Mapping the myoglobin concentration, oxygenation, and optical pathlength in heart ex vivo using near-infrared imaging

A method that provides maps of absolute concentrations of oxygenated and deoxygenated myoglobin (Mb), its oxygenation, and its near-infrared (NIR) optical pathlength in cardiac tissue was developed. These parameters are available simultaneously. The method is based on NIR diffuse reflectance spectroscopic imaging and specific processing of the NIR images, which included a first derivative of the diffuse reflectance spectrum. Mb oxygenation, total Mb concentration, and NIR light pathlength were found to be in the range of 92%, 0.3 mM, and 12.5 mm, respectively, in beating isolated buffer-perfused and arrested pig hearts. The charge-coupled device camera enables sub-millimeter spatial resolution and spectroscopic imaging in 1.5 to 2.0 min. The technique is noninvasive and nondestructive. The equipment has no mechanical contact with the tissue of interest, leaving it undisturbed.     

用蒙特卡罗方法模拟光在多层组织中的吸收特性

在讨论目前新颖的组织功能成像打骂能性（例如光声成像）时,光子在组织中的吸收和散射特性是一个很重要的问题,鉴于这一点,本文利用一个多层模型研究了光子在皮肤,脂肪和肌肉组织中的吸收和散射特性,得到了在组织中某一深度处光子在一个平面上的吸收分布,以及在不同吸收系数和散射系数的情况下,光子的反射,吸收和透射几率,结果表明在经过多次散射后,大部分的光子被吸收,在本文的模型中只有7.3%的光子从表面反射（包括镜面反射和漫反射）,还讨论了不同光学参灵敏对参流分布的影响。     

Variability in leaf optical properties among 26 species from a broad range of habitats

Leaves from 26 species with growth forms from annual herbs to trees were collected from open, intermediate, and shaded understory habitats in Mississippi and Kansas, USA. Leaf optical properties including reflectance, transmittance, and absorptance in visible and near infrared (NIR) wavelengths were measured along with leaf thickness and specific leaf mass (SLM). These leaf properties and internal light scattering have been reported to vary with light availability in studies that have focused on a limited number of species. Our objective was to determine whether these patterns in leaf optics and light availability were consistent when a greater number of species were evaluated. Leaf thickness and SLM varied by tenfold among species sampled, but within-habitat variance was high. Although there was a strong trend toward thicker leaves in open habitats, only SLM was significantly greater in open vs. understory habitats. In contrast, leaf optical properties were strikingly similar among habitats. Reflectance and reflectance/transmittance in the NIR were used to estimate internal light scattering and there were strong relationships (r1 > 0.65) between these optical properties and leaf thickness. We concluded that leaf thickness, which did not vary consistently among habitats, was the best predictor of NIR reflectance and internal light scattering. However, because carbon allocation to leaves was lower in understory species (low SLM) yet gross optical properties were similar among all habitats, the energy investment by shade leaves required to achieve optical equivalence with sun leaves was lower. Differences in leaf longevity and growth form within a habitat may help explain the lack of consistent patterns in leaf optics as the number of species sampled increases.     

Near‐infrared bulk optical properties of goat wound tissue and human serum: consequences for an implantable optical glucose sensor

Near‐infrared (NIR) spectroscopy offers a promising technological platform for continuous glucose monitoring in the human body. Moreover, these measurements could be performed in vivo with an implantable single‐chip based optical sensor. However, a thin tissue layer may grow in the optical path of the sensor. As most biological tissues are highly scattering, they only allow a small fraction of the collimated light to pass, significantly reducing the light throughput. To quantify the effect of a thin tissue layer in the optical path, the bulk optical properties of serum and tissue samples grown on implanted dummy sensors were characterized using double integrating sphere and unscattered transmittance measurements. The estimated bulk optical properties were then used to calculate the light attenuation through a thin tissue layer. The combination band of glucose was found to be the better option, relative to the first overtone band, as the absorptivity of glucose molecules is higher, while the reduction in unscattered transmittance due to tissue growth is less. Additionally, as the wound tissue was found to be highly scattering, the unscattered transmittance of the tissue layer is expected to be very low. Therefore, a sensor configuration which measures the diffuse transmittance and/or reflectance instead was recommended.

( a ) Dummy sensor; ( b ) explanted dummy sensor in tissue lump; ( c ) removal of dummy sensor from tissue lump; and ( d ) 900 µm slices of tissue lump.     

用于胃癌检测的漫反射光谱

胃癌正严重地威胁人类的健康,为了开发一种可以诊断早期胃癌的新型光学检测技术,建立了一套简便的组织体漫反射光谱检测装置.首先,根据组织体的光学特性,介绍光谱检测方法的基本原理.然后,利用组织癌变导致的组织体光学特性变化,建立漫反射光谱检测的实验装置.最后,利用该实验装置分别获取正常胃组织和胃癌组织的漫反射光谱.实验结果表明:癌变和正常胃组织的漫反射光谱在可见光区域有明显差别,特别是在波长为500 nm和630 nm处,但是漫反射光谱检测无法分辨来自组织不同层的光谱信息.这些结果说明漫反射光谱检测装置可用于胃癌的辅助检测.     

Multiparameter wide‐field integrated optical imaging system‐based spatially modulated illumination and laser speckles in model of tissue injuries

In this report, an integrated optical platform based on spatial illumination together with laser speckle contrast technique was utilized to measure multiple parameters in live tissue including absorption, scattering, saturation, composition, metabolism, and blood flow. Measurements in three models of tissue injury including drug toxicity, artery occlusion, and acute hyperglycemia were used to test the efficacy of this system. With this hybrid apparatus, a series of structured light patterns at low and high spatial frequencies are projected onto the tissue surface and diffuse reflected light is captured by a CCD camera. A six position filter wheel, equipped with four bandpass filters centered at wavelengths of 650, 690, 800 and 880 nm is placed in front of the camera. Then, light patterns are blocked and a laser source at 650 nm illuminates the tissue while the diffusely reflected light is captured by the camera through the two remaining open holes in the wheel. In this manner, near‐infrared (NIR) and laser speckle images are captured and stored together in the computer for off‐line processing to reconstruct the tissue's properties. Spatial patterns are used to differentiate the effects of tissue scattering from those of absorption, allowing accurate quantification of tissue hemodynamics and morphology, while a coherent light source is used to study blood flow changes, a feature which cannot be measured with the NIR structured light. This combined configuration utilizes the strengths of each system in a complementary way, thus collecting a larger range of sample properties. In addition, once the flow and hemodynamics are measured, tissue oxygen metabolism can be calculated, a property which cannot be measured independently. Therefore, this merged platform can be considered a multiparameter wide‐field imaging and spectroscopy modality. Overall, experiments demonstrate the capability of this spatially coregistered imaging setup to provide complementary, useful information of various tissue metrics in a simple and noncontact manner, making it attractive for use in a variety of biomedical applications.     

Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb + Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert-Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation (R = 0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb + Mb] was 0.9-1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700-965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF] = 2.7-2.8) in a living heart tissue.     

Investigation of the source‐detector separation in near infrared spectroscopy for healthy and clinical applications

Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations [(4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330)] each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.      

Safety assessment of near infrared light emitting diodes for diffuse optical measurements

Background  

Near infrared (NIR) light has been used widely to monitor important hemodynamic parameters in tissue non-invasively. Pulse oximetry, near infrared spectroscopy, and diffuse optical tomography are examples of such NIR light-based applications. These and other similar applications employ either lasers or light emitting diodes (LED) as the source of the NIR light. Although the hazards of laser sources have been addressed in regulations, the risk of LED sources in such applications is still unknown.     

Monte Carlo simulation of NIR diffuse reflectance in the normal and diseased human breast tissues

The spectral reflectance measurements in tissue reveal physiological meaning. Normally, functional changes like, increase in total hemoglobin concentration, decrease in oxygen saturation, etc., are observed when there is an abnormality creeping in the normal tissue. These functional changes can act together to reveal disease by non-invasive near-infrared (NIR) spectroscopy, as it influence its optical properties. In the present study, a simple two dimensional, four layer model of breast is proposed. The four layers are (i) skin (ii) adipose layer (iii) glandular tissue and (iv) muscle. Each layer is modeled with appropriate biological chromophores like hemoglobin, water, lipid and melanin. From the literature, the concentrations and molar extinction coefficients of the chromophores in various layers of the model are obtained. These values are used to calculate the wavelength dependent absorption characteristics of a particular layer. Monte Carlo simulation of diffuse reflectance (percentage of back reflected photons after multiple scattering with the broad variety of angles) are simulated for the modeled breast tissue with and without diseased condition. Near-infrared wavelengths are chosen, as the depth of penetration in tissue is more compared to UV and visible region. Simulations are carried out on the modeled breast tissue for different races (skin colors) at different NIR wavelengths. Results show significant changes in diffuse reflectance and relative absorbance for normal and diseased breast tissues for differently pigmented model. This model can be used to study the photo dynamical therapy, drug delivery and prognosis of cancer.     

In-Line Monitoring of a High-Shear Granulation Process Using the Baseline Shift of Near Infrared Spectra

Although near infrared (NIR) spectra are primarily influenced by undesired variations, i.e., baseline shifts and non-linearity, and many applications of NIR spectroscopy to the real-time monitoring of wet granulation processes have been reported, the granulation mechanisms behind these variations have not been fully discussed. These variations of NIR spectra can be canceled out using appropriate pre-processing techniques prior to spectral analysis. The present study assessed the feasibility of directly using baseline shifts in NIR spectra to monitor granulation processes, because such shifts can reflect changes in the physical properties of the granular material, including particle size, shape, density, and refractive index. Specifically, OPUSGRAN^®, a novel granulation technology, was investigated by in-line NIR monitoring. NIR spectra were collected using a NIR diffuse reflectance fiber optic probe immersed in a high-shear granulator while simultaneously examining the morphology, particle size, density, strength, and Raman images of the mixture during granulation. The NIR baseline shift pattern was found to be characteristic of the OPUSGRAN^® technology and was attributed to variations in the light transmittance, reflection, and scattering resulting from changes in the physicochemical properties of the samples during granulation. The baseline shift also exhibited an inflection point around the completion of granulation, and therefore may be used to determine the endpoint of the process. These results suggest that a specific pattern of NIR baseline shifts are associated with the unique OPUSGRAN^® granulation mechanism and can be applied to monitor the manufacturing process and determine the endpoint.     

Effect of the source and detector configuration on the detectability of breast cancer

Breast cancer causes the death of more than 150,000 women in the United States each year. Pregnant women cannot undergo mammography due to its dangerous side effects and, for younger women, a mammogram does not differentiate tumor from their dense breast tissue. Breast tumors usually become a localized absorber in the near infrared (NIR) wavelength region, because of the increased hemoglobin concentration around the area of the tumor. Therefore, NIR has a high potential to detect breast cancer without side effects. A computer simulation solving the photon transfer equation was used to study the detectability of various tumor sizes embedded in the breast model at various depths, for both reflectance and transmittance. Previous reflectance studies demonstrated that increasing the S-D separation does not necessarily allow the photons to penetrate deeper in the medium. The optimum S-D separation for breast tissue was found to be 3.0 cm, where the light penetrates up to 1.7 cm. Studies on the photon path in transmittance demonstrate that, at high modulation frequencies, (e.g. 1.0 GHz), the photon path becomes more coherent. Therefore, for transmittance measurements, high modulation frequencies can be useful to localize deep tumors. Multi frequency, multi- S-D separation reflectance can be used to provide information on tumor depth.     

Quantitative imaging of lymph function

Functional lymphatic imaging was demonstrated in the abdomen and anterior hindlimb of anesthetized, intact Yorkshire swine by using near-infrared (NIR) fluorescence imaging following intradermal administration of 100-200 microl of 32 microM indocyanine green (ICG) and 64 microM hyaluronan NIR imaging conjugate to target the lymph vascular endothelial receptor (LYVE)-1 on the lymph endothelium. NIR fluorescence imaging employed illumination of 780 nm excitation light ( approximately 2 mW/cm(2)) and collection of 830 nm fluorescence generated from the imaging agents. Our results show the ability to image the immediate trafficking of ICG from the plexus, through the vessels and lymphangions, and to the superficial mammary, subiliac, and middle iliac lymph nodes, which were located as deep as 3 cm beneath the tissue surface. "Packets" of ICG-transited lymph vessels of 2-16 cm length propelled at frequencies of 0.5-3.3 pulses/min and velocities of 0.23-0.75 cm/s. Lymph propulsion was independent of respiration rate. In the case of the hyaluronan imaging agent, lymph propulsion was absent as the dye progressed immediately through the plexus and stained the lymph vessels and nodes. Lymph imaging required 5.0 and 11.9 microg of ICG and hyaluronan conjugate, respectively. Our results suggest that microgram quantities of NIR optical imaging agents and their conjugates have a potential to image lymph function in patients suffering from lymph-related disorders.     

Fluorescence-enhanced,near infrared diagnostic imaging with contrast agents

The deep tissue propagation of near-infrared (NIR) light between 700-900 nm offers new opportunities for diagnostic imaging when employing sensitive detection techniques and NIR excitable fluorescent agents that target and report disease and metabolism. Herein, we highlight approaches for illuminating tissues and monitoring the re-emitted fluorescence for tomographic reconstruction, strategies for developing fluorescent dye constructs, and clinical opportunities for fluorescence-enhanced NIR optical imaging.     

Real‐time optical spectroscopic monitoring of nonirrigated lesion progression within atrial and ventricular tissues

Despite considerable advances in guidance of radiofrequency ablation (RFA) therapy for the treatment of cardiac arrhythmias, success rates have been hampered by a lack of tools for precise intraoperative evaluation of lesion extent. Near‐infrared spectroscopic (NIRS) techniques are sensitive to tissue structural and biomolecular properties, characteristics that are directly altered by radiofrequency (RF) treatment. In this work, a combined NIRS‐RFA catheter is developed for real‐time monitoring of tissue reflectance during RF energy delivery. An algorithm is proposed for processing NIR spectra to approximate nonirrigated lesion depth in both atrial and ventricular tissues. The probe optical geometry was designed to bias measurement influence toward absorption enabling enhanced sensitivity to changes in tissue composition. A set of parameters termed “lesion optical indices” are defined encapsulating spectral differences between ablated and unablated tissue. Utilizing these features, a model for real‐time tissue spectra classification and lesion size estimation is presented. Experimental validation conducted within freshly excised porcine cardiac specimens showed strong concordance between algorithm estimates and post‐hoc tissue assessment.      

Optical properties of Nannochloropsis sp and their application to remote estimation of cell mass

The absorption and scattering coefficients and reflectance spectra of ultra-high density Nannochloropsis occulata cultures were investigated in detail to identify the optical properties of the cultures and devise algorithms for remote estimation of dry cell mass in ultra-high cell density cultures. High-spectral resolution measurements of apparent absorption and attenuation as well as reflectance from 400 to 900 nm were carried out in relation to the dry weight, cell count, and pigment concentration in outdoor cultures. Indices calculated as (R(NIR) - R(red))/(R(NIR) + R(red)) and R(NIR)/R(red), in which R(NIR) is reflectance in the range from 750 to 800 nm and R(red) is reflectance in the range 670-680 nm, were used for remote assessment of dry cell mass. Remote estimation in the range 1 to 8 g/L was accomplished with an error of less than 0.66 g/L. A different index, i.e., (R(NIR) - R(red)) was employed for estimation of cell-chlorophyll concentration. This is the first report of in vivo specific absorption coefficient of chlorophyll-a and specific scattering coefficient per dry algal weight of Nannochloropsis sp., providing a basis for remote monitoring of dense phytoplankton masses.     

光动力治疗中光的有效吸收光剂量及其确定

光动力治疗中真正有效的光剂量是达到病变组织并且被组织中的光敏剂所吸收的那部分剂量,即有效吸收光剂量。明确组织中的有效吸收光剂量可以指导临床治疗,从而避免治疗剂量不足(治疗不彻底)或剂量过量(造成正常组织的热损伤)。而确定PDT中光的有效吸收剂量时,需测定组织中考察点的光辐射能流率。在目前计算和模拟组织中光辐射能流率的方法中,都需要首先确定所研究组织的光学特性参数。本文概述了常用的测定组织中光辐射能流率及组织光学特性参数的测量方法。     

Differential pathlength factor for diffuse photon scattering through tissue by a pulse-response method.

Although near-infrared (NIR) spectroscopy may one day provide a noninvasive measurement of oxidative metabolism in tissue, the method cannot be fully quantitative until the mean pathlength traveled by photons between reference and output detectors (i.e, optrodes) is known. In NIR spectroscopy, photons are transported primarily by diffuse scattering, and their mean pathlength can be expressed by a differential path factor (DPF) whose value is greater than the interoptrode distance. Beginning with a P1 diffusion approximation of the Boltzmann equation, one-dimensional photon currents originating from plane, line, and point photon sources were analyzed. DPF was formulated from the attenuation of light intensity generated by constant sources, and an equation for the mean time of flight of photons between reference and output optrodes, delta tau, was derived for arbitrarily pulsed sources. The results indicate that (1) the attenuation of light in tissue does not, in general, vary with interoptrode distance in the manner predicted by Beer's law; (2) the relationship between DPF and interoptrode distance is nonlinear and geometry-dependent; and (3) in spite of these nonidealities, DPF is equal to the product of delta tau and the speed of light.     

Polymorph Transformation in Paracetamol Monitored by In-line NIR Spectroscopy During a Cooling Crystallization Process

The reliable in-line monitoring of pharmaceutical processes has been regarded as a key tool toward the full implementation of process analytical technology. In this study, near-infrared (NIR) spectroscopy was examined for use as an in-line monitoring method of the paracetamol cooling crystallization process. The drug powder was dissolved in ethanol-based cosolvent at 60°C and was cooled by 1°C/min for crystallization. NIR spectra acquired by in-line measurement were interpreted by principal component analysis combined with off-line characterizations via X-ray diffraction, optical microscopy, and transmission electron microscopy. The whole crystallization process appeared to take place in three steps. A metastable form II polymorph of paracetamol was formed and transformed into the stable form I polymorph on the way to the growth of pure form I by cooling crystallization. These observations are consistent with a previous focused beam reflectance method-based study (Barthe et al., Cryst Growth Des 8:3316–3322, 2008).     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.