Line-scanning Confocal Microscopy Research Articles

Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes.

This study investigated the function of FK506-binding protein (FKBP12.6) using adenoviral-mediated gene transfer to over-express FKBP12.6 (Ad-FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a beta-galactosidase-expressing adenovirus (Ad-LacZ) was used as a control. Peak-systolic intracellular [Ca(2+)] (measured with Fura-2) was higher in the Ad-FKBP12.6 group compared to Ad-LacZ (1 Hz field stimulation at 37 degrees C). The amplitude of caffeine-induced Ca(2+) release was also greater, indicating a higher SR Ca(2+) content in the Ad-FKBP12.6 group. Voltage clamp experiments indicated that FKBP12.6 over-expression did not change L-type Ca(2+) current amplitude or Ca(2+) efflux rates via the Na(+)-Ca(2+) exchanger. Ca(2+) transients comparable to those after Ad-FKBP12.6 transfection could be obtained by enhancing SR Ca(2+) content of Ad-LacZ infected cells with periods of high frequency stimulation. Line-scan confocal microscopy (Fluo-3 fluorescence) of intact cardiomyocytes stimulated at 0.5 Hz (20-21 degrees C) revealed a higher degree of synchronicity of SR Ca(2+) release and fewer non-responsive Ca(2+) release sites in the Ad-FKBP12.6 group compared to control. Ca(2+) spark morphology was measured in beta-escin-permeabilized cardiomyocytes at a free [Ca(2+)](i) of 150 nm. The average values of the spark parameters (amplitude, duration, width and frequency) were reduced in the Ad-FKBP12.6 group. Increasing [Ca(2+)](i) to 400 nm caused coherent propagating Ca(2+) waves in the Ad-FKBP12.6 group but only limited Ca(2+) release events were recorded in the control group. These data indicate that FKBP12.6 over-expression enhances Ca(2+) transient amplitude predominately by increasing SR Ca(2+) content. Moreover, there is also evidence that FKBP12.6 can enhance the coupling between SR Ca(2+) release sites independently of SR content.

Intracellular Ca2+ measurements in live cells by rapid line scan confocal microscopy: simplified calibration methodology

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate CA2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3 AM loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretaton and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be used to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Mitochondria regulate Ca2+ wave initiation and inositol trisphosphate signal transduction in oligodendrocyte progenitors.

Mitochondria in oligodendrocyte progenitor cells (OPs) take up and release cytosolic Ca2+ during agonist-evoked Ca2+ waves, but it is not clear whether or how they regulate Ca2+ signaling in OPs. We asked whether mitochondria play an active role during agonist-evoked Ca2+ release from intracellular stores. Ca2+ puffs, wave initiation, and wave propagation were measured in fluo-4 loaded OP processes using linescan confocal microscopy. Mitochondrial depolarization, measured by tetramethyl rhodamine ethyl ester (TMRE) fluorescence, accompanied Ca2+ puffs and waves. In addition, waves initiated only where mitochondria were localized. To determine whether energized mitochondria were necessary for wave generation, we blocked mitochondrial function with the electron transport chain inhibitor antimycin A (AA) in combination with oligomycin. AA decreased wave speed and puff probability. These effects were not due to global changes in ATP. We found that AA increased cytosolic Ca2+, markedly reduced agonist-evoked inositol trisphosphate (IP3) production, and also enhanced phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the Ca2+ dependent protein gelsolin. Thus, the reduction in puff probability and wave speed after AA treatment may be explained by competition for PIP2 between phospholipase C and gelsolin. Energized mitochondria and low cytosolic Ca2+ concentration may be required to maintain PIP2, a substrate for IP3 signal transduction.

A cardiac dihydropyridine receptor II-III loop peptide inhibits resting Ca(2+) sparks in ferret ventricular myocytes.

1. We studied the effect of a peptide (Ac-10C) on cardiac ryanodine receptor (RyR) opening. This decapeptide (KKERKLARTA) is a fragment of the cardiac dihydropyridine receptor (DHPR) from the cytosolic loop between the second and third transmembrane domains (II-III loop). Studies were carried out in ferret ventricular myocytes by simultaneously applying ruptured-patch voltage clamp and line-scan confocal microscopy with fluo-3 to measure intracellular [Ca(2+)] ([Ca(2+)](i)) and Ca(2+) sparks. 2. Inclusion of Ac-10C in the dialysing pipette solution inhibited resting Ca(2+) spark frequency (due to diastolic RyR openings) by > 50 %. This occurred without changing sarcoplasmic reticulum (SR) Ca(2+) content, which was measured via the caffeine-induced Ca(2+) transient amplitude and the caffeine-induced Na(+)-Ca(2+) exchange current (I(NCX)) integral. Ac-10C also reduced slightly the size of Ca(2+) sparks. 3. Ac-10C did not alter either resting [Ca(2+)](i) (assessed by indo-1 fluorescence) or DHPR gating (measured as L-type Ca(2+) current). 4. The SR Ca(2+) fractional release was depressed by Ac-10C at relatively low SR Ca(2+) content, but not at higher SR Ca(2+) content. 5. A control scrambled peptide (Ac-10CS) did not alter any of the measured parameters (notably Ca(2+) spark frequency or SR Ca(2+) fractional release). Thus, the Ac-10C effects may be sequence or charge distribution specific. 6. Our results suggest an inhibitory regulation of RyRs at rest via the cardiac DHPR II-III loop N-terminus region. The mechanism of the effect and whether this interaction is important in cardiac excitation-contraction coupling (E-C coupling) per se, requires further investigation.

Subcellular Mechanisms of Presenilin-Mediated Enhancement of Calcium Signaling

Mutations in presenilin-1 (PS1), the leading cause of early-onset, autosomal-dominant familial Alzheimer's disease (FAD), enhance calcium signaling mediated by inositol 1,4,5-trisphosphate (IP3). To elucidate the subcellular mechanisms underlying this enhancement, we used high resolution line-scanning confocal microscopy to image elementary calcium release events (“puffs”) in Xenopus oocytes expressing wild-type or mutant PS1. Here we report that mutant PS1-rendered puffs more sensitive to IP3 and increased both the magnitude and the rate of calcium release during each event. These effects were not attributable to quantitative changes in the levels of IP3 receptors or their distribution on the ER, but were instead associated with an abnormal elevation of ER calcium stores. Together, our results suggest that the effects of mutant PS1 on calcium signaling are manifested predominantly at the level of the regulation of calcium stores rather than via perturbations in the numbers or activity of IP3-activated calcium release channels.

Fiber-coupled multiplexed confocal microscope

We describe a new parallel scanning mechanism for confocal microscopy that is inherently fiber-optic compatible and that retains the simplicity of the line scanning confocal microscope. The method works by employing an incoherent fiber-optic bundle that maps a line illumination pattern back on itself on double passing, while separating the fibers that carry photons from out-of-focus sample planes. The transformation permits efficient rejection of out-of-focus photons by a slit aperture.

Laser Line‐Scanning Confocal Fluorescence Imaging of the Photodynamic Action of Aluminum and Zinc Phthalocyanines in V79–4Chinese Hamster Fibroblasts

Confocal fluorescence microscopy, using a newly constructed laser line-scanning confocal microscope, was applied to an investigation of the early stages of photoinduced destruction of V79-4 Chinese hamster fibroblasts using aluminum and zinc phthalocyanines as photosensitizers. Results obtained in this work show that aluminum and zinc phthalocyanines, once internalized, localize in perinuclear sites that are disrupted upon light exposure resulting in fluorescence redistribution. The combination of laser-line scanning with charge-coupled device detection used in the confocal microscope developed in this work can enable rapid high-resolution sequential imaging, which is ideal for studying photoinduced intracellular fluorescence dynamics.

Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition.

1. The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA-1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (delta [Ca2+]v) were determined following single impulses, and short (5-impulse) and long (200-impulse) trains at 5 Hz. 2. All varicosities showed a significant delta [Ca2+]v in response to every single impulse. The elevated delta [Ca2+]v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 +/- 0.05 s) and a moderate phase (3.6 +/- 0.4 s). 3. Line scanning confocal microscopy revealed that the delta [Ca2+] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities. 4. Blockade of the voltage-sensitive calcium channels with Cd2+ (in calcium-free solution) completely blocked the delta [Ca2+]v on stimulation. The addition of either nifedipine (10 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin TK (100 nM) showed that 47 +/- 6% of the evoked response was mediated by N-type calcium channels. 5. Ryanodine (10 microM) did not significantly change the amplitude of delta [Ca2+]v in response to short trains. 6. Spontaneous increases in delta [Ca2+]v were observed in individual varicosities, with coupling in the increase of delta [Ca2+]v between varicosities. 7. The presynaptic alpha 2-receptor antagonist yohimbine (10 microM) increased the amplitude of delta [Ca2+]v in response to five impulses (5 Hz) by 54 +/- 14%, while the alpha 2-receptor agonist clonidine (1 microM) decreased the delta [Ca2+]v by 55 +/- 4%. 8. These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual delta [Ca2+]v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.