Confocal Line Scanning Research Articles

Confocal Theta Line-Scanning Microscope for Imaging Human Tissues in vivo

Confocal Theta Line-Scanning Microscope for Imaging Human Tissues in vivo

‘Trigger’ Events Precede Calcium Puffs in Xenopus Oocytes

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP 3Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca 2+ puffs believed to arise from several to tens of clustered IP 3Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca 2+ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca 2+ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP 3 receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP 3Rs within the cluster by a regenerative process of Ca 2+-induced Ca 2+ release. Puff amplitudes (fluorescence ratio change) are on average ∼6 times greater than that of the triggers, suggesting that at least six IP 3Rs may simultaneously be open during a puff. Trigger events have average durations of ∼12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is ∼3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 μm). All these parameters were relatively independent of IP 3 concentration, although the proportion of puffs showing resolved triggers was greatest (∼80%) at low [IP 3]. Because Ca 2+ puffs constitute the building blocks from which cellular IP 3-mediated Ca 2+ signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP 3Rs within clusters.

The Number and Spatial Distribution of IP 3 Receptors Underlying Calcium Puffs in Xenopus Oocytes

Calcium puffs are local Ca 2+ release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP 3Rs) and serve as a basic “building block” from which global Ca 2+ waves are generated. Important questions remain as to the number of IP 3Rs that open during a puff, their spatial distribution within a cluster, and how much Ca 2+ current flows through each channel. The recent discovery of “trigger” events—small Ca 2+ signals that immediately precede puffs and are interpreted to arise through opening of single IP 3R channels—now provides a useful yardstick by which to calibrate the Ca 2+ flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca 2+ release in two sequential stages; representing the trigger by the opening of a single IP 3R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP 3Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca 2+ and Ca 2+-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca 2+ buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25–35 IP 3Rs, each carrying a Ca 2+ current of ∼0.4 pA, with the channels distributed through a cluster 300–800 nm in diameter.

Faster than real-time: confocal linescan systems provide ideal conditions for millisecond-resolution physiological imaging

The LSM 5 LIVE is a new design for fast confocal imaging, which acquires images at extremely high frame rates and allows experiments not possible in the past, while maintaining the advantages of conventional scanning systems, such as adjustable confocal apertures and scalable frame formats with pan and zoom. Additionally, the LSM 5 LIVE offers fast and sensitive parallel acquisition with charge-coupled device (CCD) detectors for high quantum efficiency.

Use of confocal linescan to document ciliary beat frequency

We present a method to document ciliary beat frequency with the linescan function of a scanning confocal microscope, using ciliated tracheal cells and free-swimming rotifers as examples. Depending on the clarity of the original data, the ciliary beat frequency can be determined from the confocal linescan directly or from an intensity linescan analysis of the original data. Fast Fourier transform treatment of the data can be used to verify the derived ciliary beat frequency. The linescan approach allows analysis of simple ciliary movements displayed by the ciliated tracheal cells, as well as complex movements performed by free-swimming rotifers while feeding.

No Apparent Requirement for Neuronal Sodium Channels in Excitation-Contraction Coupling in Rat Ventricular Myocytes

The majority of Na channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2 to 6 micromol/L) "cardiac" NaV1.5 isoform; however, TTX-sensitive (KD, 1 to 25 nmol/L) "neuronal" Na channel isoforms have recently been detected in several cardiac preparations. In the present study, we determined the functional subcellular localization of Na channel isoforms (according to their TTX sensitivity) in rat ventricular myocytes by recording INa in control and detubulated myocytes. We found that TTX-sensitive INa (KD, &8.8 nmol/L) makes up 14+/-3% of total INa in control and < or =4% in detubulated myocytes and calculated that &80% of TTX-sensitive INa is located in the t-tubules, where it generates &1/3 of t-tubular INa. In contrast, TTX-resistant INa is located predominantly (&78%) at the surface membrane. We also investigated the possible contribution of TTX-sensitive INa to excitation-contraction coupling, using 200 nmol/L TTX to selectively block TTX-sensitive INa. TTX decreased the rate of depolarization of the action potential by 10% but did not delay the rise of systolic Ca2+ in the center of the cell (transverse confocal line scan), suggesting that TTX-sensitive INa does not play a role in synchronizing Ca2+ release at the t-tubules; the amplitude of the Ca2+ transient and contraction were also unchanged by 200 nmol/L TTX. The quantity of charge entering via ICa elicited by control or TTX action potential waveforms was similar, suggesting that the trigger for Ca2+ release is not altered by blocking TTX-sensitive INa. We conclude that neuronal INa is concentrated at the t-tubules, but there is no evidence of a requirement for these channels in normal excitation-contraction coupling in ventricular myocytes.

Alterations in Early Action Potential Repolarization Causes Localized Failure of Sarcoplasmic Reticulum Ca 2+ Release

Depressed contractility of failing myocytes involves a decreased rate of rise of the Ca2+ transient. Synchronization of Ca2+ release from the junctional sarcoplasmic reticulum (SR) is responsible for the rapid rise of the normal Ca2+ transient. This study examined the idea that spatially and temporally dyssynchronous SR Ca2+ release slows the rise of the cytosolic Ca2+ transient in failing feline myocytes. Left ventricular hypertrophy (LVH) with and without heart failure (HF) was induced in felines by constricting the ascending aorta. Ca2+ transients were measured in ventricular myocytes using confocal line scan imaging. Ca2+ transients were induced by field stimulation, square wave voltage steps, or action potential (AP) voltage clamp. SR Ca2+ release was significantly less well spatially and temporally synchronized in field-stimulated HF versus control or LVH myocytes. Surprisingly, depolarization of HF cells to potentials where Ca2+ currents (ICa) were maximal resynchronized SR Ca2+ release. Correspondingly, decreases in the amplitude of ICa desynchronized SR Ca2+ release in control, LVH, and HF myocytes to the same extent. HF myocytes had significant loss of phase 1 AP repolarization and smaller ICa density, which should both reduce Ca2+ influx. When normal myocytes were voltage clamped with HF AP profiles SR Ca2+ release was desynchronized. SR Ca2+ release becomes dyssynchronized in failing feline ventricular myocytes because of reductions in Ca2+ influx induced in part by alterations in early repolarization of the AP. Therefore, therapies that restore normal early repolarization should improve the contractility of the failing heart.

Calcium sparks in muscle cells: interactive procedures for automatic detection and measurements on line-scan confocal images series

In muscle cells, force development is controlled by Ca2+ ions, which are rapidly released from the sarcoplasmic reticulum (SR) during sarcolemmal depolarization. In addition to this synchronized spatially homogeneous calcium signal, localized quantal Ca2+ release events (sparks) have been recorded using laser scanning confocal fluorescence microscopy. Previously, algorithms without user intervention have been developed to automatically detect and analyse sparks on confocal line-scan (space-time: 512 x 512 pixels) single images. Here we present a computer program that we called "HARVest of Elementary Events" (HARVELE) developed to both analyse events on series of confocal images and follow sparks morphology from one site during several seconds. HARVELE, coded in the image-processing language IDL 5.3., can be applied on series of n images (512 x 512 x n) obtained from the same scanning line. Computing simultaneously entire series of images allows to measure, for each release site, the frequency and the morphology of release with the conventional amplitude, size, time and duration parameters defined for sparks analysis. The use of these procedures rapidly provides much information about the properties of calcium release sites in muscle cells population and can be applied on any elementary calcium events whatever the type of cell.

Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events.

The feasibility of determining localized Ca2+ influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca2+ fluorescence transients (SCCaFTs), due to Ca2+ entry through single openings of Ca2+-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (ΔFtotal or “signal mass”) associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca2+ influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that ΔFtotal is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca2+ influx and ΔFtotal was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca2+ was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca2+ current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca2+ current underlying Ca2+ sparks resulting from Ca2+ release from intracellular stores via ryanodine receptors in the same preparation.

The elementary events of Ca2+ release elicited by membrane depolarization in mammalian muscle.

Cytosolic [Ca(2+)] transients elicited by voltage clamp depolarization were examined by confocal line scanning of rat skeletal muscle fibres. Ca(2+) sparks were observed in the fibres' membrane-permeabilized ends, but not in responses to voltage in the membrane-intact area. Elementary events of the depolarization-evoked response could be separated either at low voltages (near -50 mV) or at -20 mV in partially inactivated cells. These were of lower amplitude, narrower and of much longer duration than sparks, similar to 'lone embers' observed in the permeabilized segments. Their average amplitude was 0.19 and spatial half-width 1.3 microm. Other parameters depended on voltage. At -50 mV average duration was 111 ms and latency 185 ms. At -20 mV duration was 203 ms and latency 24 ms. Ca(2+) release current, calculated on an average of events, was nearly steady at 0.5-0.6 pA. Accordingly, simulations of the fluorescence event elicited by a subresolution source of 0.5 pA open for 100 ms had morphology similar to the experimental average. Because 0.5 pA is approximately the current measured for single RyR channels in physiological conditions, the elementary fluorescence events in rat muscle probably reflect opening of a single RyR channel. A reconstruction of cell-averaged release flux at -20 mV based on the observed distribution of latencies and calculated elementary release had qualitatively correct but slower kinetics than the release flux in prior whole-cell measurements. The qualitative agreement indicates that global Ca(2+) release flux results from summation of these discrete events. The quantitative discrepancies suggest that the partial inactivation strategy may lead to events of greater duration than those occurring physiologically in fully polarized cells.

Reduced synchrony of Ca2+ release with loss of T-tubules-a comparison to Ca2+ release in human failing cardiomyocytes.

During cardiac excitation-contraction coupling, Ca2+ release from the sarcoplasmic reticulum (SR) occurs at the junctional complex with the T-tubules, containing the L-type Ca2+ channels. A partial loss of T-tubules has been described in myocytes from failing canine and human hearts. We examined how graded reduction of T-tubule density would affect the synchrony of Ca2+ release. Adult pig ventricular myocytes were isolated and cultured for 24 and 72 h. T-tubules, visualized with di-8-ANEPPS, and [Ca2+]i transients (Fluo-3) were recorded during confocal line scan imaging. Cultured cardiomyocytes exhibited a progressive reduction in T-tubule density. [Ca2+]i transients showed small areas of delayed Ca2+ release which gradually increased in number and size with loss of T-tubules. Local [Ca2+]i transients in the delayed regions were reduced. Due to these changes, loss of T-tubules resulted in an overall slowing of the rise of [Ca2+] along the entire line scan and transient magnitude tended to be reduced, but there was no change in SR Ca2+ content. Human myocytes isolated from failing hearts had a T-tubule density comparable to that of freshly isolated pig myocytes. The size, but not the number, of delayed release areas tended to be larger. The overall rate of rise of [Ca2+]i was significantly faster than in pig myocytes with low T-tubule density. Loss of T-tubules reduces the synchrony of SR Ca2+ release. This could contribute to reduced efficiency of excitation-contraction coupling in heart failure, though dyssynchrony in human failing cells appears to be modest.

Rhythmic Ryanodine Receptor Ca 2+ Releases During Diastolic Depolarization of Sinoatrial Pacemaker Cells Do Not Require Membrane Depolarization

Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.

FPL-64176 alters both charge movement and Ca2+ release properties in amphibian muscle fibres.

A number of recent reports have suggested that ryanodine receptor (RyR)-Ca2+ release channels are gated by tubular depolarization in skeletal muscle through their direct coupling to intramembrane dihydropyridine receptor (DHPR)-voltage sensors. The qgama charge movement, which is inhibited by DHPR antagonists, is often regarded as the electrical signature for the voltage sensing process, yet pharmacological modifications of the RyR produce reciprocal upstream kinetic effects on an otherwise conserved qgamma charge. This study investigates the effect of DHPR-specific agonists upon intramembrane charge and the release of intracellularly stored Ca2+. We empirically demonstrate kinetic effects of FPL-64176 upon charge movements that closely resemble the consequences of previous interventions directed instead at the RyR. Increases in extracellular FPL-64176 concentration from 10 to 40 microM converted delayed qgamma transients to monotonic decays indistinguishable from the exponential qbeta current component. Yet total steady-state intramembrane charge and the steepness of its dependence upon test potential closely resembled previous reports from untreated fibres. These changes accompanied an appearance of transient cytosolic [Ca2+] elevations in confocal line-scans in fluo-3-loaded fibres studied in 10mM K+ and 40, but not 10 microM, FPL-64176 that resembled elementary Ca2+ release events ('sparks'). Pharmacological manipulations of the DHPR whose effects on intramembrane charge resembled those from manoeuvres directed at the RyR can thus produce downstream effects upon Ca2+ release.

Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells.

1. The distribution and localization of Ca2+ transients and Ca2+ sparks in isolated adult rabbit Purkinje cells were examined using confocal microscopy and the Ca2+ indicator fluo-3. 2. When cells were field stimulated in 2.0 mM Ca2+ buffer, a transverse confocal line scan (500 Hz) showed that the fluorescence intensity was greatest at the cell periphery during the onset of the Ca2+ transient ([Ca2+]i). In contrast, the [Ca2+]i of ventricular cells showed a more uniform pattern of activation across the cell. Staining with di-8-ANEPPS revealed that Purkinje cells lack t-tubules, whereas ventricular cells have an extensive t-tubular system. 3. When we superfused both cell types with a buffer containing 5 mM Ca2+-1 microM isoproterenol (isoprenaline) they produced Ca2+ sparks spontaneously. Ca2+ sparks occurred only at the periphery of Purkinje cells but occurred throughout ventricular cells. Sparks in both cell types could be completely abolished by addition of the SR inhibitor thapsigargin (500 nM). Brief exposure to nifedipine (10 microM) did not reduce the number of spontaneous sparks. 4. Immunofluorescence staining of Purkinje cells with anti-ryanodine antibody revealed that ryanodine receptors (RyRs) are present at both peripheral and central locations. 5.Computer simulations of experiments in which the calcium transient was evoked by voltage clamp depolarizations suggested that the increase in calcium observed in the centre of the cell could be explained by simple buffered diffusion of calcium. These computations suggested that the RyRs deep within the cell do not contribute significantly to the calcium transient. 6. These results provide the first detailed, spatially resolved data describing Ca2+ transients and Ca2+ sparks in rabbit cardiac Purkinje cells. Both types of events are initiated only at subsarcolemmal SR Ca2+ release sites suggesting that in Purkinje cells, Ca2+ sparks only originate where the sarcolemma and sarcoplasmic reticulum form junctions. The role of the centrally located RyRs remains unclear. It is possible that because of the lack of t-tubules these RyRs do not experience a sufficiently large Ca2+ trigger during excitation-contraction (E-C) coupling to become active.

Local and cellular Ca2+ transients in smooth muscle of pressurized rat resistance arteries during myogenic and agonist stimulation.

1. Confocal laser scanning microscopy was used to visualize Ca2+ transients in the vascular smooth muscle cells (VSMC) of intact, pressurized rat mesenteric resistance arteries loaded with fluorescent calcium indicators. Vasoconstriction was assessed by measuring inner arterial diameter. All arteries were studied at 70 mmHg intralumenal pressure and 37 C. 2. In the control condition of myogenic tone the arteries were constricted to 62 % (n = 10) of their passive diameter (p.d.). The [Ca2+]i in most VSMC of these arteries was constant over time. In a small percentage (< 10 %) of cells in each artery, [Ca2+]i oscillated regularly. Local calcium transients (Ca2+ sparks) were observed in five arteries studied with confocal linescan imaging. 3. Activation of alpha-adrenoceptors by phenylephrine (PE, 1.0 microM) induced further vasoconstriction of pressurized arteries (to 27 % of p.d.). In this condition, [Ca2+]i oscillations were prominent in a large percentage (83 %) of the VSMC. The Ca2+ oscillations ranged in frequency from 4 to 22 min-1, and were usually asynchronous between cells. 4. High [KCl]o (65 mM) induced nearly comparable vasoconstriction to PE (37 % of p.d.) but [Ca2+]i oscillated in only about 13 % of cells in each artery. 5. Block of L-type Ca2+ channels (with nifedipine) in arteries activated by PE caused nearly full vasodilatation, but did not abolish the Ca2+ oscillations. Subsequent block of the sarcoplasmic reticulum Ca2+ pump (with cyclopiazonic acid) abolished Ca2+ oscillations in all cells. 6. We conclude that Ca2+ entering VSMC via L-type Ca2+ channels has an obligatory role in force development, both in myogenic tone and during alpha1-adrenoceptor activation. The oscillatory pattern of [Ca2+]i that persists in the absence of Ca2+ entry via L-type Ca2+ channels is ineffective in activating contraction.

Differential Regulation of Skeletal Muscle L-Type Ca 2+ Current and Excitation-Contraction Coupling by the Dihydropyridine Receptor β Subunit

The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca 2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR β subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR β subunit. These β-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of β-null cells with cDNAs encoding for either the homologous β 1a subunit or the cardiac- and brain-specific β 2a subunit fully restored the L-type Ca 2+ current (161 ± 17 pS/pF and 139 ± 9 pS/pF, respectively, in 10 mM Ca 2+). We compared the Boltzmann parameters of the Ca 2+ conductance restored by β 1a and β 2a, the kinetics of activation of the Ca 2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing β 2a was significantly lower than in cells expressing β 1a (2.7 ± 0.2 nC/ μF and 6.7 ± 0.4 nC/ μF, respectively). Furthermore, the amplitude of Ca 2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing β 2a. In summary, DHPR complexes that included β 2a or β 1a restored L-type Ca 2+ channels. However, a DHPR complex with β 1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the β 1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.

Amplitude Distribution of Calcium Sparks in Confocal Images: Theory and Studies with an Automatic Detection Method

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.

Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal linescan imaging.

The radial localization and properties of elementary calcium release events ("puffs") were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 micrometer. Weak photorelease of caged inositol 1,4,5-trisphosphate (InsP3) evoked puffs arising predominantly within a 6-micrometer thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

Theoretical Analysis of the Ca 2+ Spark Amplitude Distribution

A difficulty of using confocal microscopy to study Ca 2+ sparks is the uncertainty of the linescan position with respect to the source of Ca 2+ release. Random placement of the linescan is expected to result in a broad distribution of measured Ca 2+ spark amplitudes ( a) even if all Ca 2+ sparks were generated identically. Thus variations in Ca 2+ spark amplitude due to positional differences between confocal linescans and Ca 2+ release site are intertwined with variations due to intrinsic differences in Ca 2+ release properties. To separate these two sources of variations on the Ca 2+ spark amplitude, we determined the effect changes of channel current or channel open time—collectively called the source strength, α—had on the measured Ca 2+ spark amplitude histogram, N( a). This was done by 1) simulating Ca 2+ release, Ca 2+ and fluo-3 diffusion, and Ca 2+ binding reactions; 2) simulation of image formation of the Ca 2+ spark by a confocal microscope; and 3) using a novel automatic Ca 2+ spark detector. From these results we derived an integral equation relating the probability density function of source strengths, f α ( α), to N( a), which takes into account random positional variations between the source and linescan. In the special, but important, case that the spatial distribution of Ca 2+-bound fluo-3 is Gaussian, we show the following: 1) variations of Ca 2+ spark amplitude due to positional or intrinsic differences can be separated, and 2) f α ( α) can, in principle, be calculated from the Ca 2+ spark amplitude histogram since N( a) is the sum of shifted hyperbolas, where the magnitudes of the shifts and weights depend on f α ( α). In particular, if all Ca 2+ sparks were generated identically, then the plot of 1/ N( a) against a will be a straight line. Multiple populations of channels carrying distinct currents are revealed by discontinuities in the 1/ N( a) plot. 3) Although the inverse relationship between Ca 2+ spark amplitude and decay time might be used to distinguish Ca 2+ sparks from different channel populations, noise can render the measured decay times meaningless for small amplitude Ca 2+ sparks.

A continuum of InsP3-mediated elementary Ca2+ signalling events in Xenopus oocytes.

1. The elementary release events underlying inositol 1,4, 5-trisphosphate (InsP3)-mediated calcium signalling were investigated in Xenopus oocytes by means of high-resolution confocal linescan imaging together with flash photolysis of caged InsP3. 2. Weak photolysis flashes evoked localized, transient calcium signals that arose at specific sites following random latencies of up to several seconds. The duration, spatial spread and amplitude of these elementary events varied widely. Event durations (at half-maximal amplitude) were distributed exponentially between about 100 and 600 ms. Fluorescence magnitudes (F/F0 of Oregon Green 488 BAPTA-1) showed a skewed distribution with a peak at about 1.5 and a tail extending as high as 3.5. 3. Individual release sites exhibited both small events (blips) and large events (puffs). The spatiotemporal distribution of calcium signals during puffs was consistent with calcium diffusion from a point source (< a few hundred nanometres), rather than with propagation of a microscopic calcium wave. 4. Estimates of the calcium flux associated with individual events were made by integrating fluorescence profiles along the scan line in three dimensions to derive the 'signal mass' at each time point. The smallest resolved events corresponded to liberation of < 2 x 10-20 mol Ca2+, and large events to about 2 x 10-18 mol Ca2+. The rise of signal mass was more prolonged than that of the fluorescence intensity, suggesting that calcium liberation persists even while the fluorescence begins to decline. Rates of rise of signal mass corresponded to Ca2+ currents of 0.4-2.5 pA. 5. Measurements of signal mass from different events showed a continuous, exponential distribution, arising through variability in magnitude and duration of calcium flux. 6. We conclude that localized calcium transients in the oocyte represent a continuum of events involving widely varying amounts of calcium liberation, rather than falling into separate populations of 'fundamental' and 'elementary' events (blips and puffs) involving, respectively, single and multiple InsP3 receptor channels. This variability probably arises through stochastic variation in both the number of channels recruited and the duration of channel opening.