Membrane Protein Detection and Morphological Analysis of Red Blood Cells in Hereditary Spherocytosis by Confocal Laser Scanning Microscopy.

In hereditary spherocytosis (HS), genetic mutations in the cell membrane and cytoskeleton proteins cause structural defects in red blood cells (RBCs). As a result, cells are rigid and misshapen, usually with a characteristic spherical form (spherocytes), too stiff to circulate through microcirculation regions, so they are prone to undergo hemolysis and phagocytosis by splenic macrophages. Mild to severe anemia arises in HS, and other derived symptoms like splenomegaly, jaundice, and cholelithiasis. Although abnormally shaped RBCs can be identified under conventional light microscopy, HS diagnosis relies on several clinical factors and sometimes on the results of complex molecular testing. It is specially challenging when other causes of anemia coexist or after recent blood transfusions. We propose two different approaches to characterize RBCs in HS: (i) an immunofluorescence assay targeting protein band 3, which is affected in most HS cases and (ii) a three-dimensional morphology assay, with living cells, staining the membrane with fluorescent dyes. Confocal laser scanning microscopy (CLSM) was used to carry out both assays, and in order to complement the latter, a software was developed for the automated detection of spherocytes in blood samples. CLSM allowed the precise and unambiguous assessment of cell shape and protein expression.

Reflectance evaluation of eye fundus structures with a visible and near-infrared multispectral camera.

We examined the spectral reflectance of fundus structures in the visible and near-infrared (400-1300 nm) range for contributing to the medical diagnosis of fundus diseases. Spectral images of healthy eye fundus and other ocular diseases were acquired using a novel multispectral fundus camera. Reflectance metrics were computed based on contrast to analyze the spectral features. Significant differences were observed among the structures in healthy and diseased eye fundus. Specifically, near-infrared analysis allows imaging of deeper layers, such as the choroid, which, to date, has not been retrieved using traditional color fundus cameras. Pathological structures, which were hardly observable in color fundus images owing to metamerism, were also revealed by the developed multispectral fundus camera.

Optical Technologies for the Improvement of Skin Cancer Diagnosis: A Review.

The worldwide incidence of skin cancer has risen rapidly in the last decades, becoming one in three cancers nowadays. Currently, a person has a 4% chance of developing melanoma, the most aggressive form of skin cancer, which causes the greatest number of deaths. In the context of increasing incidence and mortality, skin cancer bears a heavy health and economic burden. Nevertheless, the 5-year survival rate for people with skin cancer significantly improves if the disease is detected and treated early. Accordingly, large research efforts have been devoted to achieve early detection and better understanding of the disease, with the aim of reversing the progressive trend of rising incidence and mortality, especially regarding melanoma. This paper reviews a variety of the optical modalities that have been used in the last years in order to improve non-invasive diagnosis of skin cancer, including confocal microscopy, multispectral imaging, three-dimensional topography, optical coherence tomography, polarimetry, self-mixing interferometry, and machine learning algorithms. The basics of each of these technologies together with the most relevant achievements obtained are described, as well as some of the obstacles still to be resolved and milestones to be met.

Spectroscopic Evaluation of Red Blood Cells of Thalassemia Patients with Confocal Microscopy: A Pilot Study.

Hemoglobinopathies represent the most common single-gene defects in the world and pose a major public health problem, particularly in tropical countries, where they occur with high frequency. Diagnosing hemoglobinopathies can sometimes be difficult due to the coexistence of different causes of anemia, such as thalassemia and iron deficiency, and blood transfusions, among other factors, and requires expensive and complex molecular tests. This work explores the possibility of using spectral confocal microscopy as a diagnostic tool for thalassemia in pediatric patients, a disease caused by mutations in the globin genes that result in changes of the globin chains that form hemoglobin-in pediatric patients. Red blood cells (RBCs) from patients with different syndromes of alpha-thalassemia and iron deficiency (including anemia) as well as healthy (control) subjects were analyzed under a Leica TCS SP8 confocal microscope following different image acquisition protocols. We found that diseased RBCs exhibited autofluorescence when excited at 405 nm and their emission was collected in the spectral range from 425 nm to 790 nm. Three experimental descriptors calculated from the mean emission intensities at 502 nm, 579 nm, 628 nm, and 649 nm allowed us to discriminate between diseased and healthy cells. According to the results obtained, spectral confocal microscopy could serve as a tool in the diagnosis of thalassemia.

Fast visible and extended near-infrared multispectral fundus camera.

We present a multispectral fundus camera that performs fast imaging of the ocular posterior pole in the visible and near-infrared (400 to 1300 nm) wavelengths through 15 spectral bands, using a flashlight source made of light-emitting diodes, and CMOS and InGaAs cameras. We investigate the potential of this system for visualizing occult and overlapping structures of the retina in the unexplored wavelength range beyond 900 nm, in which radiation can penetrate deeper into the tissue. Reflectance values at each pixel are also retrieved from the acquired images in the analyzed spectral range. The available spectroscopic information and the visualization of retinal structures, specifically the choroidal vasculature and drusen-induced retinal pigment epithelium degeneration, which are hardly visible in conventional color fundus images, underline the clinical potential of this system as a new tool for ophthalmic diagnosis.

Morphological study of skin cancer lesions through a 3D scanner based on fringe projection and machine learning.

The effective and non-invasive diagnosis of skin cancer is a hot topic, since biopsy is a costly and time-consuming surgical procedure. As skin relief is an important biophysical feature that can be difficult to perceive with the naked eye and by touch, we developed a novel 3D imaging scanner based on fringe projection to obtain morphological parameters of skin lesions related to perimeter, area and volume with micrometric precision. We measured 608 samples and significant morphological differences were found between melanomas and nevi (p<0.001). The capacity of the 3D scanner to distinguish these lesions was supported by a supervised machine learning algorithm resulting in 80.0% sensitivity and 76.7% specificity.

Visible and Extended Near-Infrared Multispectral Imaging for Skin Cancer Diagnosis.

With the goal of diagnosing skin cancer in an early and noninvasive way, an extended near infrared multispectral imaging system based on an InGaAs sensor with sensitivity from 995 nm to 1613 nm was built to evaluate deeper skin layers thanks to the higher penetration of photons at these wavelengths. The outcomes of this device were combined with those of a previously developed multispectral system that works in the visible and near infrared range (414 nm⁻995 nm). Both provide spectral and spatial information from skin lesions. A classification method to discriminate between melanomas and nevi was developed based on the analysis of first-order statistics descriptors, principal component analysis, and support vector machine tools. The system provided a sensitivity of 78.6% and a specificity of 84.6%, the latter one being improved with respect to that offered by silicon sensors.

Validation of a gonio-hyperspectral imaging system based on light-emitting diodes for the spectral and colorimetric analysis of automotive coatings.

In this study, a novel gonio-hyperspectral imaging system based on light-emitting diodes for the analysis of automotive coatings was validated colorimetrically and spectrally from 368 to 1309 nm. A total of 30 pearlescent, 30 metallic, and 30 solid real automotive coatings were evaluated with this system, the BYK-mac and X-Rite MA98 gonio-spectrophotometers, and also with the SPECTRO 320 spectrometer for further comparison. The results showed very precise correlations, especially in the visible range. In conclusion, this new system provides a deeper assessment of goniochromatic pigments than current approaches due to the expansion of the spectral range to the infrared.