Fine structure of spermatial surface in the red alga Antithamnion nipponicum (Rhodophyta)

In the ceramiacean red alga Antithamnion nipponicum Yamada et Inagaki, the structure of the spermatial covering and appendages was examined using confocal laser scanning microscopy, scanning and transmission electron microscopy. The liberated spermatium was subspherical, ca 4.5 μm in size with a colorless covering 2.7–3.0‐μm thick. Two flexible, ribbon‐like appendages arose from the periphery of the spermatial covering. The appendages averaged 80 μm in length and were 0.5–0.6 μm width in most parts. Each appendage consisted of a number of thin longitudinal fibrils. Concanavalin A conjugated with fluorescein isothiocyanate, colloidal gold orferritin, bound specifically with the inner layer of spermatial covering and spermatial appendages. When the liberated spermatia were incubated with mature female gametophytes, the spermatial appendages entangled around the tricho‐gyne.     

Cell adaptations of Rhodococcus rhodochrous IEGM 66 to betulin biotransformation

The findings of a comprehensive study on R. rhodochrous IEGM 66 and triterpenoid betulin interactions during its biotransformation were reported. In the presence of betulin, rhodococci were shown to form heterogeneous cell aggregates. The enhanced size of the aggregates from 12–15 μm to 25– 35 μm was consistent with the increase in betulin concentration from 0.5 to 3.0 g/L. The confocal laser scanning microscopy indicated a high (80.0%) level of rhodococcal viability during betulin biotransformation regardless of the betulin concentration. Experiments employing the combined confocal laser scanning and atomic force microscopy system confirmed that interactions between actinobacterial cells and betulin occur by direct contact. Transforming activities of the crude cell extracts from R. rhodochrous IEGM 66 were compared, and localization of enzymes catalyzing betulin oxidation to betulone was determined. Additionally the effects of betulin on fatty acid composition of rhodococci and their morphometric and morphofunctional characteristics during biotransformation were studied. Our findings could be used to develop approaches for enhanced betulin bioavailability, thus leading to improved biotransformation efficiency.     

利用转盘共聚焦显微镜进行快速实验的新方法

转盘共聚焦显微镜是快速激光共聚焦显微镜的一种,与传统的激光共聚焦显微镜相比具有一些相同点,也有其特有的优势。本文主要介绍转盘共聚焦显微镜的基本原理及如何利用转盘共聚焦显微镜进行快速实验及应用实例,并与传统激光共聚焦显微镜进行比较。转盘共聚焦显微镜具有速度快、灵敏度高、对样品光损伤和光淬灭程度低、操作灵活简单,是随着实验技术发展使用越来越广泛的实验仪器。     

角膜激光共聚焦显微镜在单眼感染性角膜炎疾病诊断中的应用

摘要 目的：探讨角膜激光共聚焦显微镜在单眼感染性角膜炎疾病诊断中的应用价值。方法：回顾性研究2020年6月到2021年6月选择在本院诊治的单眼感染性角膜炎疾病患者62例,所有患者都给予角膜激光共聚焦显微镜检查,记录影像学特征并判断诊断价值（以病原学诊断为金标准）。结果：真菌性角膜炎在角膜激光共焦显微镜下的病变区纵横交错的高反射的真菌菌丝或高反光细长颗粒状的孢子,细菌性角膜炎的病变处会聚集活化的树突状细胞及大量的炎症细胞,病毒性角膜炎的基底膜下神经纤维密度、神经主干的分支数减少,棘阿米巴性角膜炎的包囊表现为圆形高反光厚壁结构。角膜激光共聚焦显微镜判断为病毒性角膜炎17例,诊断病毒性角膜炎的敏感性与特异性为94.4 %和100.0 %;角膜激光共聚焦显微镜判断为棘阿米巴性角膜炎4例,诊断棘阿米巴性角膜炎的敏感性与特异性为94.4 %和100.0 %;角膜激光共聚焦显微镜判断为细菌性角膜炎21例,诊断细菌性角膜炎的敏感性与特异性为95.5 %和97.5 %;角膜激光共聚焦显微镜判断为真菌性角膜炎20例,诊断真菌性角膜炎的敏感性与特异性为94.4 %和93.2 %。ROC曲线分析显示角膜激光共聚焦显微镜诊断细菌性角膜炎、真菌性角膜炎、病毒性角膜炎、棘阿米巴性角膜炎的曲线下面积分别为0.525、0.579、0.777、0.731。结论：角膜激光共聚焦显微镜在单眼感染性角膜炎疾病诊断中的应用能较好的区分细菌性角膜炎、真菌性角膜炎、病毒性角膜炎、棘阿米巴性角膜炎,具有良好的诊断敏感性与特异性。     

颗粒状角膜营养不良的研究进展

颗粒状角膜营养不良是一种临床少见的常染色体显性遗传病,由于5q31染色体上的TGFBI突变使TGFBIp在角膜前弹力层和基质层异常聚集以及代谢的障碍,导致患者双侧角膜进行性出现不同程度的的浑浊,造成视力的进行性损害。目前报道的TGFBI突变至少有66种,其中至少有10种与颗粒状角膜营养不良有关,由于基因型的差异、纯合子以及杂合子的区别,患者表现型也有很大的差别。随着人们对该病认识的提高,共聚焦显微镜、基因诊疗等方法的应用,越来越多的患者得到了正确诊断,目前的治疗的方法主要有角膜移植和激光消融治疗,但由于术后复发甚至加重的原因,并不能使患者满意。由于颗粒状角膜营养不良动物模型的建立,锂或者基因治疗等方法将会有良好的应用前景。     

Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic,neurofilament immunoreactive myenteric neuron types of human small intestine

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO₄ or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO₄ or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.     

Visualization of silver-enhanced reaction products from protein- and immuno-colloidal gold probes by laser scanning confocal microscopy in leflection mode

We have employed a laser scanning confocal microscope in reflection mode to directly and indirectly visualize sites of deposition of silver-enhanced reaction products from colloidal gold probes. A direct approach was used for the localization of alpha-fetoprotein receptors in human myoblasts by incubating primary cultures with an alpha-fetoprotein-gold conjugate. For an indirect approach, cultured CEM cells, derived from a human T-lymphoma cell line, were incubated with a mouse monoclonal antibody to mature T-cells, followed by a gold-labelled antibody to mouse immunglobulins. Multiple optical sections of each sample were collected by reflection laser scanning confocal microscopy and combined into three-dimensional renderings. A (non-confocal) transmission image was generated of each field for comparative purposes. The increasing use of reflection laser scanning confocal microscopy combined with colloidal gold conjugates as biological markes will probably be of considerable advantage in cytochemical analysis.     

Decreased expression of MUC2 due to a decrease in the expression of lectins and apoptotic defects in colitis patients

IntroductionThe involvement of mucin, lectin, and apoptosis in colitis is still unclear. This study aimed to investigate changes in MUC2 expression, inflammation, and changes in lectin expression in colitis patients.MethodsA total of 17 patients were divided into two groups including 11 hemorrhoid patients as a control group and 6 colitis patients. MUC2 mutation analysis was carried out using immunofluorescent and FISH techniques. Assessment of caspase-3, Ki-67, NF-kB, and lectin expressions was also carried out by immunofluorescent technique then analyzed by confocal laser scanning microscope.ResultsThe MUC2, caspase-3, and lectin expressions were significantly lower in the colitis group than in the control group (p < 0.05).ConclusionsIt was concluded that in colitis there was a change in MUC2 expression due to changes in lectins accompanied by apoptotic defects.conclusion     

激光共聚焦显微镜在肿瘤免疫治疗检测中的应用

目的:通过激光共聚焦显微镜对肿瘤生物治疗后患者的外周血淋巴细胞进行亚群计数,为生物治疗后外周血淋巴细胞无法分群的肿瘤患者提供新的监测免疫功能状态的方法。方法:收集35例肿瘤生物治疗后患者的外周血标本,通过激光共聚焦显微镜和流式细胞仪两种方法分别对患者外周血淋巴细胞亚群进行分类计数。结果:流式细胞仪和激光共聚焦显微镜同时分类计数的患者外周血细胞标本30例,两种方法在检测CD3、CD3~+/CD4~+、CD3~+/CD8~+、CD3-/CD16~+56~+、CD3-/CD19~+细胞时均无统计学差异(P值0.05);5例流式细胞仪无法将患者外周血淋巴细胞分群的样本,通过激光共聚焦显微镜可以进行分类计数。结论:激光共聚焦显微镜亦可以用于外周血淋巴细胞的分类计数。     

内窥式激光共聚焦显微镜

为了能在活体内进行实时的细胞尺寸水平的共聚焦观测,科研工作者开展了大量的将激光共聚焦显微镜和内窥镜技术相结合的内窥式激光共聚焦显微镜的研究.本文主要列举了一些国外典型的内窥式激光共聚焦的结构及性能参数,介绍了我们在这方面取得的成果.我们建立的结构的横向分辨率为3.3 μm,轴向分辨率为16.6 μm.     

IAA-induced apogamy in Platycerium coronarium (Koenig) Desv. gametophytes cultured in vitro

Apogamous sporophytes were produced on Platycerium coronarium gametophytes cultured in the presence of indole-3-acetic acid (IAA). The percentage of apogamy as well as the total number of apogamous sporophytes produced per gametophyte clump were highest in the presence of 40 M IAA. When ethylene was allowed to accumulate in the culture vessel in the presence of an optimum level of IAA, the percentage and total number of apogamous sporophyte production decreased significantly. Using light microscope and confocal laser scanning microscope we have shown that nuclear size can be used as a quick parameter to estimate the ploidy level of P. coronarium.Abbreviations CLSM confocal laser scanning microscope - IAA Indole-3-acetic acid - MS Murashige and Skoog     

Highly efficient uptake of ultrafine mesoporous silica nanoparticles with excellent biocompatibility by Liriodendron hybrid suspension cells

The characteristics of the interactions co-cultures of ultrafine mesoporous silica nanoparticles (MSNs) and the Liriodendron hybrid suspension cells were systematically investigated using laser scanning confocal microscope (LSCM) and scanning electron microscopy (SEM). Using fluorescein isothiocyanate (FITC) labeling, the LSCM observations demonstrated that MSNs (size, 5-15nm) with attached FITC molecules efficiently penetrated walled plant cells through endocytic pathways, but free FITC could not enter the intact plant cells. The SEM measurements indicated that MSNs readily aggregated on the surface of intact plant cells, and also directly confirmed that MSNs could enter intact plant cells; this was achieved by determining the amount of silicon present. After 24 h of incubation with 1.0mg mL-1 of MSNs, the viability of the plant cells was analyzed using fluorescein diacetate staining; the results showed that these cells retained high viability, and no cell death was observed. Interestingly, after the incubation with MSNs, the Liriodendron hybrid suspension cells retained the capability for plant regeneration via somatic embryogenesis. Our results indicate that ultrafine MSNs hold considerable potential as nano-carriers of extracellular molecules, and can be used to investigate in vitro gene-delivery in plant cells.     

Enriched Au nanoclusters with mesoporous silica nanoparticles for improved fluorescence/computed tomography dual‐modal imaging

ObjectivesAu nanoclusters (AuNCs) have been used widely in fluorescence bio‐imaging because of their good fluorescence, small particle size and non‐cytotoxicity. AuNCs are also efficient in computed tomography (CT) imaging. Hence, a dual‐modal imaging probe can be constructed without any complicated modification processes by exploiting the excellent performance of AuNCs. In the present study, AuNCs were enriched with mesoporous silica nanoparticles (MSNs) to obtain enhanced fluorescence/CT dual‐modal imaging, which was capable of acquiring more imaging information for diseases compared with single‐mode imaging.Materials and methodsBiocompatible bovine serum albumin (BSA)‐capped AuNCs were prepared and loaded into amine‐functionalized MSNs to form MSN@AuNCs. BSA‐AuNCs, MSNs, and MSN@AuNCs were characterized by ultraviolet‐visible (UV‐vis) spectra, transmission electron microscopy (TEM), fluorescence spectra, and zeta potential. CT imaging was recorded using micro‐CT scanning. Fluorescence imaging was measured using confocal laser scanning microscopy and flow cytometry.ResultsThe prepared AuNCs and MSNs possessed good properties as previously reported. The fluorescence intensity and CT value of the AuNCs were enhanced after being enriched with MSNs. The nanoparticles were both non‐cytotoxic. Confocal laser scanning microscopy and flow cytometry indicated that MSN@AuNCs in CAL‐27 cells showed improved fluorescence imaging compared with simple AuNCs at the same concentration.ConclusionsThe results revealed that the strategy of enriching AuNCs with MSNs can obtain highly sensitive fluorescence/CT dual‐modal imaging, which indicated the potential of this nanoparticle in the diagnosis and treatment of disease.     

激光扫描共聚焦显微镜观察细菌生物膜形成的方法学研究

目的:建立激光扫描共聚焦显微镜观察生物膜形成过程的方法,为进一步研究生物膜的形成机制奠定基础。方法:以临床分离金葡菌X428为研究对象,在盖玻片上形成生物膜,分别于接种后的4、8、12、16、24和48h取出玻片,采用免疫荧光技术标记多糖和细菌,激光扫描共聚焦显微镜(CLSM)观察生物膜形成情况。结果:取得了生物膜形成过程的不同时间点的CLSM图像,4h时细菌在盖玻片上粘附形成小菌落,8h和12h细菌聚集成簇,多糖基质产生并逐渐增多,至16h形成成熟生物膜结构;24h和48h生物膜已经播散,其结构变小。结论:应用免疫荧光技术和激光扫描共聚焦显微镜技术研究生物膜形成过程是一种简便可行的方法。     

Otolin-1, an otolith- and otoconia-related protein,controls calcium carbonate bioinspired mineralization

BackgroundOtoliths and otoconia are calcium carbonate biomineral structures that form in the inner ear of fish and humans, respectively. The formation of these structures is tightly linked to the formation of an organic matrix framework with otolin-1, a short collagen-like protein from the C1q family as one of its major constituents.MethodsIn this study, we examined the activity of recombinant otolin-1 originating from Danio rerio and Homo sapiens on calcium carbonate bioinspired mineralization with slow-diffusion method and performed crystals characterization with scanning electron microscopy, two-photon excited fluorescence microscopy, confocal laser scanning microscopy and micro-Raman spectroscopy.ResultsWe show that both proteins are embedded in the core of CaCO₃ crystals that form through the slow-diffusion mineralization method. Both of them influence the morphology but do not change the polymorphic mineral phase. D.rerio otolin-1 also closely adheres to the crystal surface.General significanceThe results suggest, that otolin-1 is not a passive scaffold, but is directly involved in regulating the morphology of the resulting calcium carbonate biocrystals.     

组织透明化三维显示全包埋耳皮肤神经血管网（英文）

目的 结合组织学染色技术和组织透明化策略研究耳部皮肤中神经纤维和血管的空间对应关系。方法 将耳廓前面和后面的皮肤从其中间软骨仔细剥离，然后直接分别用蛋白基因产物9.5 (PGP 9.5)和鬼笔环肽对耳部皮肤的神经纤维和血管进行免疫荧光染色并进行组织透明化处理。随后，以全包埋方式将耳皮肤组织裱贴在载玻片上用于荧光显微镜和共聚焦显微镜观察。结果 本研究显示PGP 9.5阳性神经纤维伴随鬼笔环肽标记的血管一道从耳廓的基部走行到其外围区域形成耳廓的神经血管网。在传统的免疫荧光染色技术基础上，后续的组织透明化技术可以更好地展示耳部皮肤中神经纤维和血管的形态学细节。结论 从方法学的角度来看，组织透明化技术增进了耳部皮肤中免疫荧光标记的可视性，它可能成为有效的技术手段用于解析正常和病理状态下耳部神经血管网的空间结构。     

External Morphology and Muscle Arrangement of Brachionus urceolaris, Floscularia ringens, Hexarthra mira and Notommata glyphura (Rotifera, Monogononta)

We studied four monogonont rotifers (Brachionus urceolaris, Floscularia ringens, Hexarthra mira, Notommata glyphura) using two different techniques of microscopy: (1) the presence of filamentous actin was examined using phalloidin-fluorescent labelled specimens and a confocal laser scanning microscope (CLSM); (2) external morphology was investigated using a scanning electron microscope (SEM). B. urceolaris, F. ringens, and N. glyphura showed similar patterns of muscle distribution: a set of longitudinal muscles acting as head and foot retractors, and a set of circular muscles. However, the size and distribution of circular muscles differed among these species. H. mira differed from the other species in that it lacked circular muscles but possessed strong muscles that extended into each arm. The study showed that using both CLSM and SEM provides better resolution of the anatomy and external morphology of rotifers than using one of these techniques alone. This can facilitate better understanding of the complicated anatomy of these animals.     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Imaging of estrogen receptor-α in rat pial arterioles using a digital immunofluorescent microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors (1, 2, 3). Although two sub-types exist (estrogen receptor -α and β),estrogen receptor-α has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy (4). Furthermore, ER-α is located in the nuclei and in the cytoplasm of rat basilar arteries (5). The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments (6). Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-α using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 μm cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.