Recently, an ultrafast random access two-photon microscope (Sacconi et al., 2012) in combination with novel fluorinated voltage sensitive dyes with improved photostability (Yan et al., 2012), has been used to measure APs at multiple sites within the T-tubular system of a cardiomyocyte with submillisecond temporal and submicrometer spatial resolution in real time. The noise in such measurements is generally dominated by the shot noise caused by the limited number of photons being detected. For that reason, it is useful to use a dye with high photostability that can produce more photons before it is bleached, and therefore, can produce fluorescence with intrinsically better signal-to-noise ratio.

With this approach, it was possible to observe that the tight electrical coupling between different membrane domains is guaranteed only when the tubular system is intact. In fact, the AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. These findings provide insights on the relationship between abnormal TATS and asynchronous Ca²⁺ release, a major determinant of cardiac contractile dysfunction and arrhythmias. A simultaneous recording of local Ca²⁺ release and AP can be useful to unravel the consequences of electrical anomalies on the intracellular Ca²⁺ dynamics. To address this challenge, an experimental method capable of optically recording APs in several tubular elements and, simultaneously, the corresponding local Ca²⁺ transients has been recently developed (Crocini et al., 2014). Figure 1 shows an example of an optical recording of voltage and Ca²⁺ release in six different positions across the sarcolemma. In this experiment an isolated rat cardiomyocytes was stained with FluoForte GFP-certified, a Ca²⁺ indicator, and di-4-AN(F)EPPTEA. The random access multi-photon microscope (Figure 1A) was used to simultaneously excite both dyes while band-pass filters were used to select the two distinct spectral ranges of the overlapping fluorescence spectra. The acousto-optic deflectors rapidly scan lines on different membrane segments with a commutation time of 4 μ s between a line and the next. The integration time on each line is about 0.1 ms, leading to a temporal resolution of the order of 0.6 ms for acquiring 6 membrane domains. At the end of one cycle the acousto-optic deflectors return to the initial position and repeats the described cycle, thus probing each site every 0.6 ms. Then, a spectral unmixing procedure was applied to properly uncouple the Ca²⁺ and the voltage signals. As shown in Figure 1, the sensitivity of this optical method is sufficient to detect the presence of an AP and to assess the temporal features of Ca²⁺ transients in real time. The power of this methodology has been demonstrated exploring the role of single TATS elements in determining the correspondent local Ca²⁺ transients as well as a new pro-arrhythmogenic mechanism (Crocini et al., 2014). In fact, the capability of the system in detecting AP and local Ca²⁺ release simultaneously in multiple sites may solve unanswered questions about spatio-temporal relationship between TATS electrical activity and Ca²⁺ release in heart failure.

The next generation of imaging systems can be also implemented with additional ultrafast scanning head for local Ca²⁺ release and/or electrical stimulation. Local Ca²⁺ release can be performed using specific caged compounds (Gurney et al., 1989; Ji et al., 2006), i.e., compounds that release Ca²⁺ at the site of action upon photons absorption and following cleavage of “caged groups.” The photochemical reaction can be very fast, with release of the active species often completed within less than a millisecond. These photochemical reactions can be also induced by two-photon excitation producing highly localized fast Ca²⁺ transients (Rubart, 2004). On the other hand, the electrical stimulation can be achieved by using light-gated ion channels that can be transfected in the plasma membrane of the sample. For example, the photosensitive protein channelrhodopsin-2 (ChR2) has been recently used for photo-stimulating excitable cells (Arrenberg et al., 2010). In fact, under blue light illumination, the cations flux generated through ChR2 produces a rapid membrane depolarization, which can evoke an AP. The two-photon excitation to stimulate ChR2 is more challenging than Ca²⁺ uncaging, mainly due to the low conductance and the long lifetime of the conducting states of ChR2 (Rickgauer and Tank, 2009; Andrasfalvy et al., 2010; Papagiakoumou et al., 2010). In fact, the low conductance and long lifetime make difficult to drive APs by photoactivation with the standard small two-photon excitation volume even by increasing the excitation power. Recent studies suggest that promising non-linear illumination strategies to stimulate ChR2 are based on low excitation density spread over a relatively large excitation area. These requirements could be easily fulfilled using ultra-fast scanning to generate multiplexed excitation patterns that spread the illumination light across an extended area. Furthermore, improved optogenetic tools can now be easily combined with two-photon excitation, allowing light stimulation within single cells (Oron et al., 2012). These new optical implementations will be extremely useful to assess the properties of arrhythmogenic Ca²⁺ waves and to characterize the electrical defects of the tubular system in diseased myocytes. For instance, the rate of spontaneous T-tubular depolarization is rather low (Sacconi et al., 2012), thus a comprehensive assessment of their arrhythmogenic potential is difficult. A direct stimulation of remodeled TATS in cells expressing ChR2 (simulating a spontaneous depolarization) will help understanding whether localized spontaneous T-tubular events play a role in generating or modulating cellular arrhythmias (Ca²⁺ waves and DADs). Moreover, the ability of ultra-fast scanning to stimulate multiple distant TATS regions within the cell will help understanding the amount of single spontaneous events that are required to generate a cell-spanning Ca²⁺ wave.

As discussed above, Ca²⁺ waves propagation is caused by local Ca²⁺ release from the SR, followed by diffusion and subsequent activation of adjacent SR Calcium Release units, generating a cascade of events. Ca²⁺ waves are accompanied by DADs (Ter Keurs and Boyden, 2007). Since the average ventricular myocyte is directly coupled to about 11 other myocytes, an afterdepolarization would be immediately suppressed by the large “current sink” unless a sufficient number of neighboring cardiomyocytes also synchronously develop an afterdepolarization at the same beat. Propagation of Ca²⁺ waves from cell-to-cell has been shown to occur at a constant velocity (V_prop) that ranged from 0.1 to 15 mm/s, a speed that is too slow to be determined by active (1 m/s) or electrotonic membrane conduction (0.1 m/s) (Ter Keurs and Boyden, 2007) but is compatible with a facilitated Ca²⁺ diffusion. Mechanisms underlying Ca²⁺ waves propagation are still poorly understood. However, V_prop can increase under certain circumstances, such as increased RyR open probability or in presence of SR Ca²⁺ overload. The faster the V_prop the higher the probability that a large area would “nearly simultaneously” be invaded by a propagated Ca²⁺ wave, thus increasing the likelihood of “synchronized” DADs in adjacent cells. Exploiting the advantage of multi-photon excitation, multisite optical recordings can also be performed in multicellular preparation such as intact trabeculae and muscle bundles in which cell-to-cell conduction occurs through sarcolemmal gap junctions (Sacconi et al., 2012; Ferrantini et al., 2014). Figure 2 shows examples of APs recorded in SS and TATS of two adjacent cells. Trabeculae were locally stimulated in a region 2 mm apart from the recording area (Figure 2A), and an 15 ms delay was found between the stimulus and the rapid AP upstroke phase (Figure 2D), in agreement with the expected myocardium conduction velocity at room temperature.

Following a similar approach described above the same excitation wavelength can be used to simultaneously detect voltage and Ca²⁺ in different cells, opening new perspectives for the investigation of inter-cellular propagation of Ca²⁺ waves. On the other hand, the probability to observe a spontaneous Ca²⁺ wave within the limited field of view of laser scanning microscopy is extremely low. Thus, the implementation of a multi-photon Ca²⁺ uncaging system based on random access scanner can be used to induce local elevation of intracellular Ca²⁺ in different position within the scanned region. For example this approach can be exploited to explore how Ca²⁺ perturbation in a single myocyte affects Ca²⁺ propagation to the neighboring cells and how RyR open probability and cytosolic Ca²⁺ buffering affect V_prop and DADs “synchronicity” between neighboring cells. We could assess whether premature local elevation of Ca²⁺ in a single cell is able to trigger Ca²⁺ release in the neighboring cells, thus synchronizing Ca²⁺ release to generate a propagated Ca²⁺ wave. Moreover, by inducing a Ca²⁺ wave during regular pacing, we could study how unregulated Ca²⁺ release affects electrical propagation and whether this facilitates the formation of a re-entry circuit or other forms of sustained arrhythmias. Pharmacological interventions such as low dose of caffeine (to increase RyR open probability) or cyclopiazonic acid (to decrease Ca²⁺ buffering) can be administrated to enhance V_prop. Conversely, pharmacological blockers of cardiac RyR, such as JTV519 (Elliott et al., 2011), could be employed to test the consequeces of RyR desensistization on spontaneous wave propagation. Finally, multicellular preparation dissected from mice carrying mutations of the cardiac RyR with intrinsically higher or lower open probability (Liu et al., 2006; Jones et al., 2008) could be also employed to elucidate how changes of RyR open probability affect local Ca²⁺ dynamics.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.