In order to understand the relationship between accommodation and vision quality, a custom-built ultra-long scan depth spectral domain optical coherence tomography (UL-SDOCT) and a Shack-Hartmann wavefront sensor (HSWFS) were combined. The resolution and scan depth of UL-SDOCT are 6 μm and 15.6 mm, respectively, which allow for high-resolution imaging of the whole anterior segment. The HSWFS consists of a 32×32 microlens array, and is able to measure first 35th-order Zernike aberrations with (1/20)λ measurement accuracy. The integrated system succeeded in measuring the ocular anterior segment dimension parameters and the ocular monochromatic high-order aberrations simultaneously under the conditions of nonaccommodative and accommodative stimuli. This may help understand the regulatory mechanism of image quality control in the human eye.

Knowledge of the biomechanical/elastic property of the cardiac wall is of fundamental importance in improving our understanding of cardiac development, particularly the interaction between the wall dynamics and hemodynamics in the developing outflow tract (OFT). We describe a method that employs optical coherence tomography (OCT) as a means to noninvasively measure the local elastic property of the cardiac wall in vivo. The method uses a time-lapse sequence of OCT images that represent the dynamic behavior of the OFT longitudinal section to calculate the regional wall pulse wave velocity (PWV), upon which the Young's modulus of the cardiac wall is deduced by the use of the Moens-Korteweg equation. The experimental results show that the foot-to-foot PWV ranges from 3.2 to 6.6 mm/s with a mean of 4.7 mm/s, and the averaged Young's modulus is 0.36 Pa, both of which are comparable to the documented values of stage HH17 atrioventricular canal tissue. The proposed method that provides the quantitative mechanical assessment may play a significant role in the understanding of the cardiac development.

Cerebral blood flow (CBF) is critical for the maintenance of cerebral function by guaranteed constant oxygen and glucose supply to brain. Collateral channels (CCs) are recruited to provide alternatives to CBF to ischemic regions once the primary vessel is occluded during ischemic stroke. However, the knowledge of the relationship between dynamic evolution of collateral flow and the distribution of regional blood flow remains limited. In this study, laser speckle imaging was used to assess dynamic changes of CCs and regional blood flow in a rat cortex with permanent middle cerebral artery occlusion (MCAo). We found that CCs immediately provided blood flow to ischemic territories after MCAo. More importantly, there were three kinds of dynamic changes of CCs during acute stroke: persistent CC, impermanent CC, and transient CC, respectively, related to different distributions of regional blood flow. Although there was the possible occurrence of peri-infarct depolarization (PID) during ischemia, there was no obvious significance about the onset time and duration of CCs between rats with and without PID. These results suggest that the initial arising of CCs does not ensure their persistence, and that collateral flow could be varied with distribution of regional blood flow in acute ischemic stroke, which may facilitate the understanding of collateral recruitment and promote the development of collateral therapeutics in the future.

Hemoglobin (Hb), as one of main components of blood, has a unique quaternary structure. Its release of oxygen is controlled by oxygen partial pressure (PO₂). We investigate the specific spectroscopic changes in Hb under different PO₂ levels to optimize clinical methods of measuring tissue PO₂. The transmissivity of Hb under different PO₂ levels is measured with a UV/Vis fiber optic spectrometer. Its plotted absorption spectral curve shows two high absorption peaks at 540 and 576 nm and an absorption valley at 560 nm when PO₂ is higher than 100 mm Hg. The two high absorption peaks decrease gradually with a decrease in PO₂, whereas the absorption valley at 560 nm increases. When PO₂ decreases to approximately 0 mm Hg, the two high absorption peaks disappear completely, while the absorption valley has a hypochromic shift (8 to 10 nm) and forms a specific high absorption peak at approximately 550 nm. The same phenomena can be observed in visible reflectance spectra of finger-tip microcirculation. Specific changes in extinction coefficient and absorption spectra of Hb occur along with variations in PO₂, which could be used to explain pathological changes caused by tissue hypoxia and for early detection of oxygen deficiency diseases in clinical monitoring.

Raman spectroscopy (RS) and a genetic algorithm (GA) were applied to distinguish nasopharyngeal cancer (NPC) from normal nasopharyngeal tissue. A total of 225 Raman spectra are acquired from 120 tissue sites of 63 nasopharyngeal patients, 56 Raman spectra from normal tissue and 169 Raman spectra from NPC tissue. The GA integrated with linear discriminant analysis (LDA) is developed to differentiate NPC and normal tissue according to spectral variables in the selected regions of 792-805, 867-880, 996-1009, 1086-1099, 1288-1304, 1663-1670, and 1742-1752 cm^-1 related to proteins, nucleic acids and lipids of tissue. The GA-LDA algorithms with the leave-one-out cross-validation method provide a sensitivity of 69.2% and specificity of 100%. The results are better than that of principal component analysis which is applied to the same Raman dataset of nasopharyngeal tissue with a sensitivity of 63.3% and specificity of 94.6%. This demonstrates that Raman spectroscopy associated with GA-LDA diagnostic algorithm has enormous potential to detect and diagnose nasopharyngeal cancer.

Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media.

We study the surface topographical, structural, and compositional modifications induced in bovine cortical bone by femtosecond laser ablation. The tests are performed in air, with a Yb:KYW chirped-pulse-regenerative amplification laser system (500 fs, 1030 nm) at fluences ranging from 0.55 to 2.24 J/cm². The ablation process is monitored by acoustic emission measurements. The topography of the laser-treated surfaces is studied by scanning electron microscopy, and their constitution is characterized by glancing incidence x-ray diffraction, x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and micro-Raman spectroscopy. The results show that femtosecond laser ablation allows removing bone without melting, carbonization, or cracking. The structure and composition of the remaining tissue are essentially preserved, the only constitutional changes observed being a reduction of the organic material content and a partial recrystallization of hydroxyapatite in the most superficial region of samples. The results suggest that, within this fluence range, ablation occurs by a combination of thermal and electrostatic mechanisms, with the first type of mechanism predominating at lower fluences. The associated thermal effects explain the constitutional changes observed. We show that femtosecond lasers are a promising tool for delicate orthopaedic surgeries, where small amounts of bone must be cut with negligible damage, thus minimizing surgical trauma.

Red blood cell (RBC) aggregation and oxygenation are important markers for a variety of blood disorders. No current technique is capable of simultaneously measuring aggregation/oxygenation levels noninvasively. We propose using photoacoustic ultrasound spectroscopy (PAUS) for assessing both phenomena. This technique relies on frequency-domain analysis of the PA signals by extracting parameters such as the ultrasound spectral slope and the midband fit. To investigate the effect of hematocrit, aggregation, and oxygenation levels on PAUS parameters, a Monte Carlo-based theoretical model and an experimental protocol using porcine RBCs were developed. The samples were illuminated at 750 and 1064 nm and changes in the PAUS parameters were compared to the oxygen-dependent optical absorption coefficients to assess the oxygenation level. Good agreement between the theoretical and experimental spectral parameters was obtained for the spectral slope of the nonaggregated spectra ( ~ 0.3 dB / MHz). The experimental midband fit increased by ~ 5 dB for the largest aggregate size. Based on the analysis of the PA signals, the oxygen saturation level of the most aggregated sample was > 20% greater than the nonaggregated sample. The results provide a framework for using PA signals' spectroscopic parameters for monitoring the aggregation and oxygenation levels of RBCs.

To improve our understanding of lipid metabolism, Drosophila is used as a model animal, and its lipid homeostasis is monitored by coherent anti-Stokes Raman scattering microscopy. We are able to achieve in vivo imaging of larval fat body (analogous to adipose tissue in mammals) and oenocytes (analogous to hepatocytes) in Drosophila larvae at subcellular level without any labeling. By overexpressing two lipid regulatory proteins-Brummer lipase (Bmm) and lipid storage droplet-2 (Lsd-2)-we found different phenotypes and responses under fed and starved conditions. Comparing with the control larva, we observed more lipid droplet accumulation by ~twofold in oenocytes of fat-body-Bmm-overexpressing (FB-Bmm-overexpressing) mutant under fed condition, and less lipid by ~fourfold in oenocytes of fat-body-Lsd-2-overexpressing (FB-Lsd-2-overexpressing) mutant under starved condition. Moreover, together with reduced size of lipid droplets, the lipid content in the fat body of FB-Bmm-overexpressing mutant decreases much faster than that of the control and FB-Lsd-2-overexpressing mutant during starvation. From long-term starvation assay, we found FB-Bmm-overexpressing mutant has a shorter lifespan, which can be attributed to faster consumption of lipid in its fat body. Our results demonstrate in vivo observations of direct influences of Bmm and Lsd-2 on lipid homeostasis in Drosophila larvae.

The multimodal optical imaging technique, which utilizes nonlinear and linear optical processes, plays an important role in biological and biomedical research. As second-order nonlinear phenomenon, the two-photon luminescence (TPL) results from the nonlinear excitation of fluorescent molecules, while the second harmonic generation (SHG) depends on the second order nonlinear polarization, orientation, and noncentrosymmetric properties of molecules. In contrast, the linear resonance light scattering (RLS) occurs when the molecules are excited by a light beam with a wavelength close to their absorption bands. Since SHG, TPL, and RLS involve different kinds of optical processes, they might be used in parallel to provide complementary information about the structure and function of cells and tissues. Herein, we develop for the first time a multimodal optical microscopy with the capability of simultaneous SHG, TPL, and RLS imaging. We analyze theoretically and demonstrate experimentally the near-infrared irradiation-induced SHG, TPL, and RLS from the gold nanorods with nanometer spatial resolution. With the gold nanorods as the contrast agents, we further demonstrate the simultaneous SHG, TPL, and RLS imaging of A431 human epithelial skin cancer cells. This multimodal optical microscopy might provide a reliable and complementary approach for biological and biomedical research.

Unique features and the underlining hypotheses of how these features may relate to the tumor physiology in coregistered ultrasound and photoacoustic images of ex vivo ovarian tissue are introduced. The images were first compressed with wavelet transform. The mean Radon transform of photoacoustic images was then computed and fitted with a Gaussian function to find the centroid of a suspicious area for shift-invariant recognition process. Twenty-four features were extracted from a training set by several methods, including Fourier transform, image statistics, and different composite filters. The features were chosen from more than 400 training images obtained from 33 ex vivo ovaries of 24 patients, and used to train three classifiers, including generalized linear model, neural network, and support vector machine (SVM). The SVM achieved the best training performance and was able to exclusively separate cancerous from non-cancerous cases with 100% sensitivity and specificity. At the end, the classifiers were used to test 95 new images obtained from 37 ovaries of 20 additional patients. The SVM classifier achieved 76.92% sensitivity and 95.12% specificity. Furthermore, if we assume that recognizing one image as a cancer is sufficient to consider an ovary as malignant, the SVM classifier achieves 100% sensitivity and 87.88% specificity.

Near-infrared fluorescence-enhanced diffuse optical tomography (fDOT) is used to localize tumors in mice using fluorescent nanoparticles as a blood pool contrast agent. The infrared dye DiR is loaded in the lipid core of nontargeted nanoparticles (DiR-lipidots) and injected systemically via the tail vein in mice bearing U87 tumors. Distribution and time-course of DiR-lipidots are followed using in vivo fluorescence reflectance imaging and reveal enhanced fluorescent signal within the subcutaneous tumors up to seven days due to the enhanced permeability and retention effect. Tumor growth into the brain is followed using bioluminescent imaging, and tumor localization is further determined by magnetic resonance imaging. The fDOT provides three-dimensional fluorescent maps that allow for consistent localization for both subcutaneous and brain tumors.

We acquired in vivo images of the human optic nerve head (ONH) using an adaptive optics—optical coherence tomography (AO-OCT) system. In order to improve imaging of the lamina cribrosa in the ONH with high lateral resolution and sensitivity, we implemented a complex conjugate artifact-free Fourier domain OCT (Fd-OCT) acquisition scheme with a reference arm-based phase shifting method. This allowed positioning of the lamina cribrosa structures near the zero path length difference where AO-OCT imaging achieves highest sensitivity. Implementation of our complex conjugate artifact removal (CCR) method required constant phase shifts between consecutive axial scans (A-scans), generated by continuous beam path-length changes from offsetting the pivot point of the scanning mirror placed in the reference arm. Fourier transform along the transverse axis and a filtering algorithm allowed reconstruction of CCR AO-OCT images. The suppression ratio of the mirror artifact was approximately 22 dB (at 18,000 A-scans per second acquisition speed) with a paperboard test target and an optimum phase-shift value. Finally, we reconstructed the three-dimensional structure of human ONH with enhanced depth range and sensitivity using CCR AO-OCT.

We investigate morphological differences in three-dimensional (3-D) images with cellular resolution between nonmelanoma skin cancer and normal skin using Gabor domain optical coherence microscopy. As a result, we show for the first time cellular optical coherence images of 3-D features differentiating cancerous skin from normal skin. In addition, in vivo volumetric images of normal skin from different anatomic locations are shown and compared.

Total internal reflection fluorescence (TIRF) microscopy has the exciting laser beam incident beyond critical angle from the glass side of a glass/aqueous interface formed by the coverslip and aqueous sample. The aqueous side evanescent field decays exponentially with distance from the interface with penetration depth depending on incidence angle. Through-the-objective TIRF has the exciting laser focused at the back focal plane (BFP) creating a refracted parallel beam approaching the interface in the small gap between objective and coverslip, making incidence angle challenging to measure. Objective axial scanning does not affect incidence angle but translates beam and interface intersection detected by the fluorescence center of mass from fluorescent spheres attached to the aqueous side of the interface. Center of mass translation divided by the axial translation is the tangent of the incidence angle that is sampled repeatedly over objective trajectory to obtain a best estimate. Incidence angle is measured for progressively larger radial positions of the focused beam on the BFP. A through-the-objective TIRF microscope, utilizing a micrometer and relay lenses to position the focused beam at the BFP, is calibrated for incidence angle. Calibration depends on microscope characteristics and TIRF objective and is applicable to any interface or sample.

We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies.

A main field in biomedical optics research is diffuse optical tomography, where intensity variations of the transmitted light traversing through tissue are detected. Mathematical models and reconstruction algorithms based on finite element methods and Monte Carlo simulations describe the light transport inside the tissue and determine differences in absorption and scattering coefficients. Precise knowledge of the sample's surface shape and orientation is required to provide boundary conditions for these techniques. We propose an integrated method based on structured light three-dimensional (3-D) scanning that provides detailed surface information of the object, which is usable for volume mesh creation and allows the normalization of the intensity dispersion between surface and camera. The experimental setup is complemented by polarization difference imaging to avoid overlaying byproducts caused by inter-reflections and multiple scattering in semitransparent tissue.

The solution of the forward problem in fluorescence molecular imaging strongly influences the successful convergence of the fluorophore reconstruction. The most common approach to meeting this problem has been to apply the diffusion approximation. However, this model is a first-order angular approximation of the radiative transfer equation, and thus is subject to some well-known limitations. This manuscript proposes a methodology that confronts these limitations by applying the radiative transfer equation in spatial regions in which the diffusion approximation gives decreased accuracy. The explicit integro differential equations that formulate this model were solved by applying the Galerkin finite element approximation. The required spatial discretization of the investigated domain was implemented through the Delaunay triangulation, while the azimuthal discretization scheme was used for the angular space. This model has been evaluated on two simulation geometries and the results were compared with results from an independent Monte Carlo method and the radiative transfer equation by calculating the absolute values of the relative errors between these models. The results show that the proposed forward solver can approximate the radiative transfer equation and the Monte Carlo method with better than 95% accuracy, while the accuracy of the diffusion approximation is approximately 10% lower.

We examine the improvement in imaging performance, such as axial resolution and signal localization, when employing limited-projection-angle fluorescence molecular tomography (FMT) together with x-ray computed tomography (XCT) measurements versus stand-alone FMT. For this purpose, we employed living mice, bearing a spontaneous lung tumor model, and imaged them with FMT and XCT under identical geometrical conditions using fluorescent probes for cancer targeting. The XCT data was employed, herein, as structural prior information to guide the FMT reconstruction. Gold standard images were provided by fluorescence images of mouse cryoslices, providing the ground truth in fluorescence bio-distribution. Upon comparison of FMT images versus images reconstructed using hybrid FMT and XCT data, we demonstrate marked improvements in image accuracy. This work relates to currently disseminated FMT systems, using limited projection scans, and can be employed to enhance their performance.

Liver cancer is one of the most common malignant tumors worldwide. In order to enable the noninvasive detection of small liver tumors in mice, we present a parallel iterative shrinkage (PIS) algorithm for dual-modality tomography. It takes advantage of microcomputed tomography and multiview bioluminescence imaging, providing anatomical structure and bioluminescence intensity information to reconstruct the size and location of tumors. By incorporating prior knowledge of signal sparsity, we associate some mathematical strategies including specific smooth convex approximation, an iterative shrinkage operator, and affine subspace with the PIS method, which guarantees the accuracy, efficiency, and reliability for three-dimensional reconstruction. Then an in vivo experiment on the bead-implanted mouse has been performed to validate the feasibility of this method. The findings indicate that a tiny lesion less than 3 mm in diameter can be localized with a position bias no more than 1 mm; the computational efficiency is one to three orders of magnitude faster than the existing algorithms; this approach is robust to the different regularization parameters and the lp norms. Finally, we have applied this algorithm to another in vivo experiment on an HCCLM3 orthotopic xenograft mouse model, which suggests the PIS method holds the promise for practical applications of whole-body cancer detection.

Recent technologies, such as nanotechnology, provide new opportunities for next generation scintillation devices and instruments. New nanophosphor-based materials seem to be promising for further improvements in optical diffusion studies. In medical imaging, detector technology has found widespread use, offering improved signal capabilities. However, in spite of many spectacular innovations and the significant research in chemical synthesis on the detective material, improvement in signal quality is still an issue requiring further progress. Here, a sophisticated analysis is shown within the framework of Mie scattering theory and Monte Carlo simulation which demonstrates the optimum structural and optical properties of nanophosphors that are significantly promising in manufacture for further signal modulation improvement. A variety of structural and optical properties were examined: (1) phosphors of grain size (1 to 1000 nm), (2) packing density (50% to 99%), (3) light wavelength (400 to 700 nm), and (4) refractive index of nanophosphor (real part: 1.4-2.0, imaginary part: 10^-6). Results showed that for a specific thickness of nanophosphor layer, the compromise between spatial resolution and sensitivity can be achieved by optimizing the structural (200 nm ≤ grain diameter ≤ 800 nm) and optical properties of the nanophosphor (1.7 ≤ refractiveindex ≤ 2.0). Finally, high optical modulation was accomplished employing grains of high refractive index and size above 200 nm.

The imaging depth of ballistic optical imaging technologies is limited by light scattering. To study the effects of scattering on optical-resolution photoacoustic microscopy (OR-PAM), the signals were divided into target and background signals. A method to simulate the point spread function (PSF) of the PAM system considering both optical illumination and acoustic detection was proposed, then the PSF was used to calculate the contribution of each class of signal at different depths of the focal plane (z_f ). How image contrast is degraded when there is a uniformly absorbing background as well as when there are small targets densely packed in the acoustic resolution cell were studied. By using the hyperboloid-focusing-based Monte Carlo method, optical focusing into a scattering medium was simulated. It was found that the lateral resolution provided by optical focusing is degraded by only 14% when z_f= 1.1 transport mean free path (l^′_t), compared with the case of no scattering. When z_f= 1.7l^′_t, the fluence at 50 μm radial distance away from the focal point is 93% of that at the focal point, which shows optical focusing is very weak at this depth. The method to simulate the PSF of PAM can be used in the future to optimize parameters so as to improve the system performance.

Optical coherence tomography (OCT) has been increasingly used for imaging pulmonary alveoli. Only a few studies, however, have quantified individual alveolar areas, and the validity of alveolar volumes represented within OCT images has not been shown. To validate quantitative measurements of alveoli from OCT images, we compared the cross-sectional area, perimeter, volume, and surface area of matched subpleural alveoli from microcomputed tomography (micro-CT) and OCT images of fixed air-filled swine samples. The relative change in size between different alveoli was extremely well correlated (r<0.9, P<0.0001), but OCT images underestimated absolute sizes compared to micro-CT by 27% (area), 7% (perimeter), 46% (volume), and 25% (surface area) on average. We hypothesized that the differences resulted from refraction at the tissue-air interfaces and developed a ray-tracing model that approximates the reconstructed alveolar size within OCT images. Using this model and OCT measurements of the refractive index for lung tissue (1.41 for fresh, 1.53 for fixed), we derived equations to obtain absolute size measurements of superellipse and circular alveoli with the use of predictive correction factors. These methods and results should enable the quantification of alveolar sizes from OCT images in vivo.

We have developed a sphere-cylinder birefringence model (SCBM) for anisotropic media. The new model is based on a previously published sphere-cylinder scattering model (SCSM), but the spherical and cylindrical scatterers are embedded in a linearly birefringent medium. A Monte Carlo simulation program for SCBM was also developed by adding a new module to the SCSM program to take into account the effects of birefringence. Simulations of the backscattering Mueller matrix demonstrate that SCBM results in better agreement with experimental results than SCSM and is more suitable to characterize fibrous tissues such as skeletal muscle. Using Monte Carlo simulations, we also examined the characteristics of two-dimensional backscattering Mueller matrix of SCBM and analyzed the influence of linear birefringence.

Laser speckle contrast imaging (LSCI) is a contrast agent free imaging technique suited for longitudinal assessment of vascular remodeling that accompanies brain tumor growth. We report the use of LSCI to monitor vascular changes in a rodent glioma model. Ten rats are inoculated with 9L gliosarcoma cells, and the angiogenic response is monitored five times over two weeks through a thinned skull imaging window. We are able to visualize neovascularization and measure the number of vessels per unit area to assess quantitatively the microvessel density (MVD). Spatial spread of MVD reveals regions of high MVD that may correspond to tumor location. Whole-field average MVD values increase with time in the tumor group but are fairly stable in the control groups. Statistical analysis shows significant differences in MVD values between the tumor group and both saline-receiving and unperturbed control groups over the two-week period (p < 0.05). In conclusion, LSCI is suitable for investigation of tumor angiogenesis in rodent models. In addition, the statistical difference (p < 0.02) between MVD values of the tumor (24.40 ± 1.41) and control groups (15.40 ± 1.60) on the 14th day after inoculation suggests a potential use of LSCI in the clinic in distinguishing tumor environments from normal vasculature.

Endothelial cells cultured in three-dimensional (3-D) extracellular matrices spontaneously form microvessels in response to soluble and matrix-bound factors. Such cultures are common for the study of angiogenesis and may find widespread use in drug discovery. Vascular networks are imaged over weeks to measure the distribution of vessel morphogenic parameters. Measurements require micron-scale spatial resolution, which for light microscopy comes at the cost of limited field-of-view (FOV) and shallow depth-of-focus (DOF). Small FOVs and DOFs necessitate lateral and axial mechanical scanning, thus limiting imaging throughput. We present a lens-free holographic on-chip microscopy technique to rapidly image microvessels within a Petri dish over a large volume without any mechanical scanning. This on-chip method uses partially coherent illumination and a CMOS sensor to record in-line holographic images of the sample. For digital reconstruction of the measured holograms, we implement a multiheight phase recovery method to obtain phase images of capillary morphogenesis over a large FOV (24  mm ² ) with ∼1.5  μm spatial resolution. On average, measured capillary length in our method was within approximately 2% of lengths measured using a 10× microscope objective. These results suggest lens-free on-chip imaging is a useful toolset for high-throughput monitoring and quantitative analysis of microvascular 3-D networks.

Determining both bilirubin's concentration and its spatial distribution are important in disease diagnosis. Here, for the first time, we applied quantitative multiwavelength photoacoustic microscopy (PAM) to detect bilirubin concentration and distribution simultaneously. By measuring tissue-mimicking phantoms with different bilirubin concentrations, we showed that the root-mean-square error of prediction has reached 0.52 and 0.83  mg/dL for pure bilirubin and for blood-mixed bilirubin detection (with 100% oxygen saturation), respectively. We further demonstrated the capability of the PAM system to image bilirubin distribution both with and without blood. Finally, by underlaying bilirubin phantoms with mouse skins, we showed that bilirubin can be imaged with consistent accuracy down to >400  μm in depth. Our results show that PAM has potential for noninvasive bilirubin monitoring in vivo, as well as for further clinical applications.

We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

We have developed a hybrid frequency domain fluorescence tomography and magnetic resonance imaging system (MRI) for small animal imaging. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration and lifetime images using a multi-modality approach. In vivo experiments are undertaken to evaluate the system. We compare the recovered fluorescence parameters with and without MRI structural a priori information. In addition, we compare two optical background heterogeneity correction methods: Born normalization and utilizing diffuse optical tomography (DOT) functional a priori information. The results show that the concentration and lifetime of a 4.2-mm diameter indocyanine green inclusion located 15 mm deep inside a rat can be recovered with less than a 5% error when functional a priori information from DOT and structural a priori information from MRI are utilized.

The main objective of the study was to investigate the ability of frequency-domain photothermal radiometry (PTR) and modulated luminescence (LUM) to detect secondary caries lesions on the walls of restorations (wall lesions). Changes in experimental PTR-LUM signals due to sequential demineralization on entire vertical walls of sectioned tooth samples were investigated. In addition, transverse micro-radiography (TMR) analysis (used as a gold standard) was conducted to measure the degree of demineralization that occurred in each sample. Statistical correlation between TMR results and PTR-LUM signals was determined using Pearson's correlation coefficient. LUM signals were found to be dominated by the scattered component of the incident laser beam. The more clinically relevant cases of localized demineralization and remineralization on vertical walls were also investigated to examine whether PTR-LUM signals are sensitive to demineralization and remineralization of much smaller areas. The overall results demonstrated that PTR-LUM is sensitive to progressive demineralization and remineralization on vertical walls of sectioned tooth samples.

The beneficial effects of pharmacotherapy on children with attention-deficit hyperactivity disorder (ADHD) are well documented. We use near-infrared spectroscopy (NIRS) methodology to determine reorganization of brain neurovascular properties following the medication treatment. Twenty-six children with ADHD (ages six through 12) participated in a modified laboratory school protocol to monitor treatment response with lisdexamfetamine dimesylate (LDX; Vyvanse®, Shire US Inc.). All children refrained from taking medication for at least two weeks (washout period). To detect neurovascular reorganization, we measured changes in synchronization of oxy (HbO₂) and deoxy (HHb) hemoglobin waves between the two frontal lobes. Participants without medication displayed average baseline HbO₂ phase difference at about - 7-deg. and HHb differences at about 240-deg.. This phase synchronization index changed after pharmacological intervention. Medication induced an average phase changes of HbO₂ after first medication to 280-deg. and after medication optimization to 242-deg.. Instead first medication changed of the average HHb phase difference at 186-deg. and then after medication optimization to 120-deg. In agreement with findings of White et al., and Varela et al., we associated the phase synchronization differences of brain hemodynamics in children with ADHD with lobe specific hemodynamic reorganization of HbO₂- and HHB oscillations following medication status.

General application of continuous-wave (CW) laser irradiation modes in photodynamic therapy can cause thermal damage to normal tissues in addition to tumors. A new photodynamic laser therapy system using a pulse irradiation mode was optimized to reduce nonspecific thermal damage. In in vitro tissue specimens, tissue energy deposition rates were measured in three irradiation modes, CW, pulse, and burst-pulse. In addition, methods were tested for reducing variations in laser output and specific wavelength shifts using a thermoelectric cooler and thermistor. The average temperature elevation per 10 J/cm² was 0.27°C, 0.09°C, and 0.08°C using the three methods, respectively, in pig muscle tissue. Variations in laser output were controlled within ±0.2%, and specific wavelength shift was limited to ±3 nm. Thus, optimization of a photodynamic laser system was achieved using a new pulse irradiation mode and controlled laser output to reduce potential thermal damage during conventional CW-based photodynamic therapy.

Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction.

Photothermal treatment (PTT) using nanoparticles has gained attention as a promising alternative therapy for malignant tumors. One strategy for increasing the selectivity of PTT is the use of macrophages as a cellular vector for delivering nanoparticles. The aim of the present study is to examine the use of macrophages as a cellular vector for efficient PTT and determine the appropriate irradiation power and time of a near-infrared (NIR) laser using real-time phase-contrast imaging. Thermally induced injury and death of cancer cells were found to begin at 44°C to 45°C, which was achieved using the PTT effect with gold nanoshells (NS) and irradiation with a NIR laser at a power of 2 W for 5 min. The peritoneal macrophage efficiently functioned as a cellular vector for the NS, and the cancer cells surrounding the NS-loaded macrophages selectively lost their cellular viability after being irradiated with the NIR laser.

Biomedical Imaging: Principles and Applications, edited by Reiner Salzer, PhD, is a new book recently published by John Wiley & Sons. Of the 28 contributing authors, 25 are from Europe, 2 are from Canada, and 1 is from the United States. The authors have put together a comprehensive set of both well-established and emerging imaging technologies currently used in the laboratory and clinical practice. The text is well written with plenty of references and illustrations. In my opinion, it will help educate a wide audience coming into the field of biomedical imaging from various disciplines of science and engineering.