料,行3.0T高分辨率磁共振成像和数字减影血管造影(Digital Subtraction Angiography, DSA)。以DSA为金标准,分析HR-MRI检查动脉狭窄效能;对比卒中与非卒中患者出院后责任斑块高度、标准化管壁指数(normalized wall index, NWI)值。结果 HR-MRI诊断颅内中动脉粥样硬化狭窄程度与DSA相关性高(R=0.921,P<0.001),二者检验轻度、中度、重度狭窄及闭塞一致性kappa值=0.715、0.751、1.000、1.000;与非卒中患者相比,出院6月、12月时卒中患者责任斑块高度更高(P<0.05),6月、12月、24月时NWI值更高(P<0.05)。结论 3.0T HR-MRI诊断颅内动脉粥样硬化狭窄程度与DSA一致性和相关性高,NWI和责...     

SD大鼠正常胰腺MRI表现

目的 观察SD大鼠正常胰腺MRI表现.方法 对9只健康SD大鼠行MR平扫及增强扫描,观察胰腺形态、分布、位置毗邻及信号特点.之后处死大鼠,观察其胰腺解剖及组织学表现.结果 共8只大鼠顺利完成MR扫描,1只因麻醉死亡.MRI显示胰腺位于胃、脾脏及左肾之间,分为胃叶、脾叶及十二指肠叶,汇合于胰腺结合部;脾叶信号强度＞十二指...     

颞下颌关节紊乱的影像学特征：锥形束CT和MRI效能比较

目的：分析颞下颌关节紊乱（temporomandibular joint disorder, TMD）的锥形束CT(cone-beam CT, CBCT)和磁共振成像（magnetic resonance imaging, MRI）特征,对两者的影像特征进行比较。方法：收集2018年8月—2020年5月在南通市口腔医院治疗的45例TMD患者的临床资料,所有患者均于2周内完成CBCT和MRI检查,统计所有患者颞下颌关节（temporomandibular joint, TMJ）和咀嚼肌病变在CBCT和MRI上的特征（包括关节间隙狭窄、骨质破坏、骨质增生硬化、髁突变形、骨质囊样变、关节盘移位、关节囊积液和咀嚼肌病变）,并对两者的影像学特征进行比较。采用SPSS 25.0软件包进行数据统计分析。结果：TMD临床分类Ⅰ类疼痛性疾病31例（68.9%）,Ⅱ类关节疾病14例（31.1%）。45例患者90个关节中,MRI检出存在TMJ和咀嚼肌病变数（71/90）显著高于CBCT检出的病变数（58/90）(P=0.032),其中MRI检出关节盘移位（19/90）、关节囊积液（28/90）和咀嚼肌水肿（...