Intracellular Ca²⁺waves (CaWs) of cardiomyocytes are spontaneous events of Ca²⁺release from the sarcoplasmic reticulum that are regarded as an important substrate for triggered arrhythmias and delayed afterdepolarizations. However, little is known regarding whether or how CaWs within the heart actually produce arrhythmogenic membrane oscillation because of the lack of data confirming direct correlation between CaWs and membrane potentials (V_m) in the heart. On the hypothesis that CaWs evoke arrhythmogenic oscillatory depolarization when they emerge synchronously and intensively in the heart, we conducted simultaneous fluorescence recording of intracellular Ca²⁺([Ca²⁺]_i) dynamics andV_mof ventricular myocytes on subepicardial surfaces of Langendorff-perfused rat hearts using in situ dual-view, rapid-scanning confocal microscopy. In intact hearts loaded with fluo4/acetoxymethyl ester and RH237 under perfusion with cytochalasin D at room temperature, individual myocytes exhibited Ca²⁺transients and action potentials uniformly on ventricular excitation, whereas low-K⁺–perfused (2.4 mmol/L) hearts exhibited CaWs sporadically between Ca²⁺transients without discernible membrane depolarization. Further [Ca²⁺]_iloading of the heart, produced by rapid pacing and addition of isoproterenol, evoked triggered activity and subsequent oscillatoryV_m, which are caused by burst emergence of CaWs in individual myocytes. Such arrhythmogenic membrane oscillation was abolished by ryanodine or the Na⁺–Ca²⁺exchanger inhibitor SEA0400, indicating an essential role of CaWs and resultant Na⁺–Ca²⁺exchanger–mediated depolarization in triggered activity. In summary, we demonstrate a mechanistic link between intracellular CaWs and arrhythmogenic oscillatory depolarizations in the heart. Our findings provide a cellular perspective on abnormal [Ca²⁺]_ihandling in the genesis of triggered arrhythmias in the heart.

5-Aminolevulinic acid (5-ALA) is a natural amino acid and a precursor of heme and chlorophyll. Exogenously administered 5-ALA is metabolized into protoporphyrin IX (PpIX). PpIX accumulates in cancer cells because of the low activity of ferrochelatase, an enzyme that metabolizes PpIX to heme. High expression of 5-ALA influx transporters, such as peptide transporters 1/2, in cancer cells also enhances PpIX production. Because PpIX radiates red fluorescence when excited with blue/violet light, 5-ALA has been used for the visualization of various tumors. 5-ALA photodynamic diagnosis (PDD) has been shown to improve the tumor removal rate in high-grade gliomas and non-muscular invasive bladder cancers. However, 5-ALA PDD remains a challenge as a diagnostic method because tissue autofluorescence interferes with PpIX signals in cases where tumors emit only weak signals, and non-tumorous lesions, such as inflammatory sites, tend to emit PpIX fluorescence. Here, we review the current outline of 5-ALA PDD and strategies for improving its diagnostic applicability for tumor detection, focusing on optical techniques and 5-ALA metabolic pathways in both viable and necrotic tumor tissues.

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Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm⁻¹, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm⁻¹ enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.

Kawasaki Disease (KD) involves a diffuse and systemic vasculitis of unknown etiology that mainly affects infants and children. Although a considerable number of analyses of the clinical, histopathological and molecular biological details underlying the mechanism responsible for the development of coronary arterial lesions, it is still poorly understood.

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise ratio (SNR) in measurements of biological specimens is severely limited because of the small cross section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and enhanced the spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Last, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.