List of reviewers who served JBO in 2019.

JBO guest editors introduce the Special Section Celebrating Thirty Years of Multiphoton Microscopy in the Biomedical Sciences.

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.

Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a square-shaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans ∼5.6  dB for the GB and ∼1.2  dB for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase ∼20  %   area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.

Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. With a high concentration in basal cell cytoplasm, THG contrast provided by melanin overshadows other THG sources in human skin studies. For better understanding of the THG signals in keratinocytes without the influence of melanin, an in vivo THG microscopy (THGM) study was first conducted on vitiliginous skin. As a result, the THG-brightness ratio between the melanin-lacking cytoplasm of basal cells and collagen fibers is about 1.106 at the dermal–epidermal junctions of vitiliginous skin, indicating high sensitivity of THGM for the presence of melanin. We further applied the in vivo THGM to assist evaluating the therapeutic outcome from the histopathological point of view for those showed no improvement under narrowband ultraviolet B therapy based on the seven-point Physician Global Assessment score. Our clinical study indicates the high potential of THGM to assist the histopathological assessment of the therapeutic efficacy of vitiligo treatments.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

The excited state lifetime of a fluorophore together with its fluorescence emission spectrum provide information that can yield valuable insights into the nature of a fluorophore and its microenvironment. However, it is difficult to obtain both channels of information in a conventional scheme as detectors are typically configured either for spectral or lifetime detection. We present a fiber-based method to obtain spectral information from a multiphoton fluorescence lifetime imaging (FLIM) system. This is made possible using the time delay introduced in the fluorescence emission path by a dispersive optical fiber coupled to a detector operating in time-correlated single-photon counting mode. This add-on spectral implementation requires only a few simple modifications to any existing FLIM system and is considerably more cost-efficient compared to currently available spectral detectors.

Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in-vitro β-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in-situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify β-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.

Conventional techniques are insufficient precisely to describe the internal structure, the heterogeneous cell populations, and the dynamics of biological processes occurring in diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to help avoid postoperative liver failure. We analyze the progression of both acute (cholestasis) and chronic (fibrosis) liver pathology using multiphoton microscopy with fluorescence lifetime imaging and second-harmonic generation modes combined with time-of-flight secondary ion mass spectrometry chemical analysis to obtain new data about pathological changes to hepatocytes at the cellular and molecular levels. All of these techniques allow the study of cellular metabolism, lipid composition, and collagen structure without staining the biological materials or the incorporation of fluorescent or other markers, enabling the use of these methods in a clinical situation. The combination of multiphoton microscopy and mass spectrometry provides more complete information about the liver structure and function than could be assessed using either method individually. The data can be used both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique for liver quality analysis in order to plan surgery and to help avoid postoperative liver failure in clinic.

We describe the contribution of our in vivo multiphoton microscopy (MPM) studies over the last ten years with DermaInspect^® (JenLab, Germany), a CE-certified medical tomograph based on detection of fluorescent biomolecules, to the assessment of possible penetration of nanoparticulate zinc oxide in sunscreen through human skin. At the time we started our work, there was a strong movement for the precautionary principle to be applied to the use of nanoparticles in consumer products due to a lack of knowledge. The combined application of different MPM modalities, including spectral imaging, fluorescence lifetime imaging, second harmonic fluorescence generation, and phosphorescence microscopy, has provided overwhelming evidence that nanoparticle zinc oxide particles do not penetrate human skin when applied to various skin types with a range of methods of topical sunscreen application. MPM has also been used to study the viable epidermal morphology and redox state in supporting the safe use of topical zinc oxide nanoparticles. The impact of this work is emphasized by the recent proposed rule by the United States FDA on Sunscreen Drug Products for Over-the-Counter Human Use, which listed only zinc oxide and titanium dioxide of the currently marketed products to be generally recognized as safe and effective.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

The historical development of multiphoton microscopy is described, starting with a review of two-photon absorption, and including two- and three-photon fluorescence microscopies, and second- and third-harmonic generation microscopies. The effects of pulse length on signal strength and breakdown are considered. Different contrast mechanisms, including use of nanoparticles, are discussed. Two new promising techniques that can be applied to multiphoton microscopy are described.

Temporal changes in macrophage metabolism are likely crucial to their role in inflammatory diseases. Label-free two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy are well suited to track dynamic changes in macrophage metabolism. We performed TPEF imaging of human macrophages following either pro- or an anti-inflammatory stimulation. Two endogenous fluorophores, NAD(P)H and FAD, coenzymes involved in key metabolic pathways, provided contrast. We used the corresponding intensity images to determine the optical redox ratio of FAD to FAD + NAD(P)H. We also analyzed the intensity fluctuation patterns within NAD(P)H TPEF images to determine mitochondrial clustering patterns. Finally, we acquired NAD(P)H TPEF lifetime images to assess the relative levels of bound NAD(P)H. Our studies indicate that the redox ratio increases, whereas mitochondrial clustering decreases in response to both pro- and anti-inflammatory stimuli; however, these changes are enhanced in pro-inflammatory macrophages. Interestingly, we did not detect any significant changes in the corresponding NAD(P)H bound fraction. A combination of optical metabolic metrics could be used to classify pro- and anti-inflammatory macrophages with high accuracy. Contributions from alterations in different metabolic pathways may explain our findings, which highlight the potential of label-free two-photon imaging to assess nondestructively macrophage functional state.

Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets.

Two-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies for in vivo histology. We report on clinical multiphoton coherent anti-Stokes Raman spectroscopy (CARS) tomography based on two tunable ultrashort near-infrared laser beams for label-free in vivo multimodal skin imaging. The multiphoton biopsies were obtained with the compact tomograph “MPTflex-CARS” using a photonic crystal fiber, an optomechanical articulated arm, and a four-detector-360 deg measurement head. The multiphoton tomograph has been employed to patients in a hospital with diseased skin. The clinical study involved 16 subjects, 8 patients with atopic dermatitis, 4 patients with psoriasis vulgaris, and 4 volunteers served as control. Two-photon cellular autofluorescence lifetime, second harmonic generation (SHG) of collagen, and CARS of intratissue lipids/proteins have been detected with single-photon sensitivity, submicron spatial resolution, and picosecond temporal resolution. The most important signal was the autofluorescence from nicotinamide adenine dinucleotide [NAD(P)H]. The SHG signal from collagen was mainly used to detect the epidermal–dermal junction and to calculate the ratio elastin/collagen. The CARS/Raman signal provided add-on information. Based on this view on the disease-affected skin on a subcellular level, skin areas affected by dermatitis and by psoriasis could be clearly identified. Multimodal multiphoton tomographs may become important label-free clinical high-resolution imaging tools for in vivo skin histology to realize rapid early diagnosis as well as treatment control.

Two-photon microscopy (2PM) has revolutionized biomedical imaging by allowing thin optical sectioning in relatively thick biological specimens. Because dispersive microscope components in 2PM, such as objective lens, can alter temporal laser pulse width (typically being broader at the sample plane), for accurate measurements of two-photon absorption properties, it is important to characterize pulse duration at the sample plane. We present a simple modification to a two-photon microscope light path that allows for second-harmonic-generation-based interferometric autocorrelation measurements to characterize ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting (TCSPC). We show that TCSPC can be used as a simple and versatile method to estimate the zero time delay step value between two adjacent ultrafast laser pulses for these measurements. To demonstrate the utility of this modification, we measured the Coherent Chameleon-Ultra II Ti:sapphire laser pulse width at the sample plane using a 10  ×   air, 40  ×   air, or 63  ×   water-immersion objective lens. At 950-nm two-photon excitation, the measured pulse width was 154  ±  32, 165  ±  13, and 218  ±  27  fs (n  =  6, mean  ±  standard deviation), respectively.

To detect small-scale changes in tissue with optical techniques, small sampling volumes are required. Single fiber reflectance (SFR) spectroscopy has a sampling depth of a few hundred micrometers. SFR spectroscopy uses a single fiber to emit and collect light. The only available model to determine optical properties with SFR spectroscopy was derived for tissues with modified Henyey–Greenstein phase functions. Previously, we demonstrated that this model is inadequate for other tissue phase functions. We develop a model to relate SFR measurements to scattering properties for a range of phase functions, in the absence of absorption. Since the source and detector overlap, the reflectance cannot be accurately described by diffusion theory alone: SFR measurements are subdiffuse. Therefore, we describe the reflectance as a combination of a diffuse and a semiballistic component. We use the model of Farrell et al. for the diffuse component, solved for an overlapping source and detector fiber. For the semiballistic component, we derive a new parameter, p_sb, which incorporates the integrals of the phase function over 1 deg in the backward direction and 23 deg in the forward direction. Our model predicts the reflectance with a median error of 2.1%, compared to 9.0% for the currently available model.

Significance: Definitive diagnostics of many diseases is based on the histological analysis of thin tissue cuts with optical white light microscopy. Extra information on tissue structural properties obtained with polarized light would help the pathologist to improve the accuracy of his diagnosis.

Aim: We report on using Mueller matrix microscopy data, logarithmic decomposition, and polarized Monte Carlo (MC) modeling for qualitative and quantitative analysis of thin tissue cuts to extract the information on tissue microstructure that is not available with a conventional white light microscopy.

Approach: Unstained cuts of human skin equivalents were measured with a custom-built liquid-crystal-based Mueller microscope in transmission configuration. To interpret experimental data, we performed the simulations with a polarized MC algorithm for scattering anisotropic media. Several optical models of tissue (spherical scatterers within birefringent host medium, and combination of spherical and cylindrical scatterers within either isotropic or birefringent host medium) were tested.

Results: A set of rotation invariants for the logarithmic decomposition of a Mueller matrix was derived to rule out the impact of sample orientation. These invariants were calculated for both simulated and measured Mueller matrices of the dermal layer of skin equivalents. We demonstrated that only the simulations with a model combining both spherical and cylindrical scatterers within birefringent host medium reproduced the experimental trends in optical properties of the dermal layer (linear retardance, linear dichroism, and anisotropic linear depolarization) with layer thickness.

Conclusions: Our studies prove that Mueller polarimetry provides relevant information not only on a size of dominant scatterers (e.g., cell nuclei versus subwavelength organelles) but also on its shape (e.g., cells versus collagen fibers). The latter is directly related to the state of extracellular collagen matrix, which is often affected by early pathology. Hence, using polarimetric data can help to increase the accuracy of diagnosis.

Significance: Current guidelines for rheumatoid arthritis (RA) management recommend early treatment with disease modifying antirheumatic drugs (DMARDs). However, DMARD treatment fails in 30% of patients and current monitoring methods can only detect failure after 3 to 6 months of therapy.

Aim: We investigated whether joint blood flow (BF), quantified using dynamic contrast-enhanced time-resolved near-infrared spectroscopy, can monitor disease activity and treatment response in a rat model of RA.

Approach: Ankle joint BF was measured every 5 days in eight rats with adjuvant-induced arthritis (AIA) and four healthy controls. Arthritis was allowed to progress for 20 days before rats with AIA were treated with a DMARD once every 5 days until day 40.

Results: Time and group had separate significant main effects on joint BF; however, there was no significant interaction between time and group despite a notable difference in average joint BF on day 5. Comparison of individual blood flow measures between rats with AIA and control group animals did not reveal a clear response to treatment.

Conclusions: Joint BF time courses could not distinguish between rats with AIA and study controls. Heterogeneous disease response and low temporal frequency of BF measurements may have been important study limitations.

Abnormally shaped red blood cells (RBCs), called poikilocytes, can cause anemia. At present, the biochemical abnormalities in poikilocytes are not well understood. Normal RBCs and poikilocytes were analyzed using whole-blood and single-cell methods. Poikilocytes were induced in rat blood by intragastrically administering titanium dioxide (TiO₂) nanoparticles. Complete blood count and inductively coupled plasma mass spectrometry analyses were performed on whole-blood to measure average RBC morphology, blood hemoglobin (HGB), iron content, and other blood parameters. Follow-up confocal Raman spectroscopy was performed on single RBCs to analyze cell-type-specific HGB content. Two types of poikilocytes, acanthocytes and echinocytes, were observed in TiO₂ blood samples, along with normal RBCs. Acanthocytes (diameter 7.7  ±  0.5  μm) and echinocytes (7.6  ±  0.6  μm) were microscopically larger (p  <  0.05) than normal RBCs (6.6  ±  0.4  μm) found in control blood samples (no TiO₂ administration). Similarly, mean corpuscular volume was higher (p  <  0.05) in TiO₂ whole-blood (70.70  ±  1.97  fl) than in control whole-blood (67.42  ±  2.03  fl). Poikilocytes also had higher HGB content. Mean corpuscular hemoglobin was higher (p  <  0.05) in TiO₂ whole-blood (21.84  ±  0.75  pg) than in control whole-blood (20.8  ±  0.32  pg). Iron content was higher (p  <  0.001) in TiO₂ whole-blood (697.0  ±  24.5  mg  /  l) than in control whole-blood (503.4  ±  38.5  mg  /  l), which supports elevated HGB as iron is found in HGB. HGB-associated Raman bands at 1637, 1585, and 1372  cm^−  1 had higher (p  <  0.001) amplitudes in acanthocytes and echinocytes than in RBCs from control blood and normal RBCs from TiO₂ blood. Further, the 1585-cm^−  1 band had a lower (p  <  0.05) amplitude in normal RBCs from TiO₂ versus control RBCs. This represents biochemical abnormalities in normal appearing RBCs. Overall, poikilocytes, especially acanthocytes, have elevated HGB.

Objective measurement of the nasal valve region is valuable for the assessment of functional rhinoplasty surgical outcomes. Anatomical optical coherence tomography (aOCT) is an imaging modality that may be used to obtain real-time, quantitative, and volumetric scans of the nasal airway. We aim to evaluate if volumetric aOCT imaging is useful for the examination of the nasal valve region before and after functional rhinoplasty procedures. aOCT scans of the nasal valves were performed on four cadaveric heads before and after spreader graft and butterfly graft procedures. The resulting aOCT images were compared against video endoscopy images, and the segmented volumes of the nasal airway obtained from aOCT scans were compared with computed tomography (CT) derived volumes acquired under the same conditions. The aOCT-derived volumes match the CT volumes closely, with a mean Dice similarity coefficient of 0.88 and a mean Hausdorff distance of 2.3 mm. Furthermore, the aOCT images were found to represent the shape of the nasal cavity accurately. Due to its ability to perform real-time, quantitative, and accurate evaluation of the nasal airway, aOCT imaging is a promising modality for the objective assessment of the nasal valves before and after functional rhinoplasty procedures.

Significance: Spatial frequency domain imaging (SFDI) is a diffuse optical measurement technique that can quantify tissue optical absorption (μ_a) and reduced scattering (μs′) on a pixel-by-pixel basis. Measurements of μ_a at different wavelengths enable the extraction of molar concentrations of tissue chromophores over a wide field, providing a noncontact and label-free means to assess tissue viability, oxygenation, microarchitecture, and molecular content. We present here openSFDI: an open-source guide for building a low-cost, small-footprint, three-wavelength SFDI system capable of quantifying μ_a and μs′ as well as oxyhemoglobin and deoxyhemoglobin concentrations in biological tissue. The companion website provides a complete parts list along with detailed instructions for assembling the openSFDI system.

Aim: We describe the design of openSFDI and report on the accuracy and precision of optical property extractions for three different systems fabricated according to the instructions on the openSFDI website.

Approach: Accuracy was assessed by measuring nine tissue-simulating optical phantoms with a physiologically relevant range of μ_a and μs′ with the openSFDI systems and a commercial SFDI device. Precision was assessed by repeatedly measuring the same phantom over 1 h.

Results: The openSFDI systems had an error of 0  ±  6  %   in μ_a and −2  ±  3  %   in μs′, compared to a commercial SFDI system. Bland–Altman analysis revealed the limits of agreement between the two systems to be   ±  0.004  mm^−  1 for μ_a and −0.06 to 0.1  mm^−  1 for μs′. The openSFDI system had low drift with an average standard deviation of 0.0007  mm^−  1 and 0.05  mm^−  1 in μ_a and μs′, respectively.

Conclusion: The openSFDI provides a customiz

Microcirculation plays a crucial role in delivering oxygen and nutrients to living tissues and in removing metabolic wastes from the human body. Monitoring the velocity of blood flow in microcirculation is essential for assessing various diseases, such as diabetes, cancer, and critical illnesses. Because of the complex morphological pattern of the capillaries, both in-vivo capillary identification and blood flow velocity measurement by conventional optical capillaroscopy are challenging. Thus, we focused on developing an in-vivo optical microscope for capillary imaging, and we propose an in-vivo full-field flow velocity measurement method based on intelligent object identification. The proposed method realizes full-field blood flow velocity measurements in microcirculation by employing a deep neural network to automatically identify and distinguish capillaries from images. In addition, a spatiotemporal diagram analysis is used for flow velocity calculation. In-vivo experiments were conducted, and the images and videos of capillaries were collected for analysis. We demonstrated that the proposed method is highly accurate in performing full-field blood flow velocity measurements in microcirculation. Further, because this method is simple and inexpensive, it can be effectively employed in clinics.

Significance: As a promising hybrid imaging technique with x-ray excitable nanophosphors, cone-beam x-ray luminescence computed tomography (CB-XLCT) has been proposed for in-depth biological imaging applications. In situations in which the full rotation of the imaging object (or x-ray source) is inapplicable, the x-ray excitation is limited by geometry, or a lower x-ray excitation dose is mandatory, limited view CB-XLCT reconstruction would be essential. However, this will result in severe ill-posedness and poor image quality.

Aim: The aim is to develop a limited view CB-XLCT imaging strategy to reduce the scanning span and a corresponding reconstruction method to achieve robust imaging performance.

Approach: In this study, a group sparsity-based reconstruction method is proposed with the consideration that nanophosphors usually cluster in certain regions, such as tumors or major organs such as the liver. In addition, depth compensation (DC) is adopted to avoid the depth inconsistency caused by a limited view strategy.

Results: Experiments using numerical simulations and physical phantoms with different edge-to-edge distances were carried out to illustrate the validity of the proposed method. The reconstruction results showed that the proposed method outperforms conventional methods in terms of localization accuracy, target shape, image contrast, and spatial resolution with two perpendicular projections.

Conclusions: A limited view CB-XLCT imaging strategy with two perpendicular projections and a reconstruction method based on DC and group sparsity, which is essential for fast CB-XLCT imaging and for some practical imaging applications, such as imaging-guided surgery, is proposed.

Combined ultrasound and photoacoustic imaging systems are being developed for biomedical and clinical applications. One common probe configuration is to use a linear transducer array with external light delivery to produce coregistered ultrasound and photoacoustic images. The diagnostic capability of these systems is dependent on the effectiveness of light delivery to the imaging target. We use Monte Carlo modeling to investigate the optimal design geometry of an integrated probe. Simulations are conducted with multiple tissue compositions and wavelengths. The effect of a skin layer with the thickness of a mouse or a human is also considered. The model was validated using a tissue-mimicking gelatin phantom and corresponding Monte Carlo simulations. The optimal illumination angle is shallower with human skin thickness, whereas intermediate angles are ideal with mouse skin thickness. The effect of skin thickness explains differences in the results of prior work. The simulations also indicate that even with identical hardware and imaging parameters, light delivery will be up to 3  ×   smaller in humans than in mice, due to the increased scattering from thicker skin. Our findings have clear implications for the many researchers using mice to test and develop imaging methods for clinical translation.