Two-Photon Photolysis and Two-Photon Fluorescence Photobleaching Recovery
Photolabile "caged" compounds are biologically inert precursors of active molecules that, when irradiated, free the active species at the site of action. The photochemical reaction can be very fast, with release of the active species often complete within less than a millisecond. For example, liberation of calcium ions from its photolyzable chelator DM-nitrophen generates Ca2+ concentration jumps at the site of photoactivation which in turn can mediate a wide range of Ca2+-dependent processes such as gating of ion channels or modulation of Ca2+-sensitive enzymes.8,44,45 Highly localized, fast [Ca2+]i transients (eg, Ca2+ sparks46) have been shown to play distinct roles in the regulation of variety of Ca2+-dependent cellular processes. The availability of an uncaging technique with high spatial and temporal resolution would facilitate the probing of these Ca2+ microdomains. Similarly, localized photorelease of caged neurotransmitters would enable the study of single or small clusters of agonist-gated ion channels and their distribution and behavior in intact tissue. Although the lateral dimension of a tightly focused UV-laser beam in the plane of focus can be in the submicron range, the axial resolution is comparatively poor because of extension of the conically shaped excitation beam above and below the plane of focus, resulting in photoactivation throughout elongated cellular regions (see Figure 1a).47 One way to perform photolysis on a much smaller spatial, ie, subcellular, scale and at the same time maintain the high temporal resolution of conventional photolysis is TPE-uncaging. Neglecting excitation saturation, the dependence of TPE of a caged compound on the second power of laser intensity (see Equation 1) limits photoactivation to small volumes around the focal point, giving this technique its inherent three dimensional resolution. Using TPE with high numerical aperture objectives and near-infrared light from a Ti:Sapphire laser, it has been shown that rapid (within from its cages can be confined to less than femtoliter volumes, with 1 µm resolution along the z axis.7 Quantitative approaches to predict the amount of caged compound released in a given photolysis experiment and to predict the temporal behavior of the unleashed compound have been published.7,31 Among other variables, these calculations require the knowledge of the TPE action uncaging cross section, the product of the TPE absorption cross section and the quantum yield. Action cross section values for only a small number of cages have been determined previously and are usually
Numerous previous studies have exploited the true three-dimensionally resolved excitation of cages using two-photon absorption. Denk was the first to show that the highly localized liberation of carbamyolcholine from its bath-applied cage by two-photon absorption-mediated photoactivation can be used in conjunction with whole-cell current measurements to determine the distribution of functional nicotinic acetylcholine receptors in the cell membrane of cultured BH3 cells.30 Matsuzaki et al went on to use two-photon uncaging of glutamate in hippocampal slices as a means to achieve rapid, reproducible and fine three-dimensional spatial control of neurotransmitter concentration on a scale that faithfully mimics quantal release at the individual synapse.31 In another study, transfer of uncaged fluorescein between neighboring fiber cells in the periphery of the ocular lens was shown to be highly anisotropic and to occur predominately in radial direction, whereas transport appeared more isotropic in the central portion of the lens and occurred across cell columns (Figure 4c and 4d).48 The observed directionality of intercellular dye transfer was explained by a redistribution of connexin46 gap junctions along the radial axis, with clustering at the broad site of cells in the periphery and a more dispersed appearance in cells at the center (Figure 4a and 4b). Finally, TPE uncaging of Ca2+ has become an important tool in probing the role of microdomain Ca2+ in the regulation of Ca2+-sensitive cellular processes. Mulligan and MacVicar determined the impact of changes in [Ca2+]i in astrocytes on the diameter of nearby small arterioles in brain slices by using two-photon Ca2+ uncaging to increase [Ca2+]i.49 They were able to demonstrate that vascular constrictions occurred when Ca2+ waves evoked by uncaging propagated into the astrocyte endfeet, where they triggered the release of vasoactive substances. DelPrincipe et al showed that Ca2+ signals in isolated cardiomyocytes resulting from TPE-mediated focal release of Ca2+ shared many similarities with cardiac Ca2+ sparks,8 the highly localized transient elevations of intracellular calcium resulting from the coordinated opening of a small number of colocalized SR ryanodine receptors (Figure 4e).46 When these local Ca2+ signals were superimposed on global increases in Ca2+, their amplitude decreased initially, followed by a gradual recovery (Figure 4f and 4g). These results indicate that global Ca2+ signals can result in refractoriness of the local Ca2+-induced Ca2+ release process. Soeller and Cannell were also able to produce small repeatable calcium release events using DM-nitrophen in isolated cardiomyocytes, which had spatial and temporal properties very similar to those of naturally occurring Ca2+ sparks.21 In a subsequent study from the same group, artificial sparks with known underlying SR Ca2+ release fluxes were used to validate various numerical approaches to derive SR Ca2+ release flux underlying Ca2+ sparks and, thus, the number of SR ryanodine receptors contributing to a Ca2+ spark.9
Two-photon fluorescence photobleaching recovery is a technically related approach to study the three-dimensional mobility of fluorescent molecules with three-dimensional resolution at a micrometer scale.11 Short-duration TPE of fluorescent molecules with intense laser light photobleaches a fraction within the ellipsoidal TPE volume of known dimensions (see Figures 1 and 2). As unbleached fluorescent molecules diffuse in, the fluorescence is monitored with a lower laser power to measure fluorescence recovery. From the time course of fluorescence recovery, the diffusion coefficient of the fluorescent molecule can be measured. Svoboda et al used this technique to study the diffusional exchange between dendritic spines and shafts of CA1 neurons in rat hippocampal slices.10 Stroh et al determined the diffusion coefficient of rhodamine-conjugated nerve growth factor in solution using TPE fluorescence photobleaching.50 A detailed description of the mathematical equations used to fit recovery curves and to derive diffusion coefficients has been published.11
Two-photon uncaging and two-photon fluorescence photobleaching recovery have many potential applications to the cardiovascular system. The temporal and three-dimensional spatial resolutions of two-photon uncaging are adequate for mediating a wide range of intra- and intercellular events. For example, a previous in vitro study showed that cardiac fibroblasts linking strands of cardiomyocytes are capable of relaying electrical activity between strands of cardiomyocytes through electrotonic interactions.51 Impulse propagation across fibroblast strands was slow and involved gap junctions that were composed of both connexin43 and connexin45. The important question has remained as to whether functional coupling between cardiomyocytes and cardiac fibroblasts actually occurs in vivo, such as, for example, at the interface of scar tissue and surviving myocardium in the infarct border zone. Redistribution of connexin43 immune reactivity has been shown to occur in the diseased myocardium.52,53 Two-photon uncaging of gap junction permeable fluorescent compounds (eg, fluorescein) in combination with the optical sectioning properties of TPE microscopy could be exploited to directly probe the functional communication between heterologous cells in the heart and to image the functional consequences of gap-junction remodeling, respectively. Microscopic intracellular Ca2+ transients have been shown to be involved in a variety of cellular events, such as transcription.54 The capability of TPE uncaging to reproducibly generate highly localized Ca2+ elevations both in the nucleus and cytoplasm can thus be used for quantitating the relationship between frequency, amplitude, localization, and duration of local Ca2+ elevations and transcription. The availability of caged second messengers (eg, IP3, cGMP, etc) and neurotransmitters (eg, glutamate) can be exploited toward a better understanding of functional compartmentalization as well as distribution and sensitivity of agonist-activated ion channels in the cardiovascular system, respectively. Toward this end, new cages with larger two-photon absorption action cross sections and with a greater variety of active species need to be developed to further enhance this unique and powerful tool. Finally, the technique of photobleaching recovery can be exploited to determine the mobility of biologically important ions (Na+, Ca2+) and molecules in cardiovascular cells.
TPE Fluorescence Imaging Deep Within Scattering Tissue
The combination of improved tissue penetration depth and true three-dimensionally resolved fluorophore excitation has made TPE microscopy a preferred tool to provide subcellular resolution images from deep within light scattering tissues in a contextual setting. In vivo two-photon laser scanning microscopy (TPLSM) has been used to probe coupling between neuronal activity and cerebral blood flow. Measuring blood flow in individual brain capillaries with millisecond temporal resolution, Chaigneau et al demonstrated that, in the olfactory bulb superficial layers, changes in capillary flow precisely outline regions of synaptic activation.55 Kleinfeld et al were able to resolve motion of red blood cells in individual capillaries that lie several hundreds of microns deep in the somatosensory cortex in rat (Figure 5a and 5b). 26 They too found changes in the flux and velocity of red blood cells in individual capillaries in close response to stimulation of appropriate anatomical regions within the somatosensory cortex. Dunn et al have used intravital multicolor TPLSM of animals injected with fluid-phase probes to characterize bulk fluid flow through the kidney of living animals (Figure 5c and movie in the online data supplement).56 The latter study demonstrates how bulk tracers may be used to assess capillary blood flow, glomerular filtration, fluid transport, tubular solute concentration, and endocytosis by proximal tubule cells. Further examples of intravital TPE fluorescence imaging include visualization of lymphocyte trafficking in lymph nodes,57 reconstruction of tumorangiogenesis,58 and time-lapse studies of neuronal growth.6
TPE fluorescence microscopy has been particularly successful in the imaging of intracellular Ca2+ dynamics in small cellular compartments of live brain tissue (reviewed by Helmchen and Waters5). For example, using TPLSM in combination with the fluorescent calcium indicator calcium green-1, Helmchen et al were able to resolve fast-peaking Ca2+ transients in dendritic spines of pyramidal neurons down to a depth of 500 µm below the pial surface of the rat cortex in vivo (Figure 5d).4
In contrast, TPLSM has only recently begun to be used for Ca2+ imaging in intact cardiac tissue. Subcellular resolution Ca2+ imaging in intact myocardium using single-photon confocal microscopy is typically restricted to regions less than 40 µm below the surface.21,59 The capability of TPLSM to provide optical sectioning with subcellular resolution from deeper within scattering biological specimens than confocal microscopy has recently been exploited to measure [Ca2+]i-dependent changes in fluorescence intensity of the Ca2+ indicators rhod-2 and fura-2 at the single cardiomyocyte level in a buffer-perfused mouse heart preparation.25 Figure 6 demonstrates that TPLSM using a high numerical aperture objective is able to resolve single myocyte [Ca2+]i transients at depths of up to 200 µm below the epicardial surface. Direct measurements of the optical resolution,25 as well as calculations of the lateral (x,y) and axial (x,z) width of the TPE volume for a 1.2 numerical aperture objective lens (see Figure 2a), are compatible with the notion that under the imaging conditions used, both the lateral and axial dimension of the TPE volume are considerably less than the average depth, width, or length of adult murine ventricular cardiomyocytes (13, 32, and 140 µm, respectively (see Satoh et al60). Thus, the system allows selective visualization of [Ca2+]i in a volume significantly less than that of a single cardiomyocyte within the intact heart without being affected by fluorescent signals from neighboring cells.
TPE imaging of single-myocyte [Ca2+]i transients within the intact heart requires complete immobilization of the preparation to prevent regions of interest from moving out of the plane of focus. Cytochalasin D at a concentration of 50 µmol/L effectively eliminates movement artifacts in Langendorff-perfused mouse hearts during TPE imaging but does not abolish action potential-evoked [Ca2+]i transients.2,3,25 Importantly, many spatial and temporal properties of stimulated rhod-2 transients recorded from individual ventricular myocytes within the buffer-perfused mouse hearts in the presence of cytochalasin D are qualitatively identical with those of electrically evoked fluo-3 transients previously reported for single, isolated mouse ventricular cardiomyocytes in the absence of cytochalasin D but in otherwise identical experimental conditions. These properties include shortening of the transient in response to increased stimulation rates (Figure 7),25,61 as well as rapid and spatially uniform rises of [Ca2+]i (Figures 6 and 7), reflecting synchronous activation of SR ryanodine receptors secondary to Ca2+ influx through activated L-type Ca2+ channels in the t-tubular membrane.62 Thus, cytochalasin D-induced immobilization enables subcellular resolution [Ca2+]i measurements in single cells within the intact, buffer-perfused heart using TPLSM.
TPE fluorescence microscopy has recently been exploited to probe the functional integration of donor cells following intracardiac transplantation (Figure 7).2,3 Using TPE laser scanning microscopy with a high numerical aperture objective (to minimize TPE volume), EGFP-expressing donor fetal ventricular cardiomyocytes (which appeared yellow because of the overlay of red rhod-2 and green EGFP fluorescence) were shown to exhibit action potential-induced [Ca2+]i transients in synchrony with their neighboring EGFP-negative cardiomyocytes, indicating that they are functionally coupled (Figure 7a through 7c). Moreover, the kinetics of [Ca2+]i transients in transplanted ventricular cardiomyocytes were indistinguishable from those in host cardiomyocytes (Figure 7d).2 In contrast, the majority of donor-derived myocytes in hearts carrying EGFP-expressing skeletal myoblast grafts did not develop [Ca2+]i transients in response to propagating action potentials (Figure 7f), suggesting that they are functionally isolated.3 A small fraction of EGFP-expressing myocytes at the graft-host border, most likely arising from skeletal myoblast-cardiomyocyte fusion events, developed action potential–evoked [Ca2+]i transients in synchrony with their adjacent host cardiomyocytes (Figure 7f and 7g). The time courses of [Ca2+]i transients in these cells could be distinctly different from those in their neighboring host cardiomyocyte, as shown in Figure 7h.3 These observations demonstrate exemplarily that TPLSM is ideally suited to determine the functional state of individual donor cells following their intracardiac transplantation (provided they express a fluorescent marker) and is able to resolve spatial microheterogeneities of intracellular Ca2+ signaling within the intact, buffer-perfused mouse heart with high temporal resolution.
TPE-based laser scanning fluorescence microscopy allows spatially and temporally resolved visualization of Ca2+ dynamics from deeper within intact cardiac tissue than previously attainable with confocal imaging. The approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity between neighboring cardiomyocytes. For example, distinct differences in the magnitude and spatiotemporal properties of action potential-evoked [Ca2+]i transients have been found between single ventricular cardiomyocytes isolated from different transmural layers of the normal heart,63 between single ventricular cardiomyocytes and Purkinje cardiomyocytes,64,65 as well as between single ventricular cardiomyocytes adjacent to and remote from the infarct zone, respectively.66 It will be important to examine whether and how this in vitro heterogeneity is modulated at the whole heart level, where movement of calcium ions from cell to cell may attenuate intrinsic differences in cardiomyocyte Ca2+ signaling. Localized, subcellular sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks) have been shown to precede or lead to Ca2+ waves,59 which in turn trigger arrhythmogenic afterdepolarizations via modulation of [Ca2+]i -sensitive transmembrane ion channels and transporters. Analysis of the mechanisms underlying initiation, propagation, and termination of Ca2+ waves in intact cardiac tissue should provide tremendous information concerning the role of intracellular calcium in cardiac arrhythmogenesis. TPLSM is also particularly well suited to follow the functional fate of donor cells following direct intracardiac delivery,2,3 or following homing to the site of injury, provided that the donor cells can be identified on the basis of fluorescent properties. The ability to assess functional aspects at the individual cell level (as opposed to global heart function) may also permit a better discrimination between a nonspecific effect on postinjury remodeling versus a direct contribution of transplanted donor cells to a functional syncytium.
TPE Imaging of Cellular Redox State
Two-photon microscopy provides metabolic imaging with subcellular resolution by using the inherent fluorescence of NADH as an indicator of both oxidative and glycolytic energy metabolism.67,68 Although its two-photon absorption cross section is approximately two to three orders of magnitude lower than that of conventional fluorophores (700 nm),28 in vivo detection of NADH autofluorescence by two-photon microscopy is still feasible because of its high (millimolar) concentration and enhanced action cross section in the intracellular milieu compared with its aqueous solution.28 Kasischke et al exploited the inherent three-dimensional resolution and increased penetration depth of TPE fluorescence microscopy to monitor intrinsic fluorescence of NADH deep in hippocampal brain slices several hundreds of microns thick.28 They were able to demonstrate that the NADH response to focal neural stimulation is composed of an early dendritic dip (indicating increased NADH oxidation by the respiratory chain) and a late overshoot in juxtaposed astrocytes (indicating NADH production during nonoxidative glycolysis). This functional imaging study thus confirmed the ideal suitability of TPE microscopy to reveal spatiotemporal compartmentalization with high resolution in intact, highly scattering tissue. Similarly, Piston and coworkers used quantitative two-photon imaging to spatially resolve NADH signals from the cytoplasm and mitochondria in single pancreatic ß cells within intact pancreatic islets.67,68 Thus far, intrinsic NADH fluorescence has been measured in intact rat right ventricular trabeculae using conventional epifluorescence techniques,69 but no report on NADH monitoring in a multicellular cardiac preparation by means of TPE microscopy has been published. At the level of single, isolated cardiomyocytes, O’Rourke and colleagues were able to demonstrate that release of reactive oxygen species and mitochondrial depolarization in a small fraction of the volume of the cell can trigger spatiotemporally synchronized oscillations in NADH autofluorescence throughout the entire cell volume, indicative of a role of interorganellar communication in determining whole-cell function.70
Upregulation of cardiomyocyte NADH production has been shown to closely correlate with increases in mitochondrial [Ca2+], which, in turn, follow increases in global cytosolic [Ca2+], such as when the workload is increased by increased pacing frequency.69 The ability to simultaneously measure NADH and [Ca2+] with subcellular resolution in intact cardiac tissue by means of TPE microscopy would provide tremendous information concerning the spatiotemporal organization of excitation-metabolism coupling in a more contextual setting.