2.1.

Conventional Two-Photon Excitation Microscope

A multiphoton system (Radiance 2100 MP; Bio-Rad Laboratories, UK) was used for the comparison of MCube image quality with respect to an existing TPE microscopy system. The radiance system is equipped with a Tsunami mode-locked, tunable, femto-second–pulsed Ti/sapphire laser (Ti:Sa). The laser output is capable of generating 100-fs pulse trains at a rate of 82 MHz. The microscope (Eclipse E600FN; Nikon) was equipped with a Nikon objective ($40\times /0.8\mathrm{W}\mathrm{CORR}$); a direct detection system (Bio-Rad), fitted with a 500LP DC dichroic mirror and an HQ535/50 emission filter (Chroma Technology Corp.), was used for the detection of fluorescence emission signals. The LaserSharp2000 software package (Bio-Rad) was used for data acquisition.

2.2.

Acute Heart Slices Cutting and Loading

Acute ventricular heart slices were cut following the protocol described in Ref. 18. Young mice (postnatal day 7 to 10) were sacrificed and the heart was quickly excised and washed in ice-cold ${\mathrm{Ca}}^{2+}$-free Tyrode solution (composition in mM: NaCl 136, KCl 5.4, ${\mathrm{NaH}}_2{\mathrm{PO}}_4$ 0.33, ${\mathrm{MgCl}}_2$ 1, glucose 10, hepes 5; pH 7.40 with NaOH). Atria were excised by a cut on the transversal plane, also creating a basis surface for fixing the ventricular portion of the heart to the slicer support with a commercial cyanoacrylate glue. Hearts from young mice were embedded in 4% low-melting agarose (SIGMA-Aldrich) in Tyrode. Transversal slices ($450\text{-}\mu \mathrm{m}$ thickness) were cut in ice-cold ${\mathrm{Ca}}^{2+}$-free Tyrode solution with a vibratome (Leica GmBH). After cutting, slices were transferred to room temperature 1-m M  ${\mathrm{CaCl}}_2$ Tyrode for some minutes and then they were maintained in the recovery medium (DMEM/F12 supplemented with 20% Knock-out Replacement Serum, Invitrogen) in a humidified atmosphere containing 95% ${\mathrm{O}}_2$ and 5% ${\mathrm{CO}}_2$ at 37°C for at least 1 h before loading.¹⁹

After recovery, slices were loaded with the ${\mathrm{Ca}}^{2+}$-sensitive indicator Fluo-4AM ($5\mu \mathrm{M}$, Invitrogen) for 40 min at 37°C in 1-mM ${\mathrm{Ca}}^{2+}$ Tyrode solution plus 20% Pluronic F-127 (Invitrogen) and $1\text{-}\mu \mathrm{M}$ sulfinpyrazone (SIGMA-Aldrich).

2.3.

Imaging and Stimulation

Viable heart slices loaded with Fluo-4AM were transferred on a homemade perfusion chamber on the stage of the two-photon microscope and perfused with a 1 mM ${\mathrm{Ca}}^{2+}$, oxygenated Tyrode solution.

To prevent curling and movement, slices were held down by a homemade platinum holder.

Slices were field stimulated by applying directly under the microscope a voltage difference at the slice sides ($5\mathrm{V}/\mathrm{cm}$); slices were thus electrically paced by applying brief voltage square waveforms (5 ms), with the fixed frequency of 1, 2, or 5 Hz. Changes in ${\mathrm{Ca}}^{2+}$ concentration were observed as variations in the green emission of the sample upon excitation.

3.1.

Instrument Design

Multispot multiphoton microscopy (MMM) allows the increase of the image acquisition speed several fold when compared with standard laser scanning microscopes. Analogous systems based on a microlens array have already been used to monitor ${\mathrm{Ca}}^{2+}$ dynamics in isolated cardiac cells⁷ but, to the best of our knowledge, MMM has never been employed to image cellular ${\mathrm{Ca}}^{2+}$ in whole hearts or ex vivo myocardial preparations.

To obtain an MMM system suitable for high-speed imaging at the cellular level in intact organs, we sought to obtain homogeneous light distribution over the sample, while at the same time, maximize light detection from the whole field of view of the objective. The system design is thus based on a DOE in the excitation light path, a descanned configuration in the emission path, and an array of photomultiplier tubes (PMTs) [Fig. 1(a)]. A Ti:Sa pulsed infrared (IR) laser ($<150\mathrm{fs}$, 80-MHz pulses, Chameleon Ultra II, Coherent, UK) providing up to 3.6 W of optical power at 785-nm excitation wavelength was used as the excitation light source.

Fig. 1

(a) Scheme of the multispot multiphoton microscope (MMM). (b) Essential components of the MMM. (c) Image and intensity profile of a subresolution bead along the radial axis.

The DOE was inserted along the incident light path to obtain patterned illumination of the sample, obtained by splitting the laser beam into 16 parallel beamlets generating a matrix of 4 by 4 spots regularly spaced in a squared array. Such a configuration, in the preliminary tests, offered the best compromise between the imaging rate and signal-to-noise ratio. Laser power was evenly distributed throughout the spots [within a few percent due to the DOE construction, Fig. 1(b)], thus providing uniform illumination of the entire scanned area.¹⁴ With the laser power at the source of 3.6 W, the power per beamlet measured after the $4\times 4$ DOE resulted in about 45 mW.

The 16 parallel beamlet patterns were deflected along the $x$ and $y$ axes by galvanometric mirrors, while movement of the excitation plane along the $z$-axis was achieved by shifting the objective with a piezoelectric motor.

Detection of the emitted light is performed with a multianode PMT, (H7546A-20SEL from Hamamatsu), with each detecting element corresponding to a single descanned beamlet. Each PMT has its own electronic board, performing signal amplification, A/D conversion, local storage on a 2 MB onboard memory, and parallel communication with a custom-developed control software for data acquisition and storage on the PC hard drive. This configuration minimizes the optical cross-talk between different PMT elements²⁰ that may introduce undesired degradation of the image quality, particularly when acquiring fluorescence from highly scattering samples such as muscular tissue. The essential components of the MMM are shown in Fig. 1(a).

To allow experiments with living tissue samples, the MMM was adapted to an upright fluorescence microscope (DM LFSA, Leica Microsystems GmBH, Germany), equipped with a water immersion objective (Nikon, $40\times /0.8\mathrm{W}\mathrm{CORR}$).

3.2.

Optical Performance

To evaluate the optical performance of the MMM system, we determined the PSF by collecting the three-dimensional (3-D) image profile of a subresolution fluorescent bead, as shown in Fig. 1(c). From the radial intensity profile in Fig. 1(c), we obtained a radial resolution of $464\pm 10\mathrm{nm}$, a value in line with the expected theoretical resolution of 455 nm calculated for a two-photon system at the excitation wavelength of 785 nm.

Imaging of fluorescent beads embedded in agarose has previously been used to measure the PSF of microscopy systems as shown in Refs. 7 and 20. Although such a configuration is adequate for evaluating the pure optical performance of the systems, the sample is almost transparent and isotropic, and thus is not representative of the typical experimental condition. The myocardium is densely occupied by sarcomeres, formed by alternating segments of thin and thick protein filaments arranged in a highly scattering grid, inevitably reducing the performance of an optical system. To test the optical performance of the MMM system in deep tissue imaging in the heart, we evaluated the experimental PSF using a custom-generated heart phantom sample. The phantom was made by injecting $1\text{-}\mu \mathrm{m}$ fluorescent beads (Invitrogen) at different positions and depths in a $500\text{-}\mu \mathrm{m}$ fixed heart slice. We measured the fluorescence profile of a $1\text{-}\mu \mathrm{m}$ bead at a $150\mu \mathrm{m}$ depth in the tissue and estimated the system PSF in deep tissue imaging. The ePSF obtained is shown in Fig. 2(a) and the characteristics of fluorescence profiles are reported in Table 1.

Fig. 2

(a) Intensity profile of a $1\text{-}\mu \mathrm{m}$ fluorescent bead at $150\text{-}\mu \mathrm{m}$ depth in myocardial tissue. Scale bars: $2\mu \mathrm{m}$. Radial and axial views are reported as well as relative intensity profiles. (b) The same image acquired in single-line modality and multispot modality.

Table 1

Full width at half maximum (FWHM) of the measured point spread functions (PSFs) for the single-beam BIORAD MP2100 and multispot MCube systems; data refer to a fluorescent bead located 150  μm in depth in the phantom sample.

The results obtained with the MMM were compared with that of a commercial standard, the BIORAD MP2100 multiphoton microscope powered by a Ti:Sa Spectra Physics Millennia pulsed IR laser. We obtained, in both cases, a PSF for TPE microscopy comparable with that reported in other papers,⁷ with a better resolution for the MMM in both the axial and radial directions. The improvement in the optical resolution of the MMM can be dependent, at least in part, on the spatial filtering of the DOE apparatus. To determine the effect of the DOE insertion, we next compared the quality of the images acquired in either single-line mode or multispot mode of the same field of view with a comparable laser power. In a single-line mode, the laser power was thus attenuated down to 45 mW on the sample plane. As shown in Fig. 2(b), the image resolution and S/N are comparable, demonstrating that the DOE insertion does not cause image deterioration. Notably, the acquisition with the DOE inserted is 16 times faster than the single-line modality. In the inset, a magnification is shown.

3.3.

Multispot Multiphoton Microscopy Imaging of Calcium Dynamics in Thick Myocardial Slices

The system was tested on viable acute cardiac slices from mouse heart, by performing ${\mathrm{Ca}}^{2+}$ imaging using the loaded fluorescent calcium indicator Fluo-4AM. Acute myocardial slices are an experimental system well representing the intact myocardium, allowing the use of state-of-the art imaging methods to explore ${\mathrm{Ca}}^{2+}$ signals from the subcellular to the multicellular range.

We used the MMM system to perform two different sets of experiments. In the first, ${\mathrm{Ca}}^{2+}$ imaging was performed at 16-Hz rate during field stimulation in a $137\times 137\mu \mathrm{m}$ wide area of the myocardial slice, typically including six to eight cardiomyocytes. Electrical pacing by field stimulation resulted in a synchronous increase in intracellular ${\mathrm{Ca}}^{2+}$ in the cardiomyocytes in the imaging field, as reported in a typical experiment shown in Fig. 3(a). At the acquisition rate used in the experiments, it was possible to fit the ${\mathrm{Ca}}^{2+}$ transients’ decay with a single exponential function obtaining a typical decay time in the range of 100 to 150 ms ($n=14\text{cells}$), in line with data in the literature.²¹ In the subsequent set of experiments, MMM was used to investigate ${\mathrm{Ca}}^{2+}$ dynamics with a subcellular resolution in a networked group of cells in the myocardial slices.

Fig. 3

(a) Intensity profiles of three neighboring cells in the heart slice. Cell borders are highlighted by dotted squares and the fluorescence profiles are reported. It can be seen that cell 2 crosses two subframes without affecting temporal fluorescence profiles. (b) Simultaneous onset or macrosparks in two of the three regions of interest (ROI) considered with the respective intensity profiles. (c) A travelling ${\mathrm{Ca}}^{2+}$ wave recorded in the same cell and intensity profiles of the ROIs considered.

The spatial resolution of MMM at a 16 Hz frame rate was assessed by imaging subcellular ${\mathrm{Ca}}^{2+}$ dynamics in a wide region of the slice. The elementary ${\mathrm{Ca}}^{2+}$ signals regulating the excitation–contraction coupling in heart cells are represented by ${\mathrm{Ca}}^{2+}$ “sparks,”²² i.e., transient and localized releases of ${\mathrm{Ca}}^{2+}$ from the sarcoplasmic reticulum (SR) through the ryanodine receptor ${\mathrm{Ca}}^{2+}$ channels.

A pace-stop protocol was used to increase the frequency of ${\mathrm{Ca}}^{2+}$ release events. Intracellular and local ${\mathrm{Ca}}^{2+}$ release events, both in the form of secondary “macrosparks” [Fig. 3(b)] of approximately 4 to 6 μm in diameter, and self-sustained ${\mathrm{Ca}}^{2+}$ waves traveling through the cell [Fig. 3(c)] could be imaged simultaneously in numerous cardiomyocytes in the slice. Such cell-wide ${\mathrm{Ca}}^{2+}$ waves frequently originated from the same ${\mathrm{Ca}}^{2+}$ release hotspot and, in some cases, were underlined by organized intracellular ${\mathrm{Ca}}^{2+}$ rotors (see Fig 4). Moreover, it was possible to appreciate that the neighboring cells displayed ${\mathrm{Ca}}^{2+}$ waves in a pattern of interdependency (Fig. 5).

Fig. 4

Travelling ${\mathrm{Ca}}^{2+}$ waves and rotors in the same region of Fig. 3(b) (MOV, 7.93 MB). [URL: http://dx.doi.org/10.1117/1.JBO.20.5.051016.1.]

Fig. 5

Three neighboring cells in a large field of view display spontaneous ${\mathrm{Ca}}^{2+}$ releases closely related in time.