Isuru D. Jayasinghe

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes.

We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often <50 nm, suggesting that peripheral RyR clusters may exhibit strong intercluster signaling. The wide variation of cluster size, which follows a near-exponential distribution, is compatible with a stochastic cluster assembly process. We suggest that calcium sparks may be the result of the concerted activation of several RyR clusters forming a functional “supercluster” whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels.

4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues

Background Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. Methodology/Principal Findings We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk. Conclusions/Significance Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

Organization of Ryanodine Receptors, Transverse Tubules, and Sodium-Calcium Exchanger in Rat Myocytes

Confocal and total internal reflection fluorescence imaging was used to examine the distribution of caveolin-3, sodium-calcium exchange (NCX) and ryanodine receptors (RyRs) in rat ventricular myocytes. Transverse and longitudinal optical sectioning shows that NCX is distributed widely along the transverse and longitudinal tubular system (t-system). The NCX labeling consisted of both punctate and distributed components, which partially colocalize with RyRs (27%). Surface membrane labeling showed a similar pattern but the fraction of RyR clusters containing NCX label was decreased and no nonpunctate labeling was observed. Sixteen percent of RyRs were not colocalized with the t-system and 1.6% of RyRs were found on longitudinal elements of the t-system. The surface distribution of RyR labeling was not generally consistent with circular patches of RyRs. This suggests that previous estimates for the number of RyRs in a junction (based on circular close-packed arrays) need to be revised. The observed distribution of caveolin-3 labeling was consistent with its exclusion from RyR clusters. Distance maps for all colocalization pairs were calculated to give the distance between centroids of punctate labeling and edges for distributed components. The possible roles for punctate NCX labeling are discussed.

Light-Induced Dark States of Organic Fluochromes Enable 30 nm Resolution Imaging in Standard Media

We show that high quantum efficiency fluorophores can exhibit reversible photobleaching. This observation provides the basis for an imaging technique we call reversible photobleaching microscopy. We demonstrate applicability of this technique using antibody labeled biological samples in standard aqueous (or glycerol based) media to produce far-field images at approximately 30 nm resolution. Our novel method relies on intense illumination to reversibly induce a very long-lived (>10 s) dark state from which single fluorochromes slowly return stochastically. As in other localization microscopy methods, reversible photobleaching microscopy localizes single fluorochromes, but has the advantage that specialized photoactivatible and photoswitchable molecules or special immersion/embedding media are not required.

Comparison of the organization of t‐tubules, sarcoplasmic reticulum and ryanodine receptors in rat and human ventricular myocardium

1. It is apparent from the literature that there are significant differences in excitation–contraction coupling between species, particularly in the density of calcium transporting proteins in the t‐system and sarcoplasmic reticulum (SR) Ca2+ release channels. Unfortunately, there is a lack of information as to how the principal structures that link electrical excitation to the activation of calcium‐induced calcium release (CICR) are different between human and animal models (particularly rat).

Three-dimensional high-resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes

Quantitative understanding of the Ca2+ handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high‐resolution imaging and analysis approaches to measure the three‐dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z‐line marker α‐actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling was used to relate the Z‐line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca2+ release channels) and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca2+ dynamics, we determined the three‐dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca2+ dynamics with a measured Ca2+ release unit (CRU) distribution. Calculations with this model were compared with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca2+ waves that spread axially along the cell at velocities of ∼50 μm s−1. By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca2+ dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.

Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes

The peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), were examined using single fluorophore localization-based super-resolution microscopy in rat ventricular myocytes. JPH2 was strongly associated with RyR clusters. Estimates of the colocalizing fraction of JPH labeling with RyR was ~90% within 30 nm of RyR clusters. This is comparable to fractions estimated from confocal data (~87%). Similarly, most RyRs were associated with JPH2 labeling in super-resolution images (~81% within 30 nm of JPH2 clusters). The shape of associated RyR clusters and JPH2 clusters were very similar, but not identical, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked.

Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging.

Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.

Three-dimensional reconstruction and analysis of the tubular system of vertebrate skeletal muscle

Summary Skeletal muscle fibres are very large and elongated. In response to excitation there must be a rapid and uniform release of Ca2+ throughout for contraction. To ensure a uniform spread of excitation throughout the fibre to all the Ca2+ release sites, the muscle internalizes the plasma membrane, to form the tubular (t-) system. Hence the t-system forms a complex and dense network throughout the fibre that is responsible for excitation–contraction coupling and other signalling mechanisms. However, we currently do not have a very detailed view of this membrane network because of limitations in previously used imaging techniques to visualize it. In this study we serially imaged fluorescent dye trapped in the t-system of fibres from rat and toad muscle using the confocal microscope, and deconvolved and reconstructed these images to produce the first three-dimensional reconstructions of large volumes of the vertebrate t-system. These images showed complex arrangements of tubules that have not been described previously and also allowed the association of the t-system with cellular organelles to be visualized. There was a high density of tubules close to the nuclear envelope because of the close and parallel alignment of the long axes of the myofibrils and the nuclei. Furthermore local fluorescence intensity variations from sub-resolution tubules were converted to tubule diameters. Mean diameters of tubules were 85.9±6.6 and 91.2±8.2 nm, from rat and toad muscle under isotonic conditions, respectively. Under osmotic stress the distribution of tubular diameters shifted significantly in toad muscle only, with change specifically occurring in the transverse but not longitudinal tubules.

A new twist in cardiac muscle: dislocated and helicoid arrangements of myofibrillar z-disks in mammalian ventricular myocytes.

Using deconvolved confocal microscopy of fluorescently labeled markers for z-disks, t-tubules and ryanodine receptors, we have examined sarcomere organization in cardiac myocytes from rat, rabbit and human. We show that sarcomeres exhibit dislocations in registration and occasionally more complex helicoidal topology. This organization was present at both slack ( approximately 1.8 microm) and long sarcomere lengths ( approximately 2.2 microm). Misregistrations in z-disks persisted over 15-20 sarcomere lengths and appeared to arise primarily from variations in fiber direction; particularly as myofibrils pass around nuclei. In addition, myofibrils twist along the cell length. T-tubules generally follow the sarcomere z-disks although additional elements bridging adjacent myofibrils and along the length of the myofibril are present to varying degrees in all cells. Ryanodine receptors (the sarcoplasmic reticulum Ca(2+) release channel) are generally located within 250 nm of the local plane containing t-tubules and z-disks, but a small fraction ( approximately 2%) is found on longitudinal elements of the t-system between z-disks. The results are discussed with respect to the possible role(s) of such complex z-disk organization and z-disk dislocations in the maintenance of cell structure and sarcomere assembly. In addition, the non-planar organization of z-disks may be important in the propagation of local Ca(2+) waves which may have a useful role in helping maintain the uniformity of sarcomere activation in the presence of t-tubule remodeling.