Laser Scanning Microscopy System Research Articles

Mitochondrial Membrane Potential Changes in Osteoblasts Treated with Parathyroid Hormone and Estradiol

This study assessed mitochondrial membrane potential changes in cultured osteoblasts treated with hormones known to regulate osteoblasts. A fluorescent carbocyanine dye, 5,5′, 6,6′-tetrachloro-1,1′, 3,3′-tetraethylbenzimidazolocarbocyanine iodide, also called JC-1, was used as a probe. JC-1 emits photons at 585 nm (orange–red) when the membrane potential in mitochondria is highly negative, but when the potential becomes reduced emission occurs at 527 nm (green). Osteoblasts were rinsed in serum-free medium for 5 min, then loaded with 1 × 10−6MJC-1 for 10 min. The distribution and intensity of JC-1 fluorescence were evaluated with a laser-scanning confocal microscope system. Hormone treatments included parathyroid hormone (PTH; 10−8M), 17β-estradiol (10−8M), and thyroxine (T4; 10−8M). The potassium ionophore valinomycin (10−6M) was used as a control since it is known to disrupt the electrochemical gradient of mitochondria without interfering with the pH gradient. Valinomycin caused a profound, rapid increase (22.5% above untreated values) in the green/red ratio, which indicated a lowering of the mitochondrial membrane potential in all samples evaluated. PTH caused a less pronounced, but significant (7–14%), reduction in membrane potential in all cells examined. PTH is known to affect osteoblasts in a number of ways and is inhibitory to mitochondrial respiration; the results confirm this effect. For estradiol, half of the cells responded at a significant level, with a membrane potential reduction of 6 to 13% being recorded; the other half did not respond. Thyroxine did not alter mitochondrial membrane potential. Responses were detectable within 20 s for valinomycin, but occurred at a slower rate, over 200 to 300 s, following PTH and estradiol treatment. Responses to PTH and estradiol could be due to mitochondrial uptake of cytosolic Ca2+.

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca 2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record lines-cans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

Target Cell-Induced Calcium Signals in Human Natural Killer Leukemia Cells as Revealed by Confocal Fluorescence Microscopy

The target cell-induced calcium response in natural killer (NK) cells was examined using indo-1 (a ratiometric fluorochrome), a newly developed video-rate ultraviolet laser-scanning confocal microscope system, and a human NK leukemia cell line, YTN. Susceptible target cells (JY cells), but not unsusceptible target cells (K562 cells), increased the intracellular calcium concentration ([Ca]i) in almost all indo-1-loaded YTN cells with a lag time, although the kinetics of increase in [Ca]idiffered in individual YTN cells. The increase in [Ca]iwas completely blocked by 2 mMEGTA and partially (around 50%) blocked by 10 μMnicardipine, the extents of inhibition of calcium response correlating well with those of inhibition of cytotoxic activity. Thus, the calcium response in YTN cells observed in this study may constitute a critical step in the manifestation of cytotoxic activity.

Ethanol‐Induced Apoptosis and Oxidative Stress in Hepatocytes

This short review focuses on ethanol-induced oxidative stress and hepatocyte apoptosis. Apoptosis is increasingly recognized as a fundamental biological process that impacts on an early development, maturation, and acquisition of disease states of multicellular organisms. Although the occurrence of apoptosis has been identified for many decades, relatively recent acceptance of this principle is evidenced by remarkable increases in special conferences and presentations on this topic as well as its rapidly expanding volume of scientific literature. Oxidative stress is well recognized to be a key step in the pathogenesis of ethanol-associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen reactive species and/or by decreasing the level of endogenous antioxidants. Studies in our laboratory using a confocal laser scanning microscopic system strongly suggest that agents which inhibit ethanol-induced oxidative stress effectively attenuate hepatocyte death, i.e., apoptosis and necrosis. In addition, our investigations demonstrated that inhibitors of intracellular antioxidants exaggerate ethanol-associated hepatocyte apoptosis. Although the detailed mechanism still remains unknown, it is conceivable that an oxidant-dependent mechanism is largely involved in the process for ethanol-induced hepatocyte apoptosis.

Single cell analysis of tyrosine kinase dependent and independent Ca2+ fluxes in progesterone induced acrosome reaction.

In this study we developed a single cell analysis protocol with which protein tyrosine kinase (PTK)-dependent and independent Ca2+ fluxes occurring in human spermatozoa in response to progesterone were evaluated. By recording the fluorescence emitted by fluo-3-loaded spermatozoa using a confocal laser scanning microscopy system it was possible not only to monitor relative changes in the intracellular free Ca2+ concentration ([Ca2+]i) but also to determine the time at which the acrosomal exocytosis began. The addition of progesterone produced a rapid transient [Ca2+]i increase in 35% of spermatozoa. In approximately 10% of spermatozoa, this initial [Ca2+]i increase was followed by a secondary [Ca2+]i increase beginning 2-10 min after the progesterone addition and leading to the acrosomal exocytosis in most of these spermatozoa. On the other hand, a rapid triggering of exocytosis during the initial [Ca2+]i increase was a relatively infrequent observation. The inhibition of PTK with genistein or herbimycin A did not influence the initial progesterone-induced [Ca2+]i increase but inhibited the secondary [Ca2+]i increase and the ensuing acrosomal exocytosis. The initial PTK-independent Ca2+ response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the ability to generate the secondary, PTK-dependent response developed during in-vitro capacitation.

Ethanol‐Induced Oxidative Stress in the Liver

Oxygen stress is well recognized to be a key step in the pathogenesis of ethanol-associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen-reactive species and/or by decreasing the level of endogenous antioxidants. Numerous experimental studies have emphasized the role of the ethanol-inducible cytochrome P-450 in the microsomes, as well as the molybdo-flavoenzymes xanthine oxidase in the cytosol. This review shows the putative role of ethanol-induced disturbances in iron metabolism in relation to iron as a prooxidant factor. Ethanol administration also affects the mitochondrial free radical generation. Although many previous studies suggest a role for active oxygens in ethanol-induced mitochondrial dysfunction in hepatocytes, the detailed mechanism of ethanol-induced oxidative stress on mitochondria remains to be clarified further. Studies of our laboratory using a confocal laser scanning microscopic system strongly suggest that active oxidants produced during ethanol metabolism modulate mitochondrial energy synthesis in isolated and cultured hepatocytes. In addition, our investigations implicate endogenous glutathione-glutathione peroxidase system and catalase as important antioxidants and cytoprotective machinery in the hepatocyte mitochondria exposed to ethanol. The fluorographic investigations using the confocal laser scanning microscopy may be useful to extend our knowledge and provide a new view about ethanol-associated oxidative stress and metabolic changes in hepatocytes.

Variability and Invariability in the Structure of an Identified Nonspiking Interneuron of Crayfish as Revealed by Three-Dimensional Morphometry

Abstract We have quantitatively analyzed the three-dimensional dendritic structure of an identified nonspiking interneuron, Local Directionally Selective (LDS) interneuron in the terminal abdominal ganglion of the crayfish, Procambarus clarkii Girard, using a confocal laser scanning microscopic system. Comparison of the cell size before and after fixation followed by dehydration and clearing revealed that the diameter of dendritic branches shrank by about 15% while the branch length showed no significant change. The soma and dendritic diameter in vivo were obtained from the measurement in the cleared preparation multiplied by the shrinkage factor. The total length of dendrites and their membrane area as well as their electrotonic structure were found to vary from preparation to preparation. The spatial distribution of fine dendritic branches was variable, but that of major branches was constant among preparations. The interneuron further shared bilaterally asymmetrical dendritic structure: those branches ...

Prenatal diagnosis of G M2-gangliosidosis : Immunofluorescence analysis of ganglioside G M2 in cultured amniocytes by confocal laser scanning microscopy

A confocal laser scanning microscopic system was used to detect the storage of ganglioside G M2 in Tay-Sachs fibroblasts and amniocytes. The diagnosis of the disease was confirmed by counting immunoreactive cells or by digital imaging analysis. This novel system facilitates the prenatal diagnosis of G M2-gangliosidosis.

Intracellular pH mapping with SNARF-1 and confocal microscopy. II: pH gradients within single cultured cells

Using the pH-sensitive dual-emission fluorophore, carboxy-seminaphthorhodaflour-1 (SNARF-1) coupled with a laser-scanning confocal microscope system it is now possible to map intracellular pH. We have applied this combination to investigate the pH levels and distribution within single isolated cultured proximal tubule, bovine aortic endothelial and rat aortic smooth muscle cells. The majority of cells showed a uniform cytosolic pH. However, pH gradients were evident between the cytosol and nuclear regions with the nuclear regions having a predominantly higher pH than the cytosol. High concentrations of nigericin removed this gradient and indicated that active control of intranuclear pH by the nuclear membrane occured, possibly via the K +/H + exchangers.

Confocal calcium imaging with ultraviolet laser- scanning microscopy and indo-1

We developed a newly designed ultraviolet laser-scanning confocal microscopy (UV-LSCM) system and applied it for quantitative confocal imaging of intracellular calcium concentration ([Ca 2+] i) with the dual-emission wavelength indicator indo-1. The resolution and contrast of the biological sample images obtained using the current UV system were found to be comparable to those obtained with the conventional visible LSM optics and hence the UV-transmittable optics in our LSM system employing an achromatic objective lens provides appreciable confocality in the UV range. When indo-1 is used with this UV-LSCM system, dual- imaging ratiometric measurement of [Ca 2+] i can be easily performed without requiring time-consuming geometrical decalibration of the two simultaneously obtained images. The resulting confocal images allow quantitative analysis of [Ca 2+] i in rapidly contractile cardiac cells at a high temporal resolution in line-scan and fast frame-scan modes. The combined use of the UV-LSCM and dual- emission ratiometric indicator is now practical and we anticipate its widespread application in physiological and pathological studies in living cells.

Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system

The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.

Laser scanning microscope with a differential heterodyne optical probe

A new laser scanning microscope system has been developed to observe the spatial distribution of light scattering particles or defects in a partially transparent object. The present microscope has an optical probe whose intensity is modulated by the interference effect between two crossed laser beams with slightly different frequencies. In this paper, a Zeeman laser combined with a simple polarizing optical system is used to produce two such coherent beams. Experimental results obtained by using a latex sphere and a microscale as the target show qualitatively that high image contrast is obtained by the present method even if some obscuring particles exist in front of the probe volume. Distributions of light scattering particles or defects in a LiNbO(3) and TGS single crystal can be visualized by a computer-controlled scan stage.