We describe a method to accurately measure the light scattering model parameters from forward-directed flux (FDF) measurements carried out with a fiber-optic probe (optrode). Improved determination of light scattering parameters will, in turn, permit better modeling and interpretation of optical mapping in the heart using voltage-sensitive dyes. Using our optrode-based system, we carried out high spatial resolution measurements of FDF in intact and homogenized cardiac tissue, as well as in intralipid-based tissue phantoms. The samples were illuminated with a broad collimated beam at 660 and 532 nm. Measurements were performed with a plunge fiber-optic probe (NA=0.22) at a spatial resolution of up to 10 μm. In the vicinity of the illuminated surface, the FDF consistently manifested a fast decaying exponent with a space constant comparable with the decay rate of ballistic photons. Using a Monte Carlo model, we obtained a simple empirical formula linking the rate of the fast exponent to the scattering coefficient, the anisotropy parameter g, and the numerical aperture of the probe. The estimates of scattering coefficient based on this formula were validated in tissue phantoms. Potential applications of optical fiber-based FDF measurements for the evaluation of optical parameters in turbid media are discussed.
Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5 mW/mm 2 , with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30% . At 660-nm excitation, the effect is ∼10% . These findings may have important implications for the design of optical mapping protocols in biomedical applications.
Sudden cardiac death is often caused by ventricular arrhythmias. These arrhythmias are believed to originate from the
border zones where tissue was damaged by an ischemic event involving the coronary arteries. The specific mechanisms
relating the geometry of these territories to the electrical behavior remains poorly understood. A major problem is the
lack of detailed information describing the morphology of the affected perfusion bed. We present the first perfusion MR
images of excised whole heart preparations where the irregular boundaries of perfusion territories are described. The
filling pattern and final volume of the RCA perfusion territory are clearly visualized.
Optical imaging using voltage-sensitive dyes has become an important tool for studying vortex-like electrical waves in the heart. Such waves, known as spiral or scroll waves, can spontaneously form in pathological ventricular myocardium, causing ventricular fibrillation and sudden death. Until recently, observations of scroll waves were limited to their surface manifestations, thus providing little information about the shape and location of their organizing center, the filament. We use computer modeling to assess the feasibility of visualizing filaments using dynamic transillumination imaging in conjunction with near-IR voltage-sensitive absorptive dyes (absorptive transillumination). We simulate transillumination signals produced by the intramural scroll waves in a realistic slab of ventricular tissue with trabeculated endocardial surface. The computations use a detailed ionic model of electrical excitation (LRd) coupled to a photon transport model for cardiac tissue. Our simulations show that dynamic absorptive transillumination data, with subsequent processing involving either amplitude maps, time-space plots, or power-of-the-dominant-frequency maps, can be used to reliably detect intramural scroll waves through the whole thickness (~10 mm) of the ventricular wall. Neither variations in the thickness of the myocardial wall nor noise impeded the detection of intramural filaments.
This study explores the possibility of localizing the excitation centers of electrical waves inside the heart wall using voltage-sensitive dyes (fluorescent or absorptive). In the present study, we propose a method for the 3-D localization of excitation centers from pairs of 2-D images obtained in two modes of observation: reflection and transillumination. Such images can be obtained using high-speed charge-coupled device (CCD) cameras and photodiode arrays with time resolution up to 0.5 ms. To test the method, we simulate optical signals produced by point sources and propagating ellipsoidal waves in 1-cm-thick slabs of myocardial tissue. Solutions of the optical diffusion equation are constructed by employing the method of images with Robin boundary conditions. The coordinates of point sources as well as of the centers of expanding waves can be accurately determined using the proposed algorithm. The method can be extended to depth estimations of the outer boundaries of the expanding wave. The depth estimates are based on ratios of spatially integrated images. The method shows high tolerance to noise and can give accurate results even at relatively low signal-to-noise ratios. In conclusion, we propose a novel and efficient algorithm for the localization of excitation centers in 3-D cardiac tissue.
Until recently, optical mapping of electrical activity in the heart muscle using voltage-sensitive dyes has mainly been applied to subsurface imaging. Here we present a method for the three-dimensional (3D) reconstruction of electrical activity deep inside the myocardial wall. We propose an alternative approach to diffusive optical tomography, based on ideas from binocular vision. Detection and illumination occur on opposite sides of the preparation. Staining with absorptive voltage-sensitive dyes is assumed. Data acquisition follows a paraxial scanning procedure, which modifies coaxial scanning by the introduction of a vector offset between illumination and detection axes. Pairs of 2D images are obtained corresponding to offsets of opposite signs. Those image pairs created by parallax are used as an input for the reconstruction algorithm, whose output is a 3D optical image of intramural electrical excitation. We apply this method to the slab geometry. The procedure was tested for a variety of computer-generated sources including particles, lines, bubbles, and simulated electrophysiological patterns such as scroll waves. The limitations of the method and possible improvements are discussed.
Voltage-sensitive dyes have become an important tool in visualizing electrical activity in cardiac tissue. However, there are no established methods for assessing the contribution of intramural electrical excitation to recorded optical signals. Here, we develop algorithms to calculate voltage-dependent optical signals from three-dimensional distributions of transmembrane voltage inside the myocardial wall (the forward problem). Optical diffusion theory is applied for different imaging modes including subsurface imaging or epi-illumination, transillumination and coaxial scanning. We use the solutions of the forward problem to assess these imaging methods with respect to their effectiveness in visualizing two types of 3D cardiac activity: electrical point sources and intramural scroll waves initiated at various depths. Simulations were performed both for fluorescent and absorptive voltage-sensitive dyes. In the case of point sources, we focus on the lateral optical resolution, as a function of the source depth. We find that, among the studied methods, fluorescent coaxial scanning yields the best optical resolution (<2.5 mm). In the case of scroll waves we investigate how well the filament, i.e. the organizing center, can be visualized as function of its depth. Our results show that using absorptive transillumination, filaments can be detected up to 3 mm below the recording surface. The presented results provide a powerful tool for the interpretation of experimental data and are the first step towards the development of inverse procedures.
Optical methods provide a rather precise insight into cardiac electrical activity. Voltage-sensitive dyes like di 4-ANEPPS convert the electric signal into a fluorescent signal that can be measured by standard optical methods. A realistic picture of the dynamic patterns that govern electrical activity in the human heart can be obtained only with thick tissue preparations, from large animals. We measure the fluorescence signal of an approximately 2.5 x 2.5 cm area on the surface of 8 mm thick porcine right ventricle preparations with a fast CCD camera at low magnification, and perform advanced simulations of the macroscopic dynamic features involved. To extract meaningful qualitative and quantitative data from these signals, details of the conversion from electrical to optical signal have to be known, and the problem of the 2D surface signal originating from a 3D distribution below has to be addressed.
We compare experiment to simulation results applying a composite model based on both electrical and optical tissue properties. The model predicts optical action potential upstroke morphology, involving optical point spread functions and simplified Beeler-Reuter kinetics for the electrical wave propagation. Optical point spread functions have been calculated from scattering and absorption properties applying diffusion models and Monte-Carlo simulations. First of all, the forward problem has been solved for uniform light illumination and simulations have been compared to experiments. Furthermore, we also address the question of the inverse problem and provide an analysis of the limitations for this approach.
Optical imaging of ex vivo tissue models to study heart fibrillation is normally performed using voltage-sensitive dyes. Upon stimulation by an electrode, time-dependent fluorescence or absorption signals are recorded, often in trans-illumination geometry. In order to provide quantification of the origins of these signals inside the tissue, the locally varying optical properties of the tissue have to be known and their change due to the presence of the dyes. To provide experimental input for further modeling efforts, we have performed depth dependent measurements with a fiber optic laser source inside the tissue, recording light profiles on the tissue surface, mainly in transmission geometry. From these measurements, optical properties have been extracted and the obtained profiles have been used as input into a preliminary image reconstruction scheme, together with Monte Carlo simulations. Experiments at different locations in the same sample show the variation of optical properties. Additionally, effects from the presence of heterogeneities on the signal have been investigated.
In order to provide depth resolution for bulk tissue imaging experiments using absorption signals, we have designed an internal laser point spread technique. A laser light source has been imbedded in different depths into cardiac tissue and tissue phantoms, the signal on the tissue surface detected by a CCD detector. These measurements in combination with an analytic solution of the diffusion equation allow us to estimate optical properties of the investigated tissue. We show how this information provides the core of depth quantification of fluorescence and absorption measurements in bulk tissue and investigate experimentally the transition from single scattering to diffuse photon transport in cardiac tissue and suspensions of microscopic spherical particles that serve as model systems.
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