Cytosolic and intranuclear calcium signals in rat basophilic leukemia cells as revealed by a confocal fluorescence microscope.

A confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL-2H3). After stimulation with antigen (2,4-dinitrophenol-conjugated bovine serum albumin), fluo-3-fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Time-dependent profiles of the fluo-3-fluorescence intensities resembled closely the patterns of the sequential fluorescence-ratio images of fura-2, which were used to measure the intracellular free-calcium concentration ([Ca2+]i) in individual RBL-2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo-3-fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL-2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (bisbenzimide H 33342, a DNA-specific probe) which were produced by excitation with a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.     

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.     

双重荧光染色监测听毛细胞游离钙

Calcium distribution and mobilization during mechanical stimulation in outer hair cells of the guinea pig were monitored using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Spatial calcium gradients were revealed among various subcellular areas. The ratios of the fluorescence intensity of fluo-3 and fura-red were 1.71 +/- 0.85, 1.61 +/- 0.75, 1.47 +/- 0.65 and 1.39 +/- 0.66 for the cytoplasm, the cytoplasmic membrane, the cuticular plate and the nucleus respectively, indicating that free calcium ion concentrations are the highest in the cytoplasm and the lowest in the nucleus. While the calcium concentration remained relatively constant under resting conditions, it increased during mechanical stimulation. The results show that confocal ratio imaging of fluo-3 and fura-red enables us to determine more accurately the subcellular calcium distribution and that the calcium ions make a contribution to the mechanic-electrical transduction in hair cells.     

Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy.

We describe here the use of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca2+]i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca2+]i in optically sectioned unit volumes of the cell. Images of the calcium wave obtained during propagation between paired cells revealed that the wavefront is constant in shape and propagates at constant velocity without any delay at the cell-to-cell junction. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living cells.     

增强UV-B辐照对小麦幼苗叶肉细胞内Ca2＋水平的研究

以小麦（Triticum aestivum）幼苗叶片为材料,利用提取原生质体方法在小麦幼苗叶肉细胞中成功地装载了钙离子荧光指示剂fluo-3/AM,采用激光共聚焦显微技术检测了增强UV-B辐射后小麦幼苗叶肉细胞内游离钙离子荧光强度的分布,并对[Ca2＋];进行了测定.结果显示,对照组细胞内钙离子荧光分布较均匀,主要分布于紧贴质膜处和核周围,UV-B辐射组钙离子荧光与对照组分布相似,但其原生质体表面不如对照组平滑;同时发现增强UV-B辐射组细胞内钙离子荧光强度值较对照组高,说明增强UV-B辐射组小麦幼苗叶肉细胞维持较高浓度的钙离子水平.这些变化表明Ca2＋信号有可能以一定的方式参与了小麦响应UV-B辐射胁迫的过程.     

Real-time confocal microscopy and calcium measurements in heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution

Preliminary experiments and characterization of a modified confocal fluorescence microscope have been carried out and are presented in this article. We have made use of commercially available hardware and software (having modified and extended the system) and are continuing the process of making modifications to this system to enable us to carry out investigations in living and mobile cells. We report on identified problems in measuring intracellular calcium in myocardial cells, important lessons that have been learned and new findings regarding myocardial cells. Specifically, we have found that myocardial cellular organelles (e.g. nuclei and mitochondria) are sharply defined unlike images obtained with standard epifluorescence microscopes using intracellular indicators. Furthermore, we show that the organelles can accumulate or largely exclude certain indicators relative to the concentration in the cytosol. Additionally, we have examined the use of the new calcium indicator fluo-3 in contracting heart cells and have more clearly defined certain limitations in its use in this preparation. We are working on modifications of the present equipment to enable the system to work with an ultraviolet laser as a light source. The confocal microscope offers the prospect of extraordinarily good spatial and temporal resolution under specific conditions in heart cells.     

Improved double immunofluorescence for confocal laser scanning microscopy.

Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of co-localization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems.     

A comparison of low-power helium-cadmium and argon ultraviolet lasers in commercial flow cytometers

The feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100-125 mW were compared to those obtained at 10 mW. These tests indicated that a helium-cadmium laser (He-Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He-Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He-Cd laser should provide a reasonable alternative as a UV source for flow cytometers.     

Kubelka—Munk模型下的兔血管对He—Cd激光的散射与吸收特性

测量了兔动脉和静脉夺He-Cd激光的反射和透射传输特性。实验采用两积分球系统及波长为441.6nm的He-Cd激光器,并根据测量数据及采用Kubelka-Munk模型分析和计算了兔动脉和静脉组织对该波长激光的吸收系统、散射系数及总的光强I（x）及前向散射通量i(x)和后向散射通量j(x）随厚度的变化情况。结果表明,兔动脉和静脉的温反射率和透射率有明显差别,而且,动脉对激光的吸收系数明显较静脉的要小,耐动脉对激光的散射系数却明显较静脉的要大,在动脉和静脉组织中总的光强I(x)及前向散射通量i(x)和后向散射通量j(x)随厚度的变化情况也有明显的区别。     

Anchored cell analysis and sorting (ACAS) system]

Advanced biology and recent technology have provided sophisticated and objective method for analyzing biological characteristics on cells. Following that, many new instruments have developed. Diagnostic immunocytochemistry has become an accepted diagnostic tool in cell biology. In recent years, remarkable advances in technology provide a method for quantitative and objective analyses of cell characteristics. The newly developed computer assisted laser cytometer (ACAS 570) can be applied in clinical basis as well as in research laboratory. Fluorescent intensities of ancharage-dependent cells can be automatically analyzed and make it possible to separate a subpopulation of cells. This computer controlled system principally consists of argon ion laser, phase contrast microscope. Quantitative fluorescence measurements and computer graphic images can be obtained. The present paper demonstrates multiple applications of laser cytometer for evaluation of cell biology.     

大鼠肺动脉平滑肌培养细胞内Ca~(2+)反应的多样性

用Ｃａ２＋荧光色素Ｆｌｏｕ－３／ＡＭ负荷原代培养的大鼠肺动脉平滑肌细胞,在共聚焦激光显微镜下观察细胞内Ｃａ２＋对各种缩血管物质反应的非均一性。实验结果提示：各种Ｃａ２＋通道的反应与细胞培养时间相关。８０％以上的Ｃａ２＋贮库具有ＣＩＣＲＣａ２＋通道,在肺血管管平滑肌细胞可能存在一种只具有ＣＩＣＲ通道的Ｃａ２＋贮库,ＣＩＣＲ的Ｃａ２＋释放作用强于ＩＣＲＣａ２＋通道     

Alkaline phosphatase cytochemistry in confocal scanning light microscopy for imaging the bone marrow stroma

This paper reports on the use of alkaline phosphatase cytochemistry and combined conventional and confocal reflection and fluorescence scanning light microscopic modes in the study of human marrow stroma. It was found that the end product of the enzyme reaction using Napthol AS phosphate as substrate and Fast Blue BB as coupler reflected the 633 nm (red) light from a Helium-Neon laser. Serial optical sections suitable for 3-D reconstruction and selectively depicting the marrow reticulum cells could be obtained from thick glycol methacrylate sections reacted for Alkaline phosphatase. Furthermore, the yellow background of uncoupled diazonium salt over cytochemically unreactive structures in the same specimens and fields was used for imaging haemopoietic cell mass by operating the microscope at 488 nm (argon ion laser, blue-green). These methods may offer advantages in the investigation of the bone marrow stroma and its interplay with haemopoiesis and osteogenesis in normal and disease conditions.     

Vector Alkaline Phosphatase Substrate Blue III: One Substrate for Brightfield Histochemistry and High-resolution Fluorescence Imaging by Confocal Laser Scanning Microscopy

A number of histochemical chromogenic substrates for alkaline phosphatase are commercially available and give reaction products with a range of colours for brightfield examination. Some of these reaction products are also fluorescent, exhibiting a wide excitation range and a broad emission peak. We report here that one of these substrates, Vector Blue III, yields a stable, strongly fluorescent reaction product with an excitation peak around 500 nm and a large Stokes shift to an emission peak at 680 nm. The reaction product can be excited using a mercury lamp with a fluorescein excitation filter or an argon ion laser at 488 nm or 568 nm, and the emission detected using a long-pass filter designed for Cy-5. Thus, a single substrate is suitable for brightfield imaging of tissue sections and high-resolution analysis of subcellular detail, using a confocal laser scanning microscope, in the same specimen.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.     

High concentration of calcium ions in Golgi apparatus

The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6-NBD-ceramide,and then the single cells were examined by laser scanning confocal microscopy(LSCFM) for subcellular distributions of Ca^2 and the location of Golgi apparatus.In these cells,the intracellular Ca^2 were found to be highly concentrated in the Golgi apparatus.The changes of distribution of cytosolic high Ca^2 region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium,when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin,the fluorescence of the Golgi region decreased far less than that of the cytosol.Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.     

Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells

OBJECTIVE: To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains. RESULTS: Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis. CONCLUSION: On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.     

Free intracellular calcium ([CA2+]i) in opioid sensitive cells of the porcine anterior pituitary.

A new experimental model was utilized to study calcium involvement in the mechanism of opioid influence on cultured porcine pituitary cells. The in vitro model involved interactive argon laser cytometry of pituitary cells pre-loaded by three dyes (fluo-3AM, fura-red and naloxone-conjugated to fluorescein). We compared: 1) the kinetics of free intracellular calcium ([Ca2+]i) in anterior pituitary cells of pregnant pigs (day 25-30) treated in vitro with naloxone (NAL) or gonadotrophin-releasing hormone (GnRH) and 2) the distribution of the opioid-sensitive cells by image analysis of doubly loaded cells. In experiment 1, the changes in [Ca2+]i of pituitary cells pre-loaded with fluo-3 AM (488(ex)/520(em) nm) in response to NAL (10(-6) M) or to GnRH (10(-8) M) were compared to a control cell group. Repetitive line scans across cells were performed and the fluorescence emission from individually selected cells was measured in a time-dependent manner (in 0.5 seconds intervals during periods of 50 seconds). Analysis of data indicated significant increases of [Ca2+]i in NAL- (P<0.001) and GnRH-treated cells (P<0.05) in comparison to the control group. In experiment 2, the fluorescence intensity of doubly-loaded pituitary cells (fura-red, 488(ex)/605(em) nm, as principal calcium indicator and NAL-conjugated with fluorescein, 488(ex)/520(em) nm, to distinguish opioid-sensitive cells) were measured using dual detector image analysis. We found that only approximately 8% of the entire population of anterior pituitary cells exhibited sensitivity to the opioid antagonist treatment. This paper demonstrates calcium involvement in the opioid action on anterior pituitary cells from pregnant pigs and provides a useful model for studies at the individual pituitary cell level and in time-dependent manner.     

2′,7′-Bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.     

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.