Multispot multiphoton Ca2+ imaging in acute myocardial slices

Abstract. Multiphoton microscopy has become essential for dynamic imaging in thick living tissues. High-rate, full-field image acquisition in multiphoton microscopy is achievable by parallelization of the excitation and detection pathways. We developed our approach via a diffractive optical element which splits a pulsed laser into 16 beamlets and exploits a descanned detection system consisting of an array of beamlet-associated photomultiplier tubes. The optical performance of the multiphoton multispot system (MCube) has been characterized in cardiac tissue sections and subsequently used for the first time for fluorescence imaging of cardiomyocyte Ca2+ dynamics in viable acute cardiac slices. Multispot multiphoton microscopy (MMM) has never been used before to monitor Ca2+ dynamics in thick, viable tissue samples. Acute heart slices are a powerful close-to-in vivo model of Ca2+ imaging allowing the simultaneous observation of several cells in their own tissue environment, exploiting the multiphoton excitation ability to penetrate scattering tissues. Moreover, we show that the concurrent high spatial and temporal resolutions afforded by the parallel scanning in MMM can be exploited to simultaneously assess subcellular Ca2+ dynamics in different cells in the tissue. We recorded local Ca2+ release events including macrosparks, travelling waves, and rotors.

[1]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[2]  Nathalie L Rochefort,et al.  Functional mapping of single spines in cortical neurons in vivo , 2011, Nature.

[3]  Heinrich Spiecker,et al.  The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging. , 2007, Biophysical journal.

[4]  Erich E Hoover,et al.  Advances in multiphoton microscopy technology , 2013, Nature Photonics.

[5]  J. Hescheler,et al.  Ventricular Slices of Adult Mouse Hearts - a new Multicellular In Vitro Model for Electrophysiological Studies , 2006, Cellular Physiology and Biochemistry.

[6]  Sergio Fantini,et al.  Multifocal multiphoton microscopy based on multianode photomultiplier tubes. , 2007, Optics express.

[7]  M R Taghizadeh,et al.  Multiphoton multifocal microscopy exploiting a diffractive optical element. , 2003, Optics letters.

[8]  W. Lederer,et al.  Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. , 1993, Science.

[9]  S. Houser.  Ca(2+) signaling domains responsible for cardiac hypertrophy and arrhythmias. , 2009, Circulation Research.

[10]  David S. Koos,et al.  Deep and fast live imaging with two-photon scanned light-sheet microscopy , 2011, Nature Methods.

[11]  Patrizia Camelliti,et al.  Tissue Slices from Adult Mammalian Hearts as a Model for Pharmacological Drug Testing , 2009, Cellular Physiology and Biochemistry.

[12]  Peter Kohl,et al.  Myocardial tissue slices: organotypic pseudo-2D models for cardiac research & development. , 2009, Future cardiology.

[13]  Diana Deca,et al.  LOTOS-based two-photon calcium imaging of dendritic spines in vivo , 2012, Nature Protocols.

[14]  A Diaspro,et al.  Three‐dimensional optical behaviour of a confocal microscope with single illumination and detection pinhole through imaging of subresolution beads , 1999, Microscopy research and technique.

[15]  T Nielsen,et al.  High efficiency beam splitter for multifocal multiphoton microscopy , 2001, Journal of microscopy.

[16]  C. Ward,et al.  Leaky Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice. , 2008, The Journal of clinical investigation.

[17]  C. Barclay.  Modelling diffusive O(2) supply to isolated preparations of mammalian skeletal and cardiac muscle. , 2005, Journal of muscle research and cell motility.

[18]  C. Barclay.  Modelling diffusive O2 supply to isolated preparations of mammalian skeletal and cardiac muscle , 2005, Journal of Muscle Research & Cell Motility.

[19]  Karsten Bahlmann,et al.  Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz. , 2007, Optics express.

[20]  Rüdiger Rudolf,et al.  Looking forward to seeing calcium , 2003, Nature Reviews Molecular Cell Biology.

[21]  Kenneth W Dunn,et al.  Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts. , 2003, American journal of physiology. Cell physiology.

[22]  Zeno Lavagnino,et al.  Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: characterization and application. , 2013, Optics express.

[23]  E. Neher,et al.  Highly nonlinear photodamage in two-photon fluorescence microscopy. , 2001, Biophysical journal.

[24]  D W Tank,et al.  Direct Measurement of Coupling Between Dendritic Spines and Shafts , 1996, Science.

[25]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[26]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[27]  M. Peschanski,et al.  An in vitro beating heart model for long-term assessment of experimental therapeutics. , 2009, Cardiovascular research.

[28]  M. Rubart,et al.  Cardiovascular Imaging Using Two-Photon Microscopy , 2008, Microscopy and Microanalysis.

[29]  L. Loew,et al.  Defects in T-tubular electrical activity underlie local alterations of calcium release in heart failure , 2014, Proceedings of the National Academy of Sciences.