Optical Methods for Imaging Ionic Activities

Optical fluorescence is characteristic of molecules and their environment, and dyes can be made whose fluorescence is altered by reversible binding to specific ions. By introducing these into the cytosol, fluorescence microscopy can be used to form dynamic images of ionic activities in living cells under experimental manipulation. Optical fluorescence spectra are broad-band, and if specific ion binding alters the wavelength of maximal excitation or emission, quantitative measurements can be made from the ratio of images taken at two different wavelengths, eliminating errors due to spatial variations in dye concentration and optical path-length. This method is analogous to continuum normalisation in X-ray microanalysis, and is implemented using a sensitive video camera and computer processing of digitised images. Fluorescent indicators exist for calcium, magnesium, hydrogen, sodium, zinc and chloride ions. Most imaging work has been on calcium, which is important in many cell signalling processes, and several calcium indicators are available with different spectral properties. Spatial resolution is limited to a few μm by out-of-focus blur, but repeated images can be captured with a time resolution as low as 200 msec, and by using dyes with high binding affinity, detection limits can be lower than by X-ray methods. There is a large and fast-growing literature of applications to many plant and animal cell-types

Confocal and multiphoton imaging of intracellular Ca²⁺

This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca<sup>2+</sup> within living cells. In particular, the excitation spectra of popular fluorescent Ca<sup>2+</sup> indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca<sup>2+</sup> affinity of the dye to a particular aspect of Ca<sup>2+</sup> signaling. Finally, the combined emission spectra of Ca<sup>2+</sup> and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca<sup>2+</sup> and membrane voltage measurement

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes

Development of a Plasmonic On-Chip System to Characterize Changes from External Perturbations in Cardiomyocytes

Today’s heart-on-a-chip devices are hoped to be the state-of-the-art cell and tissue characterizing tool, in clinically applicable regenerative medicine and cardiac tissue engineering. Due to the coupled electromechanical activity of cardiomyocytes (CM), a comprehensive heart-on-a-chip device as a cell characterizing tool must encompass the capability to quantify cellular contractility, conductivity, excitability, and rhythmicity. This dissertation focuses on developing a successful and statistically relevant surface plasmon resonance (SPR) biosensor for simultaneous recording of neonatal rat cardiomyocytes’ electrophysiological profile and mechanical motion under normal and perturbed conditions. The surface plasmon resonance technique can quantify (1) molecular binding onto a metal film, (2) bulk refractive index changes of the medium near (nm) the metal film, and (3) dielectric property changes of the metal film. We used thin gold metal films (also called chips) as our plasmonic sensor and obtained a periodic signal from spontaneously contracting CMs on the chip. Furthermore, we took advantage of a microfluidic module for controlled drug delivery to CMs on-chip, inhibiting and promoting their signaling pathways under dynamic flow. We identified that ionic channel activity of each contraction period of a live CM syncytium on a gold metal sensor would account for the non-specific ion adsorption onto the metal surface in a periodic manner. Moreover, the contraction of cardiomyocytes following their ion channel activity displaces the medium, changing its bulk refractive index near the metal surface. Hence, the real-time electromechanical activity of CMs using SPR sensors may be extracted as a time series we call the Plasmonic Cardio-Eukaryography Signal (P-CeG). The P-CeG signal render opportunities, where state-of-the-art heart-on-a-chip device complexities may subside to a simpler, faster and cheaper platform for label-free, non-invasive, and high throughput cellular characterization

CardIAP: calcium transients confocal image analysis tool

One of the main topics of cardiovascular research is the study of calcium (Ca2+) handling, as even small changes in Ca2+ concentration can alter cell functionality (Bers, Annu Rev Physiol, 2014, 76, 107–127). Ionic calcium (Ca2+) plays the role of a second messenger in eukaryotic cells, associated with cellular functions such as cell cycle regulation, transport, motility, gene expression, and regulation. The use of fluorometric techniques in isolated cells loaded with Ca2+-sensitive fluorescent probes allows quantitative measurement of dynamic events occurring in living, functioning cells. The Cardiomyocytes Images Analyzer Python (CardIAP) application addresses the need to analyze and retrieve information from confocal microscopy images systematically, accurately, and rapidly. Here we present CardIAP, an open-source tool developed entirely in Python, freely available and useable in an interactive web application. In addition, CardIAP can be used as a standalone Python library and freely installed via PIP, making it easy to integrate into biomedical imaging pipelines. The images that can be generated in the study of the heart have the particularity of requiring both spatial and temporal analysis. CardIAP aims to open the field of cardiomyocytes and intact hearts image processing. The improvement in the extraction of information from the images will allow optimizing the usage of resources and animals. With CardIAP, users can run the analysis to both, the complete image, and portions of it in an easy way, and replicate it on a series of images. This analysis provides users with information on the spatial and temporal changes in calcium releases and characterizes them. The web application also allows users to extract calcium dynamics data in downloadable tables, simplifying the calculation of alternation and discordance indices and their classification. CardIAP aims to provide a tool that could assist biomedical researchers in studying the underlying mechanisms of anomalous calcium release phenomena.Fil: Velez Rueda, Ana Julia. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonano, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Smith, Agustín García. Universidad Nacional de Quilmes; ArgentinaFil: Parisi, Gustavo Daniel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fornasari, Maria Silvina. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sommese, Leandro Matías. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and DNA PAINT single molecule localisation microscopy

Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models. Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has remained a mystery. Ryanodine receptors are found in clusters and their influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters. Ryanodine receptor clusters have been well characterised in electron microscopy and the fluorescence based super resolution microscopy, achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser 2808 and Ser 2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser 2808 but not Ser 2814. These differences in Ser¬ 2808 phosphorylation were then investigated in dual channel STORM super resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques. This population and sub population experiment was then translated into the new DNA PAINT technology and a direct comparison of performance between STORM and DNA PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers. DNA-PAINT is emerging as the progression of SMLM from STORM microscopy due to the greater control of imaging parameters it affords. Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA PAINT are also highlighted and discussed

New Methodologies for Studying Intercellular Connectivity and Signaling in Plant Tissues

Cells are at the core of our biosphere. Their organization into tissues, organs, organ systems, and organisms requires frequent exchange of nutrients and information to promote proper developmental patterning and organismal success. Direct physical connections that unite the cytoplasm of adjacent cells provide the most energy-efficient mechanism for moving substances from one cell to the next. Therefore, it is important to understand both the subcellular mechanisms that govern cell-to-cell transport and the supracellular processes that utilize these physical connections. In a plant organism, intercellular communication is dictated by the numerous pores in a plant cell wall that enable the unity of cytoplasm between adjacent cells within and often between tissues. These so-called plasmodesmata are vital in plant physiology at the supracellular level. As plants cannot escape either biotic or abiotic stressors, they have developed sophisticated chemical signaling processes that utilize plasmodesmata to coordinate a response, if necessary.The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern plant cell biology because it allows for a direct assessment of plasmodesmal function. However, traditional methods to introduce fluorescent probes into living cells necessarily introduce artifacts that limit our progress. Herein, I describe the diffusive injection micropipette (DIMP) which was developed to avoid several of the artifacts that impact cell function during traditional injections. I used DIMPs to successfully introduce fluorescent probes into plant, fungal, and animal cells, with a more in-depth investigation into the function of plant cell plasmodesmata. As plasmodesmata are nanoscopic channels with complex anatomical features, they are subject to certain biophysical processes that are absent at the macroscopic level. At the nanoscopic scale, surface charges of the plasma membrane generate an electric field that may be large enough to interact with molecules passing through plasmodesmata. Using the DIMP, I found that positively charged fluorescent probes were greatly impaired in their cell-to-cell movement via plasmodesmata when compared to negatively charged probes. This is intriguing because calcium signaling cascades that are vital to plant stress responses previously had been hypothesized to involve the movement of calcium through plasmodesmata. Therefore, I also investigated the propagation of calcium signals in local cell populations responding to mechanical stimuli. In response to mechanical stimuli that increase cell turgor, calcium signals depended on plasmodesmata for their propagation within the tissue. However, the signaling cascade following turgor decreases were independent of plasmodesmata and likely utilize extracellular pathways