Design of a Low-Cost Diffuse Optical Mammography System for Biomedical Image Processing in Breast Cancer Diagnosis.

Worldwide, breast cancer is the most common type of cancer that mainly affects women. Several diagnosis techniques based on optical instrumentation and image analysis have been developed, and these are commonly used in conjunction with conventional diagnostic devices such as mammographs, ultrasound, and magnetic resonance imaging of the breast. The cost of using these instruments is increasing, and developing countries, whose deaths indices due to breast cancer are high, cannot access conventional diagnostic methods and have even less access to newer techniques. Other studies, based on the analysis of images acquired by traditional methods, require high resolutions and knowledge of the origin of the captures in order to avoid errors. For this reason, the design of a low-cost diffuse optical mammography system for biomedical image processing in breast cancer diagnosis is presented. The system combines the acquisition of breast tissue photographs, diffuse optical reflectance (as a biophotonics technique), and the processing of digital images for the study and diagnosis of breast cancer. The system was developed in the form of a medical examination table with a 638 nm red-light source, using light-emitted diode technology (LED) and a low-cost web camera for the acquisition of breast tissue images. The system is automatic, and its control, through a graphical user interface (GUI), saves costs and allows for the subsequent analysis of images using a digital image-processing algorithm. The results obtained allow for the possibility of planning in vivo measurements. In addition, the acquisition of images every 30° around the breast tissue could be used in future research in order to perform a three-dimensional (3D) reconstruction and an analysis of the captures through deep learning techniques. These could be combined with virtual, augmented, or mixed reality environments to predict the position of tumors, increase the likelihood of a correct medical diagnosis, and develop a training system for specialists. Furthermore, the system allows for the possibility to develop analysis of optical characterization for new phantom studies in breast cancer diagnosis through bioimaging techniques.

Norcantharidin toxicity profile: an in vivo murine study.

Norcantharidin (NCTD) is the demethylated analog of cantharidin, with allegedly reduced toxicity. However, there is still limited information regarding its posology and potential risk in its use in cancer treatment. Healthy BDF1 mice were intraperitoneally administered with norcantharidin (0, 3, 6, 12, and 25 mg/kg) every 24 h for 6 days. Survivor mice were euthanized, and the brain, lungs, kidneys, spleen, and liver were procured for enzymatic and histopathological analysis in the liver and kidney. DL50 were 8.86 mg/kg for females and 11.77 mg/kg for males. The treatments with 3.0 mg/kg and 6.0 mg/kg significantly modified the phosphorylase, alanine transaminase, and γ-glutamyl transferase activities; however, an organ-specific response was detected. A significant dose-dependent decrease was observed in the kidney for ROS, while the liver had the opposite effect. Histopathological analysis revealed a significant elevation in hepatocytes' nuclei average size and total area (3 mg/kg), as well as centrilobular vein and adjacent sinusoidal capillaries showed a significant difference. The portal triad presented a significant difference in veins and capillarity count in 6 mg/kg. Renal samples showed cortex convoluted tubules' average size significantly augmented in both doses' groups, and tubule count was found augmented in 6 mg/kg. These physiological effects of NCTD can be exploited as treatment strategies if able to operate in an established posology and proper testing.

Combined methods of optical spectroscopy and artificial intelligence in the assessment of experimentally induced non-alcoholic fatty liver.

BACKGROUND AND OBJECTIVE: Due to the existing prevalence of nonalcoholic fatty liver disease (NAFLD) and its relation to the epidemic of obesity in the general population, it is imperative to develop detection and evaluation methods of the early stages of the disease with improved efficacy over the current diagnostic approaches. We aimed to obtain an improved diagnosis, combining methods of optical spectroscopy -diffuse reflectance and fluorescence- with statistical data analysis applied to detect early stages of NAFLD. METHODS: Statistical analysis scheme based on quadratic discriminant analysis followed by canonical discriminant analysis were applied to the diffuse reflectance data combined with endogenous fluorescence spectral data excited at one of these wavelengths: 330, 365, 385, 405 or 415 nm. The statistical scheme was also applied to the combinations of fluorescence spectrum (405 nm) with each one of the other fluorescence spectra. Details of the developed software, including the application of machine learning algorithms to the combination of spectral data followed by classification statistical schemes, are discussed. RESULTS: Steatosis progression was differentiated with little classification error (≤1.3%) by using diffuse reflectance and endogenous fluorescence at different wavelengths. Similar results were obtained using fluorescence at 405 nm and one of the other fluorescence spectra (classification error ≤1.0%). Adding the corresponding areas under the curves to the above combinations of spectra diminished errors to 0.6% and 0.3% or less, respectively. The best results for the compounded reflectance-plus-fluorescence spectra were obtained with fluorescence spectra excited at 415 nm with a total classification error of 0.2%; for the combination of the 405nm-excited fluorescence spectrum with another fluorescence spectrum, the best results were achieved for 385 nm, for which total relative classification error amounted 0.4%. The consideration of the area under the spectral curves further improved both classifiers, reducing the error to 0.0% in both cases. CONCLUSION: Spectrometric techniques combined with statistical processing are a promising tool to improve steatosis classification through a label free approach. However, statistical schemes here applied, might result complex for the everyday medical practice, the designed software including machine learning algorithms is able to render automatic classification of samples according to their steatosis grade with low error.

Fluorescence spectroscopy on paraffin-preserved human liver samples to classify several grades of fibrosis.

Nowadays, it is well established that biopsy is the gold standard for medical diagnosis of liver disease; however, recent studies have shown numerous discrepancies in biopsy assessment, even when it is evaluated by senior pathologists. Fluorescence spectroscopy is a tool that has been of utility in the diagnosis of different diseases based on biopsy analysis. Thus, fluorescence study of liver samples with five different degrees of fibrosis is presented. Paraffin-preserved human liver tissue was provided on white plastic cassettes by the Hospital General de Mexico "Dr. Eduardo Liceaga". Specimens were diagnosed by two independent-senior pathologists in a double-blind test and classified into five different groups: F0, F1, F2, F3, and F4, according to the METAVIR scale for liver fibrosis. Fluorescence spectroscopy measurements were performed using three different excitation wavelengths: 385, 405, and 450 nm. Besides, diffuse reflectance spectroscopy (DRS) measurements were taken with white light to determine morphological changes in the tissue and to compare the results with medical diagnosis. The spectral analysis at excitation wavelengths of 385 nm and 405 nm showed poor correlation with medical diagnosis. Likewise, in order to discard all possible error-sources involved in the measurements, an exhaustive study was carried out; it included the determination of the fluorescence noise produced by paraffin, cassette, and the tissue itself. At 450 nm excitation wavelength, no fluorescence by the cassette was detected and noise-subtraction methods were not required, this allows a high correlation of hepatic fibrosis stages between pathological diagnosis and spectroscopic analysis. For this excitation wavelength, 89.87% correlation with DRS measurements and 82.00% with medical diagnosis were obtained. This work demonstrates that fluorescence spectroscopy using 450 nm excitation wavelength might work as a complementary tool to grade hepatic fibrosis in human liver specimens.

Fiber-optic pulseoximeter for local oxygen saturation determination using a Monte Carlo multi-layer model for calibration.

BACKGROUND AND OBJECTIVE: Local tissue oxygenation determines the relationship between the supply and the demand for oxygen by the tissue and it is an important indicator of the physiological or pathological condition of the tissue. Moreover, some therapeutic methods strongly depend on the oxygen content of the tissue. In photodynamic therapy, when molecular oxygen is present, the irradiation of the photosensitizer with light triggers the generation of reactive oxygen species that kill the target diseased cells within the treated tissue. To ensure the best possible therapy response, the tissue must be well oxygenated; hence, oxygen concentration measurement becomes a decisive factor. In this work, the design, construction and calibration of a module to locally measure the blood oxygen saturation in tissue is presented. METHODS: The system is built using a red (660-nm) and an infrared (940-nm) light emitting diodes as light sources, a photodiode as a detector, and a homemade handheld fiber optic-based reflectance pulse oximetry sensor. In addition, the developed sensor was modeled by means of multilayered Monte Carlo simulations, to study its behavior when used in different thickness and melanin content skin. RESULTS: From the simulation reflectance values, the oxygen saturation calibration curves considering different melanin concentrations and skin thicknesses were obtained for two different skin models, one comprising three skin layers and the second, assuming seven different layers for the skin. A comparison of the performances of the developed pulse oximeter sensor with a commercial one is also presented. CONCLUSIONS: A new pulseoximeter for the measurement of local oxygenation in tissue was developed. Its calibration strongly depends on the site of measurement due to the influence of tissue thickness, vascularization, and melanin content. A three-layer skin model is proved to be suitable for the calibration of the pulseoximeter in thin and medium thickness skin.

Study of Methionine Choline Deficient Diet-Induced Steatosis in Mice Using Endogenous Fluorescence Spectroscopy.

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees of steatosis. Ex-vivo liver samples were collected from mice fed a methionine-choline deficient diet for two or eight weeks, and from a control group. The degree of hepatic steatosis was histologically evaluated, and fat content was assessed by Oil-Red O staining. On the other hand, fluorescence spectroscopy was used for the assessment of the steatosis progression. Fluorescence spectra were recorded at excitation wavelengths of 330, 365, 385, 405, and 415 nm by establishing surface contact of the fiber optic probe with the liver specimens. A multi-variate statistical approach based on principal component analysis followed by quadratic discriminant analysis was applied to spectral data to obtain classifiers able to distinguish mild and moderate stages of steatosis at the different excitation wavelengths. Receiver Operating Characteristic (ROC) curves were computed to compare classifier's performances for each one of the five excitation wavelengths and steatosis stages. Optimal sensitivity and specificity were calculated from the corresponding ROC curves using the Youden index. Intensity in the endogenous fluorescence spectra at the given wavelengths progressively increased according to the time of exposure to diet. The area under the curve of the spectra was able to discriminate control liver samples from those with steatosis and differentiate among the time of exposure to the diet for most of the used excitation wavelengths. High specificities and sensitivities were obtained for every case; however, fluorescence spectra obtained by exciting with 405 nm yielded the best results distinguishing between the mentioned classes with a total classification error of 1.5% and optimal sensitivities and specificities better than 98.6% and 99.3%, respectively.

Diffuse reflectance spectroscopy accurately discriminates early and advanced grades of fatty liver in mice.

Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis and cirrhosis. Liver biopsy, considered the gold standard to diagnose NAFLD, shows significantly high rates of interobserver variability. Thus there is a need to develop tools that accurately categorize mild and advanced grades of steatosis in order to identify patients at higher risk of developing chronic liver disease. Diffuse reflectance spectroscopy (DRS) has proved to be useful in grading liver fibrosis and cirrhosis, without having been implemented for steatosis. We aim to categorize early and advanced stages of liver steatosis in a methionine-choline deficient (MCD) mouse model. C57bl/6 mice are fed either methionine-choline control or MCD diet during 2 or 8 weeks to induce mild and advanced steatosis. Liver samples are obtained and steatosis is evaluated by oil red O staining. Diffuse reflectance spectra are directly measured on ex vivo liver specimens, in a wavelength range of 400 to 800 nm. DRS is able to discriminate between early or advanced steatosis and healthy hepatic tissue with negligible error while showing high average sensitivity and specificity (0.94 and 0.95, respectively). Our results suggest that liver steatosis can be accurately evaluated by DRS, highlighting the importance of applied spectroscopic methods in assessing NAFLD.

Fluorescence Spectroscopy as a Tool for the Assessment of Liver Samples with Several Stages of Fibrosis.

BACKGROUND: During the last years, fluorescence spectroscopy has been used as a potential tool for the evaluation and characterization of tissues with different disease conditions due to its low cost, high sensitivity, and minimally or noninvasive character. OBJECTIVE: In this study, fluorescence spectroscopy was used to study 19 paraffin blocks containing human liver tissue from biopsies. METHODS AND RESULTS: All samples were previously analyzed by two senior pathologists in a single-blind trial. After their evaluation, four liver samples were classified as nonfibrosis (F0), four as initial fibrosis (F1-F2), four as advanced fibrosis (F3), and six as cirrhosis (F4). The fluorescence was induced at different wavelengths as follows: 330, 365, and 405 nm using a portable fiber-optic system. The fluorescence spectra were recorded in the range of 400-750 nm. A distinctive correlation between the shape of each spectrum and the level of fibrosis in the liver sample was detected. A multi-variate statistical analysis based on principal component analysis followed by linear discrimination analysis was applied to develop algorithms able to distinguish different stages of fibrosis based on the characteristics of fluorescence spectra. Pairwise comparisons were performed: F0 versus F1-F2, F1-F2 versus F3, F3 versus F4, and F1-F2 versus F4. The algorithms applied to each set of data yielded values of sensitivity and specificity that were higher than 90% and 95%, respectively, in all the analyzed cases. CONCLUSIONS: With this study, it is concluded that fluorescence spectroscopy can be used as a complementary tool for the assessment of liver fibrosis in liver tissue samples, which sets the stage for subsequent clinical trials.

In Vitro Photoirradiation System for Simultaneous Irradiation with Different Light Doses at a Fixed Temperature.

OBJECTIVE: In this work an irradiance and temperature controlled in-vitro system for conducting investigations in PDT and phototherapy is presented. BACKGROUND DATA: The development of new light sources has caused a considerable increase in research and application of several photodynamic (PDT) therapeutic methods, as well as other light-based therapeutic techniques. However, further work is needed to fully understand and elucidate the mechanisms as well as to increase the effectiveness of PDT. Nowadays, there are no commercial systems to perform automated light exposure experiments with cultured cells. Also, there are very few reports of similar photoirradiation systems. MATERIALS AND METHODS: The system is composed of 24 independent light-emitting diodes that can be used to irradiate separate wells in a microwell plate. The system includes a module to measure changes in temperature within each irradiated well without contact. The light sources are placed on a plate that can easily be changed in order to irradiate at different wavelengths. The performance of the system is fully controlled with a computer, and all the experimental data are properly recorded. RESULTS: The design, construction, operation, and a full characterization of the system are presented. CONCLUSIONS: A novel fully automated photoirradiation system has been developed. The system allows the design of the experiments in this area with precise dosimetry, temperature, and irradiation regime controls reducing manipulation of the samples and saving time.

Spectroscopic and Imaging Characteristics of Pigmented Non-Melanoma Skin Cancer and Melanoma in Patients with Skin Phototypes III and IV.

INTRODUCTION: Non-melanoma skin cancer is the most common malignancy worldwide. Differentiating between malignant and benign skin tumors, however, can be challenging. As a result, various auxiliary tools have been developed to aid in the diagnosis of cutaneous neoplasms. Here, skin tumors were investigated through analysis of their digital image histograms and spectroscopic response under ultraviolet (UV) and white light-emitting diodes (LEDs). METHODS: Fifty tumoral lesions were spectroscopically and histologically studied. For optical studies, UV at 375 nm and white LEDs were used to illuminate the lesions. Commercial cameras were used for imaging, and a miniature spectrometer with a bifurcated optical fiber was used for spectroscopic measurements. RESULTS: In this study, the intensity histograms of the images taken under white and UV illumination and the spectroscopic response under white light showed clear differences between pigmented basal cell carcinoma (BCC), intradermal melanocytic nevus (IDN), and melanoma lesions for skin phototypes III and IV. However, there was little difference in their spectroscopic response to the UV LED. CONCLUSION: We found differences in the intensity and shape of diffuse reflectance spectra of pigmented BCC, IDN, and melanoma lesions in patients with skin phototypes III and IV. Also, images taken under UV and white light were helpful for differentiation of these pigmented lesions. Additional research is needed to ascertain the clinical utility of these tools for skin cancer diagnosis.

Diffuse reflectance spectroscopy as a possible tool to complement liver biopsy for grading hepatic fibrosis in paraffin-preserved human liver specimens.

A diffuse reflectance spectroscopy-based method to score fibrosis in paraffin-preserved human liver specimens has been developed and is reported here. Paraffin blocks containing human liver tissue were collected from the General Hospital of Mexico and included in the study with the patients' written consent. The score of liver fibrosis was determined in each sample by two experienced pathologists in a single-blind fashion. Spectral measurements were acquired at 450-750 nm by establishing surface contact between the optical probe and the preserved tissue. According to the histological evaluation, four liver samples showed no evidence of fibrosis and were categorized as F0, four hepatic specimens exhibited an initial degree of fibrosis (F1-F2), five liver specimens showed a severe degree of fibrosis (F3), and six samples exhibited cirrhosis (F4). The human liver tissue showed a characteristic diffuse reflectance spectrum associated with the progressive stages of fibrosis. In the F0 liver samples, the diffuse reflection intensity gradually increased in the wavelength range of 450-750 nm. In contrast, the F1-F2, F3, and F4 specimens showed corresponding 1.5-, 2-, and 5.5-fold decreases in the intensity of diffuse reflectance compared to the F0 liver specimens. At 650 nm, all the stages of liver fibrosis were clearly distinguished from each other with high sensitivity and specificity (92-100%). To our knowledge, this is the first study reporting a distinctive diffuse reflectance spectrum for each stage of fibrosis in paraffin-preserved human liver specimens. These results suggest that diffuse reflectance spectroscopy may represent a complementary tool to liver biopsy for grading fibrosis.