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.
Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.
Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias.
Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits
the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+
waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the
extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies
environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a
tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton
microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired
arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of
the intact heart and correlate these with electrophysiology.
We report on the characterization and use of a Multispot Multiphoton Microscope, to investigate calcium dynamics at
intracellular level. Time resolution of a few milliseconds, even in full frame images at 512×512 pixels, is achieved, in
order to get the most information on the evolution and propagation of ionic calcium waves across adjacent cells in an
intact cardiac tissue.
We report on the characterization and use of a Multispot Multiphoton Microscope, to investigate calcium dy-
namics at intracellular level. We apply this technique to obtain a time resolution of a few milliseconds, even
in full frame images at 512x512 pixels, in order to get the most information on the evolution and propagation
of ionic calcium waves across adjacent cells in an intact cardiac tissue. Further we report on the progress of
development of a Random Access microscope for very high speed all optical electrophysiological signal acquisition
in cell networks. Our study opens the way to the investigation of arrhythmogenic disease in animal models at
cellular level.
High rate, full field image acquisition in multiphoton imaging is achievable by parallelization of the excitation and of the
detection paths. Via a Diffractive Optical Elements (DOEs) which splits a pulsed laser, and a spatial resolved descanned
detection path, a new approach to microscopy has been developed. By exploiting the three operating mode, single beam,
16 beamlets or 64 beamlets, the best experimental conditions can be found by adapting the power per beamlet. This
Multiphoton Multispot system (MCube) has been characterized in thick tissue samples, and subsequently used for the
first time for Ca2+ imaging of acute heart slices. A test sample with fixed mice heart slices with embedded sub-resolution
fluorescent beads has been used to test the capability of optical axial resolution up to ~200 microns in depth. Radial and
axial resolutions of 0.6 microns and 3 microns have been respectively obtained with a 40X water immersion objective,
getting close to the theoretical limit. Then images of heart slices cardiomyocites, loaded with Fluo4-AM have been
acquired. The formation of Ca2+ waves during electrostimulated beating has been observed, and the possibility of easily
acquire full frame images at 15 Hz (16 beamlets) has been demonstrated, towards the in vivo study of time resolved
cellular dynamics and arrhythmia trigger mechanisms in particular. A very high speed two-photon Random Access
system for in vivo electrophysiological studies, towards the correlation of voltage and calcium signals in arrhythmia
phenomena, is now under developing at Light4tech.
We present in this paper recent results on Light - Induced Atom Desorption (LIAD) in sealed and open coated cells.
LIAD is defined via the description of an experiment on rubidium atoms stored in a dry - film coated cell, where a few
milliwatts of even non coherent and non resonant light are able to increase the alkali atomic density for more than one
order of magnitude. Modeling of the effect is given. New features become relevant in the case of LIAD in porous
glasses: in fact, although the photodesorption efficiency per unit area in bare glass is much lower, photoatomic sources
can be prepared, due to the huge inner surface of porous samples. We applied LIAD from organic coatings to the
stabilization of alkali densities out of equilibrium: sodium case is here discussed. Finally, we report on fully original,
preliminary measurements of rubidium Magneto - Optical Trap loading via LIAD from a dry - film coated cell.
We report on multiphoton imaging of biological samples performed with continuum infrared source generated
in photonic crystal fibers (PCFs). We studied the spectra generated in PCFs with dispersion profiles designed
to maximize the power density in the 700-1000 nm region, where the two-photon absorption cross sections of
the most common dyes lie. Pumping in normal dispersion region, with <140 femtosecond pulses delivered by a
tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), results in a limitation of nonlinear broadening up to a
mean power density above 2 mW/nm. Axial and lateral resolution obtained with a scanning multiphoton system
has been measureed to be near the theoretical limit. The possibility of simultaneous two-photon excitation of
different dyes in the same sample and high image resolution are demonstrated at tens of microns in depth.
Signal-to-noise ratio and general performances are found to be comparable with those of a single wavelength
system, used for comparison.
We describe the realization and characterization of a broadband, high power density and fully spectrally controllable
source, suitable for multiphoton imaging of biological samples. We used a photonic crystal fiber (PCF)
with selected dispersive and non-linear properties, in order to generate, when pumped with <140 femtosecond
pulses delivered by a tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), a smooth continuum in the
700nm-950nm region, with average power density grater than 2mW/nm. Time distribution of the generated
spectrum has been measured with autocorrelation technique. Axial and lateral resolution obtained with a scanning
multiphoton system has been determined to be near the theoretical limit. The possibility of two-photon
excitation of different dyes in the same sample and high image resolution are demonstrated at tens of microns
in depth. Future developments and different applications are also discussed.
S. N. Atutov, R. Calabrese, L. Corradi, A. Dainelli, C. de Mauro, A. Khanbekyan, E. Mariotti, P. Minguzzi, L. Moi, S. Sanguinetti, G. Stancari, L. Tomassetti
The apparatus for the production and trapping of francium is described and its performances are reported. Latest results
on magneto-optical trapping of Francium are summarized: trapping of 209, 210, 211 Francium isotopes, measurements
of their trapping frequencies, measurements of diffusion parameters of Francium ions in yttrium. Future experiments on
fundamental physics are presented.
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