Algorithmic corrections for localization microscopy with sCMOS cameras - characterisation of a computationally efficient localization approach.

Modern sCMOS cameras are attractive for single molecule localization microscopy (SMLM) due to their high speed but suffer from pixel non-uniformities that can affect localization precision and accuracy. We present a simplified sCMOS non-uniform noise model that incorporates pixel specific read-noise, offset and sensitivity variation. Using this model we develop a new weighted least squared (WLS) fitting method designed to remove the effect of sCMOS pixel non-uniformities. Simulations with the sCMOS noise model, performed to test under which conditions sCMOS specific localization corrections are required, suggested that pixel specific offsets should always be removed. In many applications with thick biological samples photon fluxes are sufficiently high that corrections of read-noise and sensitivity correction may be neglected. When correction is required, e.g. during fast imaging in thin samples, our WLS fit procedure recovered the performance of an equivalent sensor with uniform pixel properties and the fit estimates also attained the Cramer-Rao lower bound. Experiments with sub-resolution beads and a DNA origami test sample confirmed the results of the simulations. The WLS localization procedure is fast to converge, compatible with 2D, 3D and multi-emitter localization and thus provides a computationally efficient sCMOS localization approach compatible with most SMLM modalities.

Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes.

The contractile properties of cardiac myocytes depend on the calcium (Ca(2+)) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~57% of the perimeter of each myofibril. The average size of internal couplons is ~63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥80% of the total RyRs were detected in clusters containing ≥100 receptors. ~56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca(2+)-induced Ca(2+) release (100nm) and were grouped into 'superclusters'. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca(2+) levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2clusters/µm(3)) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca(2+) signalling.

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.

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

Quantitative understanding of the Ca(2+) 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 alpha-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 Ca(2+) 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 Ca(2+) 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 Ca(2+) dynamics with a measured Ca(2+) 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 Ca(2+) waves that spread axially along the cell at velocities of approximately 50 mum 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 Ca(2+) dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200-400 nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart.

Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.

The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.

Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.

Super-resolution fluorescence imaging to study cardiac biophysics: α-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices.

A major motivation for the use of super-resolution imaging methods in the investigation of cardiac biophysics has been the insight from biophysical considerations and detailed mathematical modeling that the spatial structure and protein organisation at the scale of nanometres can have enormous implications for calcium signalling in cardiac muscle. We illustrate the use of dSTORM based super-resolution in optically thick (∼10 µm) tissue slices of rat ventricular tissue to visualize proteins at the cardiac Z-disk and compare those images with confocal (diffraction-limited) as well as electron microscopy (EM) data which still provides a benchmark in terms of resolution. α-actinin is an abundant protein target that effectively defines the Z-disk in striated muscle and provides a reference structure for other proteins at the Z-line and the transverse tubules. Using super-resolution imaging α-actinin labelling provides very detailed outlines of the contractile machinery which we have used to study the properties of Z-disks and the distribution of α-actinin itself. We determined the local diameters of the myo-fibrillar and non-myofibrillar space using α-actinin labelling. Comparison between confocal and super-resolution based myofibrillar masks suggested that super-resolution data was able to segment myofibrils accurately while confocal approaches were not always able to distinguish neighbouring myofibrillar bundles which resulted in overestimated diameters. The increased resolution of super-resolution methods provides qualitatively new information to improve our understanding of cardiac biophysics. Nevertheless, conventional diffraction-limited imaging still has an important role to play which we illustrate with correlative confocal and super-resolution data.

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.