Mobile-phone-based Rheinberg microscope with a light-emitting diode array.

Mobile phone technology has led to implementation of portable and inexpensive microscopes. Light-emitting diode (LED) array microscopes support various multicontrast imaging by flexible illumination patterns of the LED array that can be achieved without changing the optical components of the microscope. Here, we demonstrate a mobile-phone-based LED array microscope to realize multimodal imaging with bright-field, dark-field, differential phase-contrast, and Rheinberg illuminations using as few as 37 LED bulbs. Using this microscope, we obtained high-contrast images of living cells. Furthermore, by changing the color combinations of Rheinberg illumination, we were able to obtain images of living chromatic structures with enhanced or diminished contrast. This technique is expected to be a foundation for high-contrast microscopy used in modern field studies.

Evaluation of dual-wavelength excitation autofluorescence imaging of colorectal tumours with a high-sensitivity CMOS imager: a cross-sectional study.

BACKGROUND: It is important to devise efficient and easy methods of detecting colorectal tumours to reduce mortality from colorectal cancer. Dual-wavelength excitation autofluorescence intensity can be used to visualize colorectal tumours. Therefore, we evaluated dual-wavelength excitation autofluorescence images of colorectal tumours obtained with a newly developed, high-sensitivity complementary metal-oxide-semiconductor (CMOS) imager. METHODS: A total 107 colorectal tumours (44 adenomas, 43 adenocarcinomas with intramucosal invasion, and 20 sessile serrated adenoma/polyps [SSA/Ps]) in 98 patients who underwent endoscopic tumour resection were included. The specimens were irradiated with excitation light at 365 nm and 405 nm, and autofluorescence images measured with a 475 ± 25-nm band pass filter were obtained using a new, high-sensitivity CMOS imager. Ratio images (F365ex/F405ex) were created to evaluate the lesion brightness compared with that of normal mucosa, and specimens were categorized into a no signal or high signal group. RESULTS: Adenomas and adenocarcinomas were depicted in 87 ratio images, with 86.2% (n = 75) in the High signal group. SSA/P was depicted in 20 ratio images, with 70.0% (n = 14) in the High signal group. CONCLUSIONS: Dual-wavelength excitation autofluorescence images of colorectal tumours can be acquired using our high-sensitivity CMOS imager, and are useful in detecting colorectal tumours.

Oximetry of retinal capillaries by multicomponent analysis.

Retinal oximetry of capillaries was performed for early detection of retinal vascular abnormalities, which are caused predominantly by complications of systemic circulatory diseases. As the conventional method for determining absorbance is not applicable to capillaries, multicomponent analysis was used to estimate the absorbance spectra of the retinal blood vessels. In this analysis, the capillary spectrum was classified as intermediate between those of the retinal arteries and veins, enabling relative estimation of oxygen saturation in the capillaries. This method could be useful for early recognition of disturbances in the peripheral circulation. Furthermore, a spectroscopic ophthalmoscope system based on the proposed method was developed to examine the human retina. A clinical trial of this system demonstrated that oximetry of the retinal capillaries may be an improvement over the present diagnosis for patients of malignant hypertension.

Temperature imaging of water in a microchannel using thermal sensitivity of near-infrared absorption.

This paper presents a remote and preparation-free method of temperature imaging of aqueous solutions in microchannels of microfluidic chips. The principle of this method is based on the temperature dependency of the near-infrared (NIR) absorption band (ν(2) + ν(3) band) of water. Temperature images were constructed from absorbances in a narrow wavelength range including 1908 nm, the most sensitive to temperature in the band, measured by using an NIR camera and an optical narrow-bandpass filter. Calculation and calibration results demonstrated a linear relationship between the absorption coefficient and temperature with a temperature coefficient of 1.5 × 10(-2) K(-1) mm(-1). Temperature images of 50 µm thick water in a Y-shaped PDMS microchannel locally heated by a neighboring hot wire were obtained, in which thermal diffusion processes in the microchip were visualized. Temperature resolution was estimated to be approximately 0.2 K according to the temperature coefficient and noise level.

Temperature measurements of turbid aqueous solutions using near-infrared spectroscopy.

We report a method that uses near-infrared spectroscopy and multivariate analysis to measure the temperature of turbid aqueous solutions. The measurement principle is based on the fact that the peak wavelength of the water absorption band, with its center near 1440 nm, shifts with changes in temperature. This principle was used to measure the temperatures of 1 mm thick samples of aqueous solutions containing Intralipid (2%), which are often used as optical phantoms for biological tissues due to similar scattering characteristics. Temperatures of pure water and aqueous solutions containing glucose (100 mg/ml and 200 mg/ml) were also measured for comparison. For the turbid Intralipid solutions, the absorbance spectrum varied irregularly with time due to the change in scattering characteristics. However, by making use of the difference between the absorbance at 1412 nm and the temperature-independent absorbance at 1440 nm, we obtained SEPs (standard error of prediction) of 0.3 degrees C and 0.2 degrees C by univariate linear regression and partial least squares regression, respectively. These accuracies were almost the same as those for the transparent samples (pure water and glucose solution).

Retinal blood oxygen saturation mapping by multispectral imaging and morphological angiography.

We report on an experiment for mapping two-dimensional blood oxygen saturation distribution by measuring multispectral images in the wavelength range from 500 to 650 nm with the resolution of 7 nm. The multispectral images of the retina are acquired with the originally designed imaging system equipped with the tunable spectral filter. To separate retinal blood vessels from other tissue area, morphological image processing is adopted. The small flick motion is also compensated. After prepossessing, the partial least squares regression model for the oxygen saturation is built by sampling typical spectra reflected from artery and vein. Applying the regression model for all the point extracted by the morphological processing allows the two-dimensional oxygen saturation map.

Measurement of Temperature Differences between Micro-regions in Water Using Near-Infrared Spectroscopy.

We have developed a method for measuring the temperature of micro-regions in aqueous solutions using near-infrared spectroscopy that enables us to measure the temperature of biological cells, tissues, and biochemical solutions in vitro. The measurement principle is based on the fact that the peak wavelength of the water absorption band with its center near 1450 nm shifts with changes in temperature. The measurement system, which consists of a biological microscope and two spectrophotometers, can measure respective absorbance spectra for two areas that are each 80 microm in diameter. We formed the temperature distribution in a 500-microm thick water film by heating an immersed Nichrome wire and measured the temperature difference between the two areas. The results of the measurement were compared to a calculated temperature distribution.

Estimation of water content distribution in the skin using dualband polarization imaging.

BACKGROUND/PURPOSE: A new enhancement imaging technique for visualizing two-dimensional distribution of water content in human tissue is proposed based on optical properties such as reflectance spectra and the scattering from the skin surface. METHODS: The method consists of two fundamental functions: spectral filtering for estimating skin hydration and polarization control for the appropriate measurement depth. Spectral filtering is simply achieved by utilizing the bandpass filters, whereas the measurement depth control is performed by changing the polarization directions in front of a light source and a detector. RESULTS: The combined method of spectral filtering and polarization imaging effectively visualized the water content distribution in skin as a shading on a composed black and white image. The result was compared with the measured value of the capacitance method, and the result of the proposed method was validated. CONCLUSION: Our results suggest that the proposed method enhances the scattering property in the skin according to the water content. Detailed discussions are given.

New methodology to obtain a calibration model for noninvasive near-infrared blood glucose monitoring.

This paper reports new methodology to obtain a calibration model for noninvasive blood glucose monitoring using diffuse reflectance near-infrared (NIR) spectroscopy. Conventional studies of noninvasive blood glucose monitoring with NIR spectroscopy use a calibration model developed by in vivo experimental data sets. In order to create a calibration model, we have used a numerical simulation of light propagation in skin tissue to obtain simulated NIR diffuse reflectance spectra. The numerical simulation method enables us to design parameters affecting the prediction of blood glucose levels and their variation ranges for a data set to create a calibration model using multivariate analysis without any in vivo experiments in advance. By designing the parameters and their variation ranges appropriately, we can prevent a calibration model from chance temporal correlations that are often observed in conventional studies using NIR spectroscopy. The calibration model (regression coefficient vector) obtained by the numerical simulation has a characteristic positive peak at the wavelength around 1600 nm. This characteristic feature of the regression coefficient vector is very similar to those obtained by our previous in vitro and in vivo experimental studies. This positive peak at around 1600 nm also corresponds to the characteristic absorption band of glucose. The present study has reinforced that the characteristic absorbance of glucose at around 1600 nm is useful to predict the blood glucose level by diffuse reflectance NIR spectroscopy. We have validated this new calibration methodology using in vivo experiments. As a result, we obtained a coefficient of determination, r2, of 0.87 and a standard error of prediction (SEP) of 12.3 mg/dL between the predicted blood glucose levels and the reference blood glucose levels for all the experiments we have conducted. These results of in vivo experiments indicate that if the parameters and their vibration ranges are appropriately taken into account in a numerical simulation, the new calibration methodology provides us with a very good calibration model that can predict blood glucose levels with small errors without conducting any experiments in advance to create a calibration model for each individual patient. This new calibration methodology using numerical simulation has promising potential for NIR spectroscopy, especially for noninvasive blood glucose monitoring.

Multispectral polarization imaging for observing blood oxygen saturation in skin tissue.

We propose a new technique that combines two-dimensional (2D) multispectral imaging and polarization gating for observing the blood oxygen saturation (SpO2) level in human skin tissue. The spectral decomposition of the skin tissue image provides the principal information on blood oxygenation. The polarization gating selects the measurement depth according to the relative orientation of the two polarizers that are placed on a camera and a light source. The combination of these two methods yields multispectral images of the superficial and deep layers of the skin tissue separately. In order to evaluate the blood oxygen, we focus on the multispectral images of the deep site. The SpO2 levels at each image pixel are calculated by means of the partial least squares regression with respect to each reflectance spectrum. The reassignment of the predicted responses retrieves an image whose pixel values represent the relative SpO2 levels. A demonstration experiment for acquiring the multispectral polarization images is performed in the spectral range of 500 to 680 nm, and the SpO2 distributions are obtained.

Regional difference of water content in human skin studied by diffuse-reflectance near-infrared spectroscopy: consideration of measurement depth.

Diffuse reflectance (DF) spectra in the 1250-2500 nm region were measured in vivo for the skin of the forehead, cheek, jaw, elbow, volar forearm, palm, knee, and heel of seven healthy volunteers, using a Fourier transform near-infrared (FT-NIR) spectrophotometer with a fiber-optic probe. Apparent regional differences of water content in the skin, as estimated from the diffuse reflectance NIR spectra, are discussed in relation to the influence of measurement depth. The NIR spectra were collected with or without a 300 microm gap between the fiber-optic probe and the skin surface. For comparison, in vitro NIR spectra of stratum corneum sheets equilibrated at 41, 50, 63, and 81% relative humidity, at 25 degrees C, were also obtained. There was a difference in the ratio of the two water bands centered near 1450 nm and 1900 nm between the contact and non-contact measurements. In addition, regional differences of water content calculated from the peak height of the 1900 nm water band, which was normalized to the peak height of the 2175 nm amide band, were compared. The results of Monte Carlo simulation indicated that the apparent regional differences arise at least in part from differences in the measurement depth due to differences in specular reflection at the skin surface and in the thickness of the stratum corneum.

Measurement of 2-D SpO2 distribution in skin tissue by multispectral imaging with depth selectivity control.

Two-dimensional hemoglobin oxygen saturation measurement is demonstrated by using the combination technique of multispectral imaging and the polarization control. Multispectral images are acquired at the wavelength range from 500 to 680 nm to observe the wavelength-dependent diffusely reflected light from the skin tissue. For eliminating the superficially reflected light from the skin, the skin tissue is illuminated by linearly polarized light and the polarization analyzer whose orientation is perpendicular to the illumination light is inserted in front of an imaging camera. The hemoglobin oxygen saturation levels corresponding to all image pixels are estimated by the partial least squares regression method with respect to each reflection spectrum. Mapping all the estimated values enables the oxygen saturation map across the observed tissue area.

Noninvasive near-infrared blood glucose monitoring using a calibration model built by a numerical simulation method: Trial application to patients in an intensive care unit.

We have applied a new methodology for noninvasive continuous blood glucose monitoring, proposed in our previous paper, to patients in ICU (intensive care unit), where strict controls of blood glucose levels are required. The new methodology can build calibration models essentially from numerical simulation, while the conventional methodology requires pre-experiments such as sugar tolerance tests, which are impossible to perform on ICU patients in most cases. The in vivo experiments in this study consisted of two stages, the first stage conducted on healthy subjects as preliminary experiments, and the second stage on ICU patients. The prediction performance of the first stage was obtained as a correlation coefficient (r) of 0.71 and standard error of prediction (SEP) of 28.7 mg/dL. Of the 323 total data, 71.5% were in the A zone, 28.5% were in the B zone, and none were in the C, D, and E zones for the Clarke error-grid analysis. The prediction performance of the second stage was obtained as an r of 0.97 and SEP of 27.2 mg/dL. Of the 304 total data, 80.3% were in the A zone, 19.7% were in the B zone, and none were in the C, D, and E zones. These prediction results suggest that the new methodology has the potential to realize a noninvasive blood glucose monitoring system using near-infrared spectroscopy (NIRS) in ICUs. Although the total performance of the present monitoring system has not yet reached a satisfactory level as a stand-alone system, it can be developed as a complementary system to the conventional one used in ICUs for routine blood glucose management, which checks the blood glucose levels of patients every few hours.

Depth profile of diffuse reflectance near-infrared spectroscopy for measurement of water content in skin.

BACKGROUND/PURPOSE: The penetration depth of light in diffuse reflectance near-infrared spectroscopy for measuring water content in skin is assessed both from theoretical and experimental points of view. METHODS: The Monte Carlo simulation was implemented to investigate the dependencies of the light penetration depth on a source-detector distance. To compare with the simulation results, an in vivo experiment for water contents of skin was performed introducing two different optical fiber probes. RESULTS: It is found that the minimum separation between a source and detector fibers influences largely the measurement depth. The larger separation leads to a deeper measurement depth at a particular wavelength. The measurement depth is also influenced fairly by the absorption coefficient of the tissue. The larger absorption coefficient results in a shallower measurement depth. CONCLUSION: The correlations between the water contents measured by the optical and capacitance techniques were discussed. The dependencies of the light penetration depth on the source-detector geometry and wavelength are presented.

Visualization technique for water content distribution of skin tissue by dualband polarization imaging.

The advanced imaging technique for visualizing two-dimensional water content distribution in the stratum corneum of human skin is proposed. The method involves two elemental principles of spectral filtering and imaging with polarization control. It is found in the in vivo experiment in the visible and the near-infrared range that the reflectance spectra of skin tissue in the wavelength range shorter than 600 nm are affected largely by water content. The polarization imaging technique is, on the other hand, introduced for emphasizing the subsurface reflection from skin tissue. The superficial reflection can be separated from the deeply penetrated light by controlling the orientations of the polarizer and the analyzing polarizer put in front of the light source and the imaging device, respectively. The combination of spectral bandpass filtering and polarization imaging enables us to acquire the water content distribution of the stratum corneum.

Non-contact skin moisture measurement based on near-infrared spectroscopy.

Non-contact skin moisture measurement based on near-infrared (NIR) spectroscopy is proposed in the spectral range from 1300 to 2000 nm. A gap is introduced between the optical fiber probe and the skin surface in order to avoid occluding surface vapor. In vitro and in vivo experiments for measuring the water content of skin are implemented. The measured absorbance spectra are processed by multivariate analyses. Processed results are compared with the water content values obtained by a capacitance method. The correlations between the optical method and the capacitance method obtained by partial-least squares regression are higher than those obtained by multiple linear regression. In addition, a Monte Carlo simulation is implemented to evaluate measurement depths of the optical methods. It is presented that the measurement depth of the optical method depends largely on the water absorption. The simulation result also shows that the measurement depth of the optical method is much deeper than the depth of the capacitance method, especially in the spectral range where water absorption is relatively weak.

Simulation study of in vitro glucose measurement by NIR spectroscopy and a method of error reduction.

The effects of some important factors on the blood glucose measurements by NIR spectroscopy are investigated by numerical simulation, and a method is proposed to significantly reduce the prediction errors induced by these effects. The changes in the absorbance spectra with the changes in the glucose concentration, temperature and scattering characteristics of background tissue are obtained by a Monte Carlo simulation of light propagation for the wavelength range from 1200 nm to 1800 nm. The glucose concentration is predicted by applying a multivariate analysis to the numerically simulated spectra. This process estimates the errors in the prediction of the glucose concentration induced by the temperature and scattering changes. It has been found that only 1 C change in the temperature or only 1% change in the scattering coefficient induces about 500 mg dl(-1) or 300 mg dl(-1) errors, respectively, in the prediction of the glucose concentration. These errors can be significantly reduced to less than 20 mg dl(-1) of the glucose concentration by incorporating the effects of the temperature and scattering characteristics on the spectra to the multivariate analysis.