构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应

目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。     

心肌细胞钙瞬变和细胞收缩的激光共聚焦成像研究

目的：游离钙离子参与机体的多种重要生理功能。本研究着重探讨如何利用激光共聚焦显微镜线扫描成像技术同时记录正常情况下心肌细胞的钙瞬变以及由此引起的细胞收缩过程。方法与结果：本研究以分离的心室肌细胞为对象,通过局部场刺激诱发细胞的钙瞬变和收缩,同时配合使用激光共聚焦显微镜成像系统,以线扫描方式记录实验结果。结果表明,钙瞬变先于细胞收缩发生(约早31ms),而收缩最大处远落后于钙瞬变峰值发生处(约慢346ms)。结论：激光共聚焦显微镜线扫描成像技术具有较好的时问分辨率和空间分辨率,其实验结果直观、明确、可靠,是较理想的研究钙瞬变和细胞收缩的光学记录方法。     

激光扫描共聚焦显微镜在医学研究中的应用

激光扫描共聚焦显微镜（Confocal laser scanning microscope,CLSM）具有高分辨率、高灵敏度、三维重建、动态分析等优点,使图像更为精确清晰和数字化。该仪器现已广泛应用于细胞生物学、生理学、病理学、遗传学和药理学等研究领域中。本文简述了激光扫描共聚焦显微镜的结构、工作原理并归纳了其在医学各领域研究中的应用。     

心肌细胞钙信号研究进展

近年自激光共聚焦显微镜使用以来,结合膜片钳技术及分子生物学方法,在心肌细胞内的钙信号种类以及在心脏兴奋－收缩偶联研究方面取得了突破性进展。本文介绍了心肌细胞的钙信号研究进展,包括在心肌细胞内可以观察到的钙闪烁,钙微粒,钙波以及由心肌细胞膜上电除极而诱发的瞬时性钙增高等几种心脏细胞内钙变化的形式,意义以及局部调控兴奋－收给偶联的机制。     

激光共焦显微成像的最新进展及其在生命科学研究中的应用

激光扫描共聚焦显微镜近年来得到了迅速发展,是近代最先进的细胞生物医学分析仪器之一。通过它可以对观察样品进行无创断层扫描和成像,在生物学和医学研究诊断的各个方面都得到了广泛的应用。本文主要介绍了激光扫描共焦显微镜的基本原理和发展状况,并着重介绍了在共焦荧光显微镜中采用薄荧光层和切片成像特性图来表征成像状态的功能。这种方法一般用于表征共聚焦和多光子显微镜的成像特性,是比较显微镜切片成像条件、成像质量等相关性能的重要依据。     

荧光成像技术及其在生命科学中的应用

近年来,荧光成像技术发展迅速,其成像系统通常为目前最先进的分析检测仪器之一的激光共聚焦显微镜,荧光探针是荧光成像技术的核心之一。作为新兴光学成像技术,荧光成像技术在生命科学领域中应用广泛,可用于蛋白质及金属离子检测,肿瘤疾病的诊断,并为药物新剂型的研究提供了新思路。     

大鼠脑片中细胞内游离钙动态变化的研究

目的：探索大鼠急性脑片中电刺激诱发的细胞内钙的动态变化规律。方法：采用表面灌流的急性脑片模型,结合电生理和激光共聚焦技术,利用细胞内钙荧光探针进行细胞内游离钙标记,观察电刺激诱发的脑片中神经细胞内游离钙的变化情况。结果：急性脑片组织中,钙标记染料的神经细胞内钙探针荧光强度,电刺激后出现显著增强,且具有波样特征,而Suramin明显抑制此反应,表现为钙探针荧光强度下降和钙反应时间出现延迟,两组之间差异具有统计学意义（P〈0．05）.结论：刺激诱发的大鼠急性脑片中瞬时动态钙信号变化具有一定的时空发生特征,且这种钙信号的时空变化过程可能与嘌呤能信号的作用有关。     

嗜中性白细胞呼吸爆发与胞内外钙信号的关系研究

以膜受体激动剂ｆＭＬＰ和ＰＫＣ激活剂ＰＭＡ为刺激剂,ｆｕｒａ－２为荧光探针,分别用化学发光和荧光方法研究了嗜中性白细胞呼吸爆发与胞内外钙信号的关系。并以ｆｌｕｏ－３为荧光探针,在激光共聚焦显微镜上观测了呼吸爆发时的胞内钙信号的时间与空间变化。ｆＭＬＰ能够迅速引起胞内钙变化,而ＰＭＡ不引起胞内钙变化。在呼吸爆发启动时间上,ｆＭＬＰ明显短于ＰＭＡ,且呼吸爆发的强度更高,持续时间较短。比较胞内钙信号与呼吸爆发,胞内钙变化在启动时间,到达峰值时间和持续时间上均短于呼吸爆发时间。胞内无钙时,呼吸爆发完全被抑制。胞外无钙时,呼吸爆发强度比有钙时低７０％左右。激光共聚焦显微镜观测发现：细胞在ｆＭＬＰ作用之后胞内钙库释放钙进入胞浆并向胞外流动。当胞外有钙时,胞内钙浓度降至最低后,由于外钙内流会使其再次缓慢上升;当胞外无钙时,胞浆钙浓度降至最低后不会再回升。结果提示胞内钙信号对细胞进入呼吸爆发有重要控制作用,而胞外钙主要用于维持细胞的呼吸爆发。     

心肌胞内钙瞬变的记录方法——快速二维扫描法与线扫描法

目的:采用共聚焦显微镜快速二维扫描方式和线扫描方式记录心肌胞内钙瞬变,并分析其优缺点。方法:标本为急性分离的SD大鼠心肌单细胞,胞内钙信号由钙指示剂fluo4-AM标记,其变化由共聚显微镜(LSM510META系统)记录。钙瞬变由局部场刺激诱发,刺激器和共聚焦成像系统之间通过触发连接同步工作。结果:快速二维扫描方式可在二维平面上反映全细胞范围内钙瞬变的动态过程,空间信息较全面;特别地,当心肌细胞由于药物或病理状态的改变而出现胞内钙稳态失衡时,快速二维扫描的结果更有利于了解胞内钙变化;其结果可制成动画,真实而直观地再现心肌细胞胞内钙瞬变的动态过程。线扫描方式的时间分辨率较高,也有一定的空间分辨率,可反映钙瞬变的时空特征,并可分析细胞收缩的情况。二种扫描方式所得的结果在实质上是一致的,但各有其侧重点和优缺点,在反映心肌细胞功能状态方面具有互补作用。结论:两种扫描方式所得的结果综合起来更有利于对胞内钙信号变化的特征和意义进行正确解读。     

共聚焦激光扫描显微镜技术

自1985年共聚焦激光扫描显微镜问世以来,该项技术一直在飞速发展和完善。近年来一系列新型的共聚焦显微镜相继研制成功和投入使用,如视频共聚焦激光扫描显微镜、双光子显微镜、4Pi显微镜、荧光寿命成像显微镜等,它们较传统共聚焦显微镜有着各自独特的优势,了解它们的基本性能和特点有助于其在生物学领域更为广泛深入的应用。     

Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research.     

Confocal laser scanning microscopy

Many technological advancements of the past decade have contributed to improvements in the photon efficiency of the confocal laser scanning microscope (CLSM). The resolution of images from the new generation of CLSMs is approaching that achieved by the microscope itself because of continued development in digital imaging methods, laser technology and the availability of brighter and more photostable fluorescent probes. Such advances have made possible novel experimental approaches for multiple label fluorescence, live cell imaging and multidimensional microscopy.     

丙肝病毒抗原在肝细胞癌组织中的定位——共聚焦激光扫描显微镜观察

应用共聚焦激光扫描显微镜（ＣｏｎｆｏｃａｌＬａｓｅｒＳｃａｎｎｉｎｇＭｉｃｒｏｓｃｏｐｅ,ＣＬＳＭ）观察免疫荧光标记的丙型肝炎病毒核心抗原（ＨＣＶＣＰ－１０）在肝癌组织中的表达,与免疫组化普通光镜观察结果比较,显示ＨＣＶＣＰ－１０抗原在肝细胞癌和癌周肝组织的定位特点,介绍ＣＬＳＭ在病理学中的应用。研究结果表明：ＨＣＶＣＰ－１０抗原大部分存在于胞浆,少部分存在于胞核,ＨＣＶ的表达有核内过程;ＨＣＶＣＰ－１０抗原在癌周肝组织的表达明显强于癌组织;ＣＬＳＭ比普通光镜在确定抗原定位上有更大的优越性     

Confocal microscopy: a tool to visualise differentiation of stem cells into cardiomyocytes

Confocal microscopy offers important advantages compared to conventional epifluorescence microscopy. It works as an "optical microtome" leading to a accurate image resolution of a defined focal plane. Furthermore, the addition of a Nipkow disk on the confocal microscope greatly accelerates the image acquisition, up to 30 frames per second. Nevertheless, the software-assisted mathematical restoration of images acquired using a wide-field microscope allows to get images with a resolution similar to the one obtained in confocal microscopy. These imaging technologies allowed us to monitor on line cardiac differentiation of murine embryonic stem (ES) cells within 3D structures called embryoid bodies. The high rate acquisition of images using the confocal microscope equipped with a Nipkow disk allows to monitor calcium spiking in differentiating cardiomyocytes within embryoid bodies.     

Three-dimensional reconstruction of Paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

The oral apparatus of the ciliate protozoan Paramecium primaurelia, a single-celled eukaryotic organism, is a highly organized structure whose arrangement is of important taxonomic, phylogenetic and developmental significance. This paper analyses oral structures by means of a confocal laser scanning optical microscope (CLSM), which allows their three-dimensional visualization and measurement. The extraction of the intrinsic three-dimensional information related to the biological objects under investigation can in turn be related to their functional state, according to the classical paradigms of structure to function relationship identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection. For comparison with conventional microscopy, two-dimensional images were acquired via a standard TV camera coupled to the microscope itself. The volumes obtained by piling up the slices were rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARCstation 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the three-dimensional structures, under UNIX and X-Window, according to a ray-tracing algorithm.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.     

Axially‐confined in vivo single‐cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes

Green‐to‐red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave‐length illumination with blue and red/near‐infrared light. By modifying a confocal laser‐scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide‐spread use of this method. Here, we investigated whether spatially‐restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half‐width half‐maximum for the axial dimension was below 5 μm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single‐cell resolution.     

SIMS ion microscopy in cancer research: single cell isotopic imaging for chemical composition, cytotoxicity and cell cycle recognition.

A secondary ion mass spectrometry (SIMS) based isotopic imaging technique was used for studies of i/ total calcium stored in cancerous and normal cell lines and ii/ intracellular chemical composition (total K, Na, and Ca) in relation to DNA staining patterns in taxol-treated breast cancer cells. A Cameca IMS-3f ion microscope with 0.5 microm spatial resolution was used. Observations were made on frozen freeze-dried cells. In MCF-10A non-tumorigenic breast epithelial cells, the nucleus contained 0.6 +/- 0.10 mM and the cytoplasm 1.1 +/- 0.30 mM total calcium per unit volume (mean +/- S.D.). MCF-7 tumorigenic breast epithelial cells revealed an abnormal total calcium distribution. Their nuclei and cytoplasm were not significantly different in stored calcium concentrations (0.5 +/- 0.08 mM total calcium in the nucleus and 0.6 +/- 0.07 mM in the cytoplasm). Furthermore, in MCF-7 cells the cytoplasmic total calcium is significantly less than in MCF-10A cells. Both cell lines contained approximately 150 mM intracellular potassium and 13 mM sodium. As 80% of the cytoplasmic total calcium pool in MCF-10A cells could be released with thapsigargin, it is plausible that the calcium storage capacity of the endoplasmic reticulum in tumorigenic MCF-7 cells is compromised. Correlative SIMS and confocal laser scanning microscopy (CLSM) revealed an increase in intracellular sodium and a redistribution of calcium in taxol-arrested M-phase cells prior to any noticeable DNA fragmentation. This novel correlative approach opens new avenues of research for understanding intracellular ionic composition in relation to therapeutic cytotoxicity. Other valuable features of SIMS for cancer research shown in this study include subcellular imaging of calcium influx using 44Ca, 127I from iododeoxyuridine for S-phase recognition, and 19F from fluorinated deoxyglucose.     

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.