Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.     

Photoacoustic tomography of foreign bodies in soft biological tissue

In detecting small foreign bodies in soft biological tissue, ultrasound imaging suffers from poor sensitivity (52.6%) and specificity (47.2%). Hence, alternative imaging methods are needed. Photoacoustic (PA) imaging takes advantage of strong optical absorption contrast and high ultrasonic resolution. A PA imaging system is employed to detect foreign bodies in biological tissues. To achieve deep penetration, we use near-infrared light ranging from 750 to 800 nm and a 5-MHz spherically focused ultrasonic transducer. PA images were obtained from various targets including glass, wood, cloth, plastic, and metal embedded more than 1 cm deep in chicken tissue. The locations and sizes of the targets from the PA images agreed well with those of the actual samples. Spectroscopic PA imaging was also performed on the objects. These results suggest that PA imaging can potentially be a useful intraoperative imaging tool to identify foreign bodies.     

基于时域后向投影的光声谱成像系统研究

光声谱成像是一种新的生物组织成像方法,它结合光学成像和超声成像的特点,可提供高分辨率和对比度的图像。采用波长532nm、重复频率10Hz的脉冲激光作为激励源,宽带PVDF非聚焦超声探测器频率响应范围为200kHz～15MHz,探测器以圆周扫描的方式采集样品的时域光声信号,并采用时域后向投影算法重建样品内部的二维光学吸收分布图像。实验表明,系统成像的空间分辨率小于1mm,重建的图像与原始样品完全吻合。     

Can Molecular Imaging Enable Personalized Diagnostics? An Example Using Magnetomotive Photoacoustic Imaging

The advantages of photoacoustic (PA) imaging, including low cost, non-ionizing operation, and sub-mm spatial resolution at centimeters depth, make it a promising modality to probe nanoparticle-targeted abnormalities in real time at cellular and molecular levels. However, detecting rare cell types in a heterogeneous background with strong optical scattering and absorption remains a big challenge. For example, differentiating circulating tumor cells in vivo (typically fewer than 10 cells/mL for an active tumor) among billions of erythrocytes in the blood is nearly impossible. In this paper, a newly developed technique, magnetomotive photoacoustic (mmPA) imaging, which can greatly increase the sensitivity and specificity of sensing targeted cells or molecular interactions, is reviewed. Its primary advantage is suppression of background signals through magnetic enrichment/manipulation with simultaneous PA detection of magnetic contrast agent targeted objects. Results from phantom and in vitro studies demonstrate the capability of mmPA imaging to differentiate regions targeted with magnetic nanoparticles from the background, and to trap and sensitively detect targeted cells at a concentration of a single cell per milliliter in a flow system mimicking a human peripheral artery. This technique provides an example of the ways in which molecular imaging can potentially enable robust molecular diagnosis and treatment, and accelerate the translation of molecular medicine into the clinic.     

Multimodality optical imaging of embryonic heart microstructure

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.     

Imaging of joints with laser-based photoacoustic tomography: an animal study

Photoacoustic tomography (PAT), a nonionizing, noninvasive, laser-based technology was adapted to joint imaging for the first time. Pulsed laser light in the near-infrared region was directed toward a joint with resultant ultrasonic signals recorded and used to reconstruct images that present the optical properties in subsurface joint tissues. The feasibility of this joint imaging system was validated on a Sprague Dawley rat tail model and verified through comparison with histology. With sufficient penetration depth, PAT realized tomographic imaging of a joint as a whole organ noninvasively. Based on the optical contrast, various intra- and extra-articular tissues, including skin, fat, muscle, blood vessels, synovium and bone, were presented successfully in images with satisfactory spatial resolution that was primarily limited by the bandwidth of detected photoacoustic signals rather than optical diffusion as occurs in traditional optical imaging. PAT, with its intrinsic advantages, may provide a unique opportunity to enable the early diagnosis of inflammatory joint disorders, e.g., rheumatoid arthritis, and to monitor therapeutic outcomes with high sensitivity and accuracy.     

Dual imaging-guided photothermal/photodynamic therapy using micelles

We report a type of photosensitizer (PS)-loaded micelles integrating cyanine dye as potential theranostic micelles for precise anatomical tumor localization via dual photoacoustic (PA)/near-infrared fluorescent (NIRF) imaging modalities, and simultaneously superior cancer therapy via sequential synergistic photothermal therapy (PTT)/photodynamic therapy (PDT). The micelles exhibit enhanced photostability, cell internalization and tumor accumulation. The dual NIRF/PA imaging modalities of the micelles cause the high imaging contrast and spatial resolution of tumors, which provide precise anatomical localization of the tumor and its inner vasculature for guiding PTT/PDT treatments. Moreover, the micelles can generate severe photothermal damage on cancer cells and destabilization of the lysosomes upon PTT photoirradiation, which subsequently facilitate synergistic photodynamic injury via PS under PDT treatment. The sequential treatments of PTT/PDT trigger the enhanced cytoplasmic delivery of PS, which contributes to the synergistic anticancer efficacy of PS. Our strategy provides a dual-modal cancer imaging with high imaging contrast and spatial resolution, and subsequent therapeutic synergy of PTT/PDT for potential multimodal theranostic application.     

Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment

Cancer metastasis involves complex cell behavior and interaction with the extracellular matrix by metabolically active cells. To observe invasion and metastasis with sub-cellular resolution in vivo, multiphoton microscopy (MPM) allows imaging more deeply into tissues with less toxicity, compared with other optical imaging methods. MPM can be combined with second harmonic generation (SHG), fluorescent lifetime imaging microscopy (FLIM), and spectral-lifetime imaging microscopy (SLIM). SHG facilitates imaging of stromal collagen and tumor–stroma interactions, including the architecture and remodeling of the tumor microenvironment. FLIM allows characterization of exogenous and endogenous fluorophores, such as the metabolites FAD and NADH to score for metabolic state and provide optical biomarkers. SLIM permits additional identification and separation of endogenous and exogenous fluorophores by simultaneously collecting their spectra and lifetime, producing an optical molecular “fingerprint”. Both FLIM and SLIM also serve as an improved method for the assessment of Förster (or fluorescence) resonance energy transfer (FRET). Hence, the use and further development of these approaches strongly enhances the visualization and quantification of tumor progression, invasion, and metastasis. Herein, we review recent developments of multiphoton FLIM and SLIM to study 2D and 3D cell migration, invasion into the tumor microenvironment, and metastasis.     

In vivo real-time visualization of tissue blood flow and angiogenesis using Ag2S quantum dots in the NIR-II window

Improving the tissue penetration depth and spatial resolution of fluorescence-based optical nanoprobes remains a grand challenge for their practical applications in in vivo imaging, due to the scattering and absorption and endogenous autofluorescence of living tissues. Here, we present that Ag₂S quantum dots (QDs), containing no toxic ions, exhibiting long circulation time and high stability, act as a new kind of fluorescent probes in the second near-infrared window (NIR-II, 1000–1350 nm) which enable in vivo monitoring of lymphatic drainage and vascular networks with deep tissue penetration and high spatial and temporal resolution. In addition, NIR-II fluorescence imaging with Ag₂S QDs provide ultrahigh spatial resolution (∼40 μm) that permits us to track angiogenesis mediated by a tiny tumor (2–3 mm in diameter) in vivo. Our results indicate that Ag₂S QDs are promising NIR-II fluorescent nanoprobes that could be useful in surgical treatments such as sentinel lymph node (SLN) dissection as well in assessment of blood supply in tissues and organs and screening of anti-angiogenic drugs.     

Dynamic imaging of PEGylated indocyanine green (ICG) liposomes within the tumor microenvironment using multi-spectral optoacoustic tomography (MSOT)

Multispectral optoacoustic tomography (MSOT) is a powerful modality that allows high-resolution imaging of photo-absorbers deep within tissue, beyond the classical depth and resolution limitations of conventional optical imaging. Imaging of intrinsic tissue contrast can be complemented by extrinsically administered gold nanoparticles or fluorescent molecular probes. Instead, we investigated herein generation of re-engineered clinically-used PEGylated liposomes incorporating indocyanine green (LipoICG) as a contrast strategy that combines materials already approved for clinical use, with strong photo-absorbing signal generation available today only from some metallic nanoparticles (e.g. gold nanorods). Using MSOT we confirmed LipoICG as a highly potent optoacoustic agent and resolved tissue accumulation in tumor-bearing animals over time with high-sensitivity and resolution using two tumor models of different vascularisation. We further showcase a paradigm shift in pharmacology studies and nanoparticle investigation, by enabling detailed volumetric optical imaging in vivo through the entire tumor tissue non-invasively, elucidating never before seen spatiotemporal features of optical agent distribution. These results point to LipoICG as a particle with significant advantageous characteristics over gold nanoparticles and organic dyes.     

Deformation-compensated averaging for clutter reduction in epiphotoacoustic imaging in vivo

Photoacoustic imaging, based on ultrasound detected after laser irradiation, is an extension to diagnostic ultrasound for imaging the vasculature, blood oxygenation and the uptake of optical contrast media with promise for cancer diagnosis. For versatile scanning, the irradiation optics is preferably combined with the acoustic probe in an epi-style arrangement avoiding acoustically dense tissue in the acoustic propagation path from tissue irradiation to acoustic detection. Unfortunately epiphotoacoustic imaging suffers from strong clutter, arising from optical absorption in tissue outside the image plane, and from acoustic backscattering. This limits the imaging depth for useful photoacoustic image contrast to typically less than one centimeter. Deformation-compensated averaging (DCA), which takes advantage of clutter decorrelation induced by palpating the tissue with the imaging probe, has previously been proposed for clutter reduction. We demonstrate for the first time that DCA results in reduced clutter in real-time freehand clinical epiphotoacoustic imaging. For this purpose, combined photoacoustic and pulse-echo imaging at 10-Hz frame rate was implemented on a commercial scanner, allowing for ultrasound-based motion tracking inherently coregistered with photoacoustic frames. Results from the forearm and the neck confirm that contrast is improved and imaging depth increased by DCA.     

生物磁光声联合成像的研究现状

超声(US)成像、光学相干断层(OCT)成像和磁共振成像(MRI)是临床常用的医学成像手段。此外,光声层析(PAT)和磁声(MA)成像是近年来新兴的多物理场耦合功能成像手段。将两种或者两种以上的成像手段结合起来形成多模态联合成像,可以使各成像模态的优点得到充分展现,对目标进行高精度和高分辨率的成像,精确识别病变组织,并对其功能成分进行定性和定量的分析。对US-PAT、US-OCT、PAT-OCT、US-PAT-OCT、磁光以及磁光声(MPA)联合成像,特别是对血管内联合成像的研究进展和临床应用前景进行综述,总结目前存在的问题,并展望未来可能的发展方向。     

Optical techniques for tracking multiple myeloma engraftment, growth, and response to therapy

Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivo flow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivo at the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods.     

Lenses and effective spatial resolution in macroscopic optical mapping

Optical mapping of excitation dynamically tracks electrical waves travelling through cardiac or brain tissue by the use of fluorescent dyes. There are several characteristics that set optical mapping apart from other imaging modalities: dynamically changing signals requiring short exposure times, dim fluorescence demanding sensitive sensors and wide fields of view (low magnification) resulting in poor optical performance. These conditions necessitate the use of optics with good light gathering ability, i.e. lenses having high numerical aperture. Previous optical mapping studies often used sensor resolution to estimate the minimum spatial feature resolvable, assuming perfect optics and infinite contrast. We examine here the influence of finite contrast and real optics on the effective spatial resolution in optical mapping under broad-field illumination for both lateral (in-plane) resolution and axial (depth) resolution of collected fluorescence signals.     

Dual-mode imaging with radiolabeled gold nanorods

Many nanoparticle contrast agents have difficulties with deep tissue and near-bone imaging due to limited penetration of visible photons in the body and mineralized tissues. We are looking into the possibility of mediating this problem while retaining the capabilities of the high spatial resolution associated with optical imaging. As such, the potential combination of emerging photoacoustic imaging and nuclear imaging in monitoring of antirheumatic drug delivery by using a newly developed dual-modality contrast agent is investigated. The contrast agent is composed of gold nanorods (GNRs) conjugated to the tumor necrosis factor (TNF-α) antibody and is subsequently radiolabeled by (125)I. ELISA experiments designed to test TNF-α binding are performed to prove the specificity and biological activity of the radiolabeled conjugated contrast agent. Photoacoustic and nuclear imaging are performed to visualize the distribution of GNRs in articular tissues of the rat tail joints in situ. Findings from the two imaging modalities correspond well with each other in all experiments. Our system can image GNRs down to a concentration of 10 pM in biological tissues and with a radioactive label of 5 μCi. This study demonstrates the potential of combining photoacoustic and nuclear imaging modalities through one targeted contrast agent for noninvasive monitoring of drug delivery as well as deep and mineralized tissue imaging.     

Three-dimensional imaging of xenograft tumors using optical computed and emission tomography

The physical basis and preliminary applications of optical computed tomography (optical-CT) and optical emission computed tomography (optical-ECT) are introduced, as new techniques with potential to provide unique 3D information on a variety of aspects of tumor structure and function. A particular focus here is imaging tumor micro-vasculature, and the spatial distribution of viable tumor cells, although the techniques have the potential for much wider application. The principle attractiveness of optical-CT and optical-ECT are that high resolution (<20 microm) and high contrast co-registered 3D images of structure and function can be acquired for relatively large intact samples. The unique combination of high contrast and resolution offers advantages over micro-CT and micro-MRI, and the lack of requirement for sectioning offers advantages over confocal microscopy, conventional microscopy, and histological sectioning techniques. Optical-CT/ECT are implemented using in-house custom apparatus and a commercial dissecting microscope capable of both transmission and fluorescence imaging. Basic studies to characterize imaging performance are presented. Negligible geometrical distortion and accurate reconstruction of relative attenuation coefficients was observed. Optical-CT and optical-ECT are investigated here by application to high resolution imaging of HCT116 xenograft tumors, about 1 cc in dimension, which were transfected with constitutive red fluorescent protein (RFP). Tumor microvasculature was stained in vivo by tail vein injection of either passive absorbing dyes or active fluorescent markers (FITC conjugated lectin). Prior to imaging, the tumors were removed (ex vivo) and optically cleared in a key process to make the samples amenable to light transmission. The cleared tumors were imaged in three modes (i) optical-CT to image the 3D distribution of microvasculature as indicated by absorbing dye, (ii) optical-ECT using the FITC excitation and emission filter set, to determine microvasculature as indicated by lectin-endothelial binding, and (iii) optical-ECT using the DSRed2 filter set to determine the 3D distribution of viable tumor as indicated by RFP emission. A clear correlation was observed between the independent vasculature imaging modes (i) and (ii) and postimaging histological sections, providing substantial validation of the optical-CT and optical-ECT techniques. Strong correlation was also observed between the RFP imaging of mode iii, and modes i and ii, supporting the intuitive conclusion that well-perfused regions contain significant viable tumor. In summary, optical-CT and optical-ECT, when combined with new optical clearing techniques, represent powerful new imaging modalities with potential for providing unique information on the structure and function of tumors.     

Deep in vivo two-photon imaging of blood vessels with a new dye encapsulated in pluronic nanomicelles

Our purpose is to test if Pluronic? fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.     

Tumor Functional and Molecular Imaging Utilizing Ultrasound and Ultrasound-Mediated Optical Techniques

Tumor functional and molecular imaging has significantly contributed to cancer preclinical research and clinical applications. Among typical imaging modalities, ultrasonic and optical techniques are two commonly used methods; both share several common features such as cost efficiency, absence of ionizing radiation, relatively inexpensive contrast agents, and comparable maximum-imaging depth. Ultrasonic and optical techniques are also complementary in imaging resolution, molecular sensitivity, and imaging space (vascular and extravascular). The marriage between ultrasonic and optical techniques takes advantages of both techniques. This review introduces tumor functional and molecular imaging using microbubble-based ultrasound and ultrasound-mediated optical imaging techniques.Tumor in vivo and noninvasive imaging has significantly contributed to cancer preclinical research and clinical applications.¹ With the introduction of contrast agents, imaging techniques have been extended from conventional structural (anatomical) imaging to functional (physiological) and molecular (biochemical) imaging.^1,2 Although structural information is important for tumor localization and assessment of size, shape, and boundary, functional/molecular imaging can evaluate tumor physiological status such as abnormal microenvironments, hypoxia and angiogenesis, metabolism, malignancy, and metastasis, which is critical, not only for clinical applications, but also for understanding cancer’s underlying mechanisms (via investigating gene expression, signaling pathways, receptors, apoptosis, etc.).^1,2Currently, several techniques are widely used for preclinical and clinical cancer imaging, such as computed tomography (CT), single-photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, and optical techniques.^1,2 These imaging modalities have been very successful in tumor imaging. Unfortunately, none of them is perfect. Although imaging depth is not a problem, clinically used CT and MRI have a spatial resolution of ∼1 mm, and clinically used PET and SPECT provide an even worse spatial resolution (>3 mm). Micro-CT (∼20 to 50 μm) and micro-MRI (∼50 μm) can provide high-resolution images by reducing imaging volume and increasing exposure dosage. However, CT or micro-CT may not be an excellent tool to differentiate different soft tissues. MRI and micro-MRI have been used for tissue molecular imaging. One of the limitations is that the sensitivity to imaging contrast agents is low compared with PET, SPECT, and optical techniques. In addition, due to the complexity of cancer, no single imaging modality is sufficient to obtain all of the desired information.³ Contrast-enhanced techniques and multimodality imaging are expected to offer new and synergistic advantages over one modality on its own.^3–5Ultrasound and optical techniques share many common features.^5–7 For example, both are cost efficient and safe. Also, their maximum imaging depths are comparable and can reach down to tens of millimeters with fast imaging speed,⁸ which is excellent for studying small animals and some human organs, such as the breast and prostate. The contrast agents of both techniques have been well developed and are generally inexpensive (for example, microbubbles for ultrasound and dyes or nanoparticles for optical imaging).Besides the common features, the two imaging techniques are also complementary.⁶ Although optical techniques suffer from low spatial resolution (2 to 5 mm for diffuse optical tomography) due to light scattering in deep tissues (20 to 50 mm), ultrasound is much less scattered by tissue and features with relatively higher resolutions, tens to hundreds of microns depending on the frequency, at similar imaging depth. Conversely, ultrasound alone is insensitive to tissue functional and molecular information, and requires the use of exogenous contrast agents.⁵ Microbubbles are the most efficacious contrast agent for ultrasound imaging. Because of the relatively large size (a few microns in diameter), microbubbles are usually limited to inside the tumor’s vascular system for detecting blood perfusion, velocity, and intravascular molecular targets.^5,8 By contrast, optical techniques are not only sensitive to tissue functional/molecular events and capable of simultaneous imaging of multiple molecular events via spectroscopic techniques, but also can image both vascular and extravascular molecular targets.⁹ Accordingly, microbubble-based ultrasound and ultrasound-mediated optical imaging techniques have been intensively developed during the past years for tumor imaging.^5,6,8,10 As expected, the marriage between ultrasound and optical techniques takes advantages of both techniques, and significant progress has been achieved recently.^6,10In this review, microbubble-enhanced ultrasound tumor imaging will be introduced first. It is followed by ultrasound-mediated optical imaging techniques that include: i) ultrasound-guided diffuse optical tomography (UG-DOT)¹⁰; ii) photoacoustic tomography/microscopy (PAT/PAM)⁶; and iii) ultrasound-modulated optical tomography/microscopy (UMOT/UMOM).¹¹     

Boundary conditions in photoacoustic tomography and image reconstruction

Recently, the field of photoacoustic tomography has experienced considerable growth. Although several commercially available pure optical imaging modalities, including confocal microscopy, two-photon microscopy, and optical coherence tomography, have been highly successful, none of these technologies can penetrate beyond approximately 1 mm into scattering biological tissues because all of them are based on ballistic and quasiballistic photons. Consequently, heretofore there has been a void in high-resolution optical imaging beyond this depth limit. Photoacoustic tomography has filled this void by combining high ultrasonic resolution and strong optical contrast in a single modality. However, it has been assumed in reconstruction of photoacoustic tomography until now that ultrasound propagates in a boundary-free infinite medium. We present the boundary conditions that must be considered in certain imaging configurations; the associated inverse solutions for image reconstruction are provided and validated by numerical simulation and experiment. Partial planar, cylindrical, and spherical detection configurations with a planar boundary are covered, where the boundary can be either hard or soft. Analogously to the method of images of sources, which is commonly used in forward problems, the ultrasonic detectors are imaged about the boundary to satisfy the boundary condition in the inverse problem.     

Simulation of voltage-sensitive optical signals in three-dimensional slabs of cardiac tissue: application to transillumination and coaxial imaging methods

Voltage-sensitive dyes are an important tool in visualizing electrical activity in cardiac tissue. Until today, they have mainly been applied in cardiac electrophysiology to subsurface imaging. In the present study, we assess different imaging methods used in optical tomography with respect to their effectiveness in visualizing 3D cardiac activity. To achieve this goal, we simulate optical signals produced by excitation fronts initiated at different depths inside the myocardial wall and compare their properties for various imaging modes. Specifically, we consider scanning and broad-field illumination, including trans- and epi-illumination. We focus on the lateral optical resolution and signal intensity, as a function of the source depth. Optical diffusion theory is applied to derive a computationally efficient approximation of the point-spread function and to predict voltage-sensitive signals. Computations were performed both for fluorescent and absorptive voltage-sensitive dyes. Among all the above-mentioned methods, fluorescent coaxial scanning yields the best resolution (<2.5 mm) and gives the most information about the intramural cardiac activity.