In Vivo Transcutaneous Monitoring of Hemoglobin Derivatives Using a Red-Green-Blue Camera-Based Spectral Imaging Technique.

Cyanosis is a pathological condition that is characterized by a bluish discoloration of the skin or mucous membranes. It may result from a number of medical conditions, including disorders of the respiratory system and central nervous system, cardiovascular diseases, peripheral vascular diseases, deep vein thrombosis, and regional ischemia. Cyanosis can also be elicited from methemoglobin. Therefore, a simple, rapid, and simultaneous monitoring of changes in oxygenated hemoglobin and deoxygenated hemoglobin is useful for protective strategies against organ ischemic injury. We previously developed a red-green-blue camera-based spectral imaging method for the measurements of melanin concentration, oxygenated hemoglobin concentration (CHbO), deoxygenated hemoglobin concentration (CHbR), total hemoglobin concentration (CHbT) and tissue oxygen saturation (StO2) in skin tissues. We leveraged this approach in this study and extended it to the simultaneous quantifications of methemoglobin concentration (CmetHb), CHbO, CHbR, and StO2. The aim of the study was to confirm the feasibility of the method to monitor CmetHb, CHbO, CHbR, CHbT, and StO2. We performed in vivo experiments using rat dorsal skin during methemoglobinemia induced by the administration of sodium nitrite (NaNO2) and changing the fraction of inspired oxygen (FiO2), including normoxia, hypoxia, and anoxia. Spectral diffuse reflectance images were estimated from an RGB image by the Wiener estimation method. Multiple regression analysis based on Monte Carlo simulations of light transport was used to estimate CHbO, CHbR, CmetHb, CHbT, and StO2. CmetHb rapidly increased with a half-maximum time of less than 30 min and reached maximal values nearly 60 min after the administration of NaNO2, whereas StO2 dramatically dropped after the administration of NaNO2, indicating the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Time courses of CHbT and StO2, while changing the FiO2, coincided with well-known physiological responses to hyperoxia, normoxia, and hypoxia. The results indicated the potential of this method to evaluate changes in skin hemodynamics due to loss of tissue viability and vitality.

Effects of spatiotemporal averaging processes on the estimation of spectral reflectance in color digital holography using speckle illuminations.

We present the effects of spatiotemporal averaging processes on an estimation of spectral reflectance in color digital holography using speckle illuminations. In this technique, speckle fields emitted from a multimode fiber are used as both a reference wave and a wavefront illuminating an object. The interference patterns of two coherent waves for three wavelengths are recorded as digital holograms on a CCD camera. Speckle fields are changed by vibrating the multimode fiber using a vibrator, and a number of holograms are acquired to average reconstructed images. After performing an averaging process, which we refer to as a temporal averaging process in this study, using images reconstructed from multiple holograms, a spatial averaging process is applied using a smoothing window function. For the estimation of spectral reflectance in reconstructed images, we use the Wiener estimation method. The effects of the averaging processes on color reproducibility are evaluated by a chromaticity diagram, the root-mean-square error, and color differences.

Estimation of melanin and hemoglobin using spectral reflectance images reconstructed from a digital RGB image by the Wiener estimation method.

A multi-spectral diffuse reflectance imaging method based on a single snap shot of Red-Green-Blue images acquired with the exposure time of 65 ms (15 fps) was investigated for estimating melanin concentration, blood concentration, and oxygen saturation in human skin tissue. The technique utilizes the Wiener estimation method to deduce spectral reflectance images instantaneously from an RGB image. Using the resultant absorbance spectrum as a response variable and the extinction coefficients of melanin, oxygenated hemoglobin and deoxygenated hemoglobin as predictor variables, multiple regression analysis provides regression coefficients. Concentrations of melanin and total blood are then determined from the regression coefficients using conversion vectors that are numerically deduced in advance by the Monte Carlo simulations for light transport in skin. Oxygen saturation is obtained directly from the regression coefficients. Experiments with a tissue-like agar gel phantom validated the method. In vivo experiments on fingers during upper limb occlusion demonstrated the ability of the method to evaluate physiological reactions of human skin.

Measuring and imaging of transcutaneous bilirubin, hemoglobin, and melanin based on diffuse reflectance spectroscopy.

Significance: Evaluation of biological chromophore levels is useful for detection of various skin diseases, including cancer, monitoring of health status and tissue metabolism, and assessment of clinical and physiological vascular functions. Clinically, it is useful to assess multiple different chromophores in vivo with a single technique or instrument. Aim: To investigate the possibility of estimating the concentration of four chromophores, bilirubin, oxygenated hemoglobin, deoxygenated hemoglobin, and melanin from diffuse reflectance spectra in the visible region. Approach: A new diffuse reflectance spectroscopic method based on the multiple regression analysis aided by Monte Carlo simulations for light transport was developed to quantify bilirubin, oxygenated hemoglobin, deoxygenated hemoglobin, and melanin. Three different experimental animal models were used to induce hyperbilirubinemia, hypoxemia, and melanogenesis in rats. Results: The estimated bilirubin concentration increased after ligation of the bile duct and reached around 18 mg/dl at 50 h after the onset of ligation, which corresponds to the reference value of bilirubin measured by a commercially available transcutaneous bilirubin meter. The concentration of oxygenated hemoglobin and that of deoxygenated hemoglobin decreased and increased, respectively, as the fraction of inspired oxygen decreased. Consequently, the tissue oxygen saturation dramatically decreased. The time course of melanin concentration after depilation of skin on the back of rats was indicative of the supply of melanosomes produced by melanocytes of hair follicles to the growing hair shaft. Conclusions: The results of our study showed that the proposed method is capable of the in vivo evaluation of percutaneous bilirubin level, skin hemodynamics, and melanogenesis in rats, and that it has potential as a tool for the diagnosis and management of hyperbilirubinemia, hypoxemia, and pigmented skin lesions.

Noninvasive spectral imaging of skin chromophores based on multiple regression analysis aided by Monte Carlo simulation.

In order to visualize melanin and blood concentrations and oxygen saturation in human skin tissue, a simple imaging technique based on multispectral diffuse reflectance images acquired at six wavelengths (500, 520, 540, 560, 580 and 600 nm) was developed. The technique utilizes multiple regression analysis aided by Monte Carlo simulation for diffuse reflectance spectra. Using the absorbance spectrum as a response variable and the extinction coefficients of melanin, oxygenated hemoglobin, and deoxygenated hemoglobin as predictor variables, multiple regression analysis provides regression coefficients. Concentrations of melanin and total blood are then determined from the regression coefficients using conversion vectors that are deduced numerically in advance, while oxygen saturation is obtained directly from the regression coefficients. Experiments with a tissue-like agar gel phantom validated the method. In vivo experiments with human skin of the human hand during upper limb occlusion and of the inner forearm exposed to UV irradiation demonstrated the ability of the method to evaluate physiological reactions of human skin tissue.

Estimation of wavelength difference using scale adjustment in two-wavelength digital holographic interferometry.

We propose a method for an estimation of wavelength difference using scale adjustment in two-wavelength digital holographic interferometry. To estimate wavelength difference, two holograms recorded with different wavelengths are reconstructed on the basis of the Fresnel diffraction integral, and pixel sizes in the reconstruction plane, which depend on the wavelength in recording hologram, are analyzed. In the analysis, a zero-padding method and an intensity correlation function are used to adjust pixel sizes in the reconstruction plane and then obtain a wavelength difference given by a difference between the pixel sizes. Theoretical predictions and experimental results are shown to indicate the usefulness of the proposed method in this paper.

Transcutaneous monitoring of hemoglobin derivatives during methemoglobinemia in rats using spectral diffuse reflectance.

SIGNIFICANCE: Untreated methemoglobinemia may cause severe hypoxemia and even death when methemoglobin levels in the blood stream exceed 70%. Although CO-oximetry can be used to monitor the response to treatment for methemoglobinemia, it is costly and requires an invasive procedure for collecting blood samples from patients. A pulse CO-oximeter with a contact probe can be used to continuously and non-invasively measure the percentage of methemoglobin, as well as the percutaneous oxygen saturation. In terms of the prevention of infectious diseases, however, it is desirable to monitor methemoglobin and oxygen saturation levels in a non-contact manner. Diffuse reflectance spectral imaging is promising as a non-contact, non-invasive, and cost-effective clinical diagnostic tool for methemoglobinemia. AIM: To demonstrate the feasibility of visible spectral diffuse reflectance for in vivo monitoring of hemoglobin derivatives and evaluating methemoglobin production and reduction as well as hypoxemia during methemoglobinemia in rats. APPROACH: A new imaging approach based on the multiple regression analysis aided by Monte Carlo simulations for light transport was developed to quantify methemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin using a hyperspectral imaging system. An in vivo experiment with rats exposed to sodium nitrite (NaNO2) at different doses was performed to confirm the feasibility of the method for evaluating the dynamics of methemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin during methemoglobinemia. Systemic physiological parameters, including the percutaneous arterial oxygen saturation, heart rate (HR), and pulse distention, were measured by a commercially available pulse oximeter, and the results were compared to those obtained by the proposed method. RESULTS: Both the methemoglobin concentration and methemoglobin saturation rapidly increased with a half-maximum time of <20 min. They reached their maximal values nearly 60 min after the administration of NaNO2. Tissue oxygen saturation dramatically dropped to a minimum of 33.7 % ± 0.4 % , 23.1 % ± 5.6 % , 8.8 % ± 1.7 % , and 9.7 % ± 5.1 % on average for NaNO2 doses of 25, 37.5, 50, and 75 mg/kg, respectively. Changes in methemoglobin concentration and tissue oxygen saturation are indicative of the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Profound increases in the HR and pulse distention implied an elevated cardiac output caused by tachycardia and the resultant increase in peripheral blood volume to compensate for the hypoxia and hypoxemia during methemoglobinemia. This was in agreement with the time course of the peripheral hemoglobin volume concentration obtained by the proposed method. CONCLUSIONS: The proposed method is capable of the in vivo non-contact simultaneous evaluation of methemoglobin levels and hypoxemia during methemoglobinemia, and that it has potential as a tool for the diagnosis and management of methemoglobinemia.

Special Series Guest Editorial: Biomedical Imaging and Sensing III.

Guest editors Toyohiko Yatagai, Osamu Matoba, Yoshihisa Aizu, Yasuhiro Awatsuji, and Yuan Luo introduce the articles in the Special Series on Biomedical Imaging and Sensing.

Special Section Guest Editorial: Biomedical Imaging and Sensing II.

Guest editors introduce the special section of Journal of Biomedical Optics Volume 25, Issue 3, entitled "Biomedical Imaging and Sensing II," a collection of papers related to the topics of the conference "Biomedical Imaging and Sensing Conference 2019" (BISC'19), which was held in April 2019, in Yokohama, Japan.

Simple and affordable imaging of multiple physiological parameters with RGB camera-based diffuse reflectance spectroscopy.

We propose a simple and affordable imaging technique to evaluate transcutaneously multiple physiological parameters by using a digital red-green-blue camera. In this method, the RGB-values were converted into tristimulus values in the CIE (Commission Internationale de l'Eclairage) XYZ color space, which is compatible with the common color spaces. Monte Carlo simulation for light transport in biological tissue was then performed to specify the relationship among the XYZ-values and the concentrations of oxygenated hemoglobin, deoxygenated hemoglobin, bilirubin, and melanin. The concentration of total hemoglobin and tissue oxygen saturation were also calculated from the estimated concentrations of oxygenated and deoxygenated hemoglobin. In vivo experiments with bile duct ligation in rats demonstrated that the estimated bilirubin concentration increased after ligation of the bile duct and reached around 22 mg/dl at 116 h after the onset of ligation, which corresponds to the ground truth value of bilirubin measured by a commercially available transcutaneous bilirubinometer. Experiments with rats while varying the fraction of inspired oxygen demonstrated that oxygenated hemoglobin and deoxygenated hemoglobin decreased and increased, respectively, as the fraction of inspired oxygen decreased. Consequently, tissue oxygen saturation dramatically decreased. We further extended the method to a non-contact imaging photo-plethysmograph and estimation of the percutaneous oxygen saturation. An empirical formula to estimate percutaneous oxygen saturation was derived from the pulse wave amplitudes of oxygenated and deoxygenated hemoglobin. The estimated percutaneous oxygen saturation dropped remarkably when a faction of inspired oxygen was below 19%, indicating the onset of hypoxemia due to hypoxia, whereas the tissue oxygen saturation decreased gradually according to the reduction of the faction of inspired oxygen. The results in this study indicate the potential of this method for imaging of multiple physiological parameters in skin tissue and evaluating an optical biomedical imaging technique that enables cost-effective, easy-to-use, portable, remotely administered, and/or point-of-care solutions.

Non-contact imaging of peripheral hemodynamics during cognitive and psychological stressors.

Peripheral hemodynamics, measured via the blood volume pulse and vasomotion, provide a valuable way of monitoring physiological state. Camera imaging-based systems can be used to measure these peripheral signals without contact with the body, at distances of multiple meters. While researchers have paid attention to non-contact imaging photoplethysmography, the study of peripheral hemodynamics and the effect of autonomic nervous system activity on these signals has received less attention. Using a method, based on a tissue-like model of the skin, we extract melanin [Formula: see text] and hemoglobin [Formula: see text] concentrations from videos of the hand and face and show that significant decreases in peripheral pulse signal power (by 36% ± 29%) and vasomotion signal power (by 50% ± 26%) occur during periods of cognitive and psychological stress. Via three experiments we show that similar results are achieved across different stimuli and regions of skin (face and hand). While changes in peripheral pulse and vasomotion power were significant the changes in pulse rate variability were less consistent across subjects and tasks.

Special Section Guest Editorial: Biomedical Imaging and Sensing.

This guest editorial introduces the special section on Biomedical Imaging and Sensing.

Three-dimensional quantitative phase imaging of blood coagulation structures by optical projection tomography in flow cytometry using digital holographic microscopy.

Blood coagulation is an important role in the hemostasis process. In the observation using microscopies, an aggregation structure of red blood cells indicates the degree of blood coagulation. Recently, it has been proposed that digital holographic microscopy (DHM) is a powerful tool for biomedical cell imaging on the basis of quantitative phase information. DHM has the advantage in that the real-time and three-dimensional (3-D) quantitative phase imaging can be realized in the wide field of view, which means that the 3-D morphological parameters of biological cells without a staining process are obtained in real time. We report the complete 3-D quantitative phase imaging of blood coagulation structures by optical projection tomography in a flow cytometry using DHM.

Analysis of blood coagulation process based on fractality and dynamic characteristic of laser speckle pattern.

The reflection and transmission of coherent light from a biological system can yield information about its condition. In the case of blood exposed to the air, there is a change in the properties of the speckle patterns observed in the coagulation process. This can be studied by means of the rate of temporal variation, the contrast, and also the fractality of patterns. The fractality of the speckle pattern can be investigated by a fractal dimension, which can quantify a level of the complexity of platelet aggregation structure and a fibrin network formed in the process of blood coagulation. In addition, dynamic characteristics of a movement in blood also contain information on the progress of the coagulation process. Fractality and dynamic characteristics are investigated simultaneously for speckle patterns observed in the coagulation process of stored horse blood. Experimental results show the feasibility of the proposed method for detecting hemolysis and formation of platelet aggregation structure and the fibrin network during the coagulation process.

Effects of Bright Light with Reduced Blue Light on Sleepiness on Rising: A Small Exploratory Study.

Bright light therapy is a treatment modality for seasonal affective disorder and circadian rhythm disorders in which artificial light of 2,500 lux or higher at the eye is effective. Although short-wavelength visible light is more effective than long-wavelength visible light, it may be hazardous to the retina. Recently, light-emitting diodes (LEDs) have been used as the light source in bright light therapy apparatuses. We developed goggles for bright light therapy equipped with LEDs as the light source. The aim of this study was to examine the efficacy and safety of our goggles when emitting 10,000-lux light with its short-wavelength light content reduced by 30% or 50% (denoted as 30%-cut and 50%-cut light, respectively, henceforth). Six healthy young males participated in this study. They were administered no light, 50%-cut light, and 30%-cut light for 30 min early in the morning for 4 days each. Subjective sleepiness and sleep quality were evaluated by the Stanford Sleepiness Scale (SSS) and the Oguri-Shirakawa-Azumi sleep inventory MA version (OSA-MA), respectively. Subjective sleepiness evaluated by the SSS and the subscale of the OSA-MA significantly decreased with 30%-cut light compared with no light. Psychomotor performance evaluated by a calculation task improved with the 30%-cut light, although not significant after multiple comparisons were considered. No abnormality was found by ophthalmoscopy and the vision test. In conclusion, our goggles with 30%-cut light may be safe and have an awakening effect.

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy.

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum µa(λ) and the reduced scattering coefficient spectrum µs'(λ) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Visualizing depth and thickness of a local blood region in skin tissue using diffuse reflectance images.

A method is proposed for visualizing the depth and thickness distribution of a local blood region in skin tissue using diffuse reflectance images at three isosbestic wavelengths of hemoglobin: 420, 585, and 800 nm. Monte Carlo simulation of light transport specifies a relation among optical densities, depth, and thickness of the region under given concentrations of melanin in epidermis and blood in dermis. Experiments with tissue-like agar gel phantoms indicate that a simple circular blood region embedded in scattering media can be visualized with errors of 6% for the depth and 22% for the thickness to the given values. In-vivo measurements on human veins demonstrate that results from the proposed method agree within errors of 30 and 19% for the depth and thickness, respectively, with values obtained from the same veins by the conventional ultrasound technique. Numerical investigation with the Monte Carlo simulation of light transport in the skin tissue is also performed to discuss effects of deviation in scattering coefficients of skin tissue and absorption coefficients of the local blood region from the typical values of the results. The depth of the local blood region is over- or underestimated as the scattering coefficients of epidermis and dermis decrease or increase, respectively, while the thickness of the region agrees well with the given values below 1.2 mm. Decreases or increases of hematocrit value give over- or underestimation of the thickness, but they have almost no influence on the depth.

Estimation of melanin and hemoglobin in skin tissue using multiple regression analysis aided by Monte Carlo simulation.

To estimate the concentrations of melanin and blood and the oxygen saturation in human skin tissue, we propose a method using a multiple regression analysis aided by a Monte Carlo simulation for diffuse reflectance spectra from the skin tissue. By using the absorbance spectrum as a response variable and the extinction coefficients of melanin, oxygenated hemoglobin, and deoxygenated hemoglobin as predictor variables, the multiple regression analysis gives regression coefficients. The concentrations of melanin and blood are determined from the regression coefficients using conversion vectors that are estimated numerically in advance, while the oxygen saturation is obtained directly from the regression coefficients. Numerical and experimental investigations were performed for layered skin tissue models and phantoms. Measurements of human skin were also carried out to monitor variations in the melanin and blood contents and oxygenation during cuff occlusion. The results confirmed the usefulness of the proposed method.

Visualization of peripheral vasodilative indices in human skin by use of red, green, blue images.

We propose a method to visualize the arterial inflow, the vascular resistance, and the venous capacitance in the skin tissue from red, green, blue (RGB) digital color images. The arterial inflow and the venous capacitance in the skin tissue are visualized based on an increase in the rate of change in the total blood concentration and the change of the total blood concentration during upper limb occlusion at a pressure of 50 mmHg. The resultant arterial inflow with the measured mean arterial pressure also provides an image of the vascular resistance in human skin. The arterial inflow, the vascular resistance, and the venous capacitance acquired by the method are well correlated with those obtained from the conventional strain-gauge plethysmograph. The correlation coefficients R between the estimated values by the method and the measurements by the SPG are calculated to be 0.83 (P < 0.001) for the arterial inflow, 0.77 (P < 0.01) for the vascular resistance, and 0.77 (P < 0.01) for the venous capacitance. The arterial inflow and the venous capacitance in the skin tissue are significantly higher in active subjects compared with the sedentary subjects, whereas the vascular resistance was significantly lower in the active subjects compared with the sedentary subjects. The results of the present study indicate the possibility of using the proposed method for evaluating the peripheral vascular functions in human skin.

Visualization of peripheral vasodilative indices in human skin by use of red, green, blue images.

We propose a method to visualize the arterial inflow, the vascular resistance, and the venous capacitance in the skin tissue from red, green, blue (RGB) digital color images. The arterial inflow and the venous capacitance in the skin tissue are visualized based on an increase in the rate of change in the total blood concentration and the change of the total blood concentration during upper limb occlusion at a pressure of 50 mmHg. The resultant arterial inflow with the measured mean arterial pressure also provides an image of the vascular resistance in human skin. The arterial inflow, the vascular resistance, and the venous capacitance acquired by the method are well correlated with those obtained from the conventional strain-gauge plethysmograph. The correlation coefficients R between the estimated values by the method and the measurements by the SPG are calculated to be 0.83 (P<0.001) for the arterial inflow, 0.77 (P<0.01) for the vascular resistance, and 0.77 (P<0.01) for the venous capacitance. The arterial inflow and the venous capacitance in the skin tissue are significantly higher in active subjects compared with the sedentary subjects, whereas the vascular resistance was significantly lower in the active subjects compared with the sedentary subjects. The results of the present study indicate the possibility of using the proposed method for evaluating the peripheral vascular functions in human skin.