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.

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.

In Vitro Antimicrobial Photodynamic Therapy for Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA) Inhibition Using a Green Light Source.

Photodynamic therapy (PDT) has been based on using photosensitizers (PS) and applying light of a specific wavelength. When this technique is used for treating infections, it is known as antimicrobial photodynamic therapy (aPDT). Currently, the use of lighting sources for in vitro studies using aPDT is generally applied in multiwell cell culture plates; however, depending on the lighting arrangement, there are usually errors in the application of the technique because the light from a well can affect the neighboring wells or it may be that not all the wells are used in the same experiment. In addition, one must be awarded high irradiance values, which can cause unwanted photothermal problems in the studies. Thus, this manuscript presents an in vitro antimicrobial photodynamic therapy for a Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA) inhibition study using an arrangement of thermally isolated and independently illuminated green light source systems for eight tubes in vitro aPDT, determining the effect of the following factors: (i) irradiance level, (ii) exposure time, and (iii) Rose Bengal (RB) concentration (used as a PS), registering the Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA) inhibition rates. The results show that in the dark, RB had a poor antimicrobial rate for P. aeruginosa, finding the maximum inhibition (2.7%) at 30 min with an RB concentration of 3 µg/mL. However, by applying light in a correct dosage (time × irradiance) and the adequate RB concentration, the inhibition rate increased by over 37%. In the case of MRSA, there was no significant inhibition with RB in complete darkness and, in contrast, the rate was 100% for those experiments that were irradiated.

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.

Theranostic-PDT with the antibody anti isoform 4 SOD mitocondrial labeled with PpIX in the lung cancer cell line A-549.

BACKGROUND: In this work, a drug product composed of an IgM antibody derived from a hybridoma subclone 4C1F6D5G7B8 was prepared and further labeled with PpIX to be used in cell lines A-549 and MRC-5. The aim of this work was to evaluate the potential theranostic activity of the obtained product together with photodynamic therapy (PDT). METHODS: The IgM antibody labeled with PpIX was used in different concentrations to perform theranostics with PDT in cell lines A-549 and MRC-5 in order to identify the specificity of IgM antibody in lung cancer cells by means of a LED-irradiation system set at 630 nm. The location of the conjugate was further determined by confocal microscopy. RESULTS: The theranostic with conjugate Ab-PpIX in the A-549 cell lines showed fluorescence by confocal microscopy, whereas the MRC-5 cell line showed no reactivity. The PDT with the conjugate in the cell line A-549 decreased its viability 70% compared to the control. On the contrary, with the MRC-5 cell line no viability diference was shown. The confocal microscopy applied to the cell line A-549 showed that the Ab-PpIX was majorly located at the cytoplasm. CONCLUSION: Ab-PpIX showed therapeutical potential in lung cancer cells A-549 and had no activity in non-cancerous lung cells (MCR-5).

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.

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.

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.

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.