Multiplex immunofluorescence is a novel, high-content imaging technique that allows simultaneous in situ labeling of multiple tissue antigens. This technique is of growing relevance in the study of the tumor microenvironment, and the discovery of biomarkers of disease progression or response to immune-based therapies. Given the number of markers and the potential complexity of the spatial interactions involved, the analysis of these images requires the use of machine learning tools that rely for their training on the availability of large image datasets, extremely laborious to annotate. We present Synplex, a computer simulator of multiplexed immunofluorescence images from user-defined parameters: i. cell phenotypes, defined by the level of expression of markers and morphological parameters; ii. cellular neighborhoods based on the spatial association of cell phenotypes; and iii. interactions between cellular neighborhoods. We validate Synplex by generating synthetic tissues that accurately simulate real cancer cohorts with underlying differences in the composition of their tumor microenvironment and show proof-of-principle examples of how Synplex could be used for data augmentation when training machine learning models, and for the in silico selection of clinically relevant biomarkers. Synplex is publicly available at https://github.com/djimenezsanchez/Synplex.

Understanding the spatial interactions between the elements of the tumor microenvironment -i.e. tumor cells. fibroblasts, immune cells- and how these interactions relate to the diagnosis or prognosis of a tumor is one of the goals of computational pathology. We present NaroNet, a deep learning framework that models the multi-scale tumor microenvironment from multiplex-stained cancer tissue images and provides patient-level interpretable predictions using a seamless end-to-end learning pipeline. Trained only with multiplex-stained tissue images and their corresponding patient-level clinical labels, NaroNet unsupervisedly learns which cell phenotypes, cell neighborhoods, and neighborhood interactions have the highest influence to predict the correct label. To this end, NaroNet incorporates several novel and state-of-the-art deep learning techniques, such as patch-level contrastive learning, multi-level graph embeddings, a novel max-sum pooling operation, or a metric that quantifies the relevance that each microenvironment element has in the individual predictions. We validate NaroNet using synthetic data simulating multiplex-immunostained images where a patient label is artificially associated to the -adjustable- probabilistic incidence of different microenvironment elements. We then apply our model to two sets of images of human cancer tissues: 336 seven-color multiplex-immunostained images from 12 high-grade endometrial cancer patients; and 382 35-plex mass cytometry images from 215 breast cancer patients. In both synthetic and real datasets, NaroNet provides outstanding predictions of relevant clinical information while associating those predictions to the presence of specific microenvironment elements.

MOTIVATION: Recent advances in multiplex immunostaining and multispectral cytometry have opened the door to simultaneously visualizing an unprecedented number of biomarkers both in liquid and solid samples. Properly unmixing fluorescent emissions is a challenging task, which normally requires the characterization of the individual fluorochromes from control samples. As the number of fluorochromes increases, the cost in time and use of reagents becomes prohibitively high. Here, we present a fully unsupervised blind spectral unmixing method for the separation of fluorescent emissions in highly mixed spectral data, without the need for control samples. To this end, we extend an existing method based on non-negative Matrix Factorization, and introduce several critical improvements: initialization based on the theoretical spectra, automated selection of 'sparse' data and use of a re-initialized multilayer optimizer. RESULTS: Our algorithm is exhaustively tested using synthetic data to study its robustness against different levels of colocalization, signal to noise ratio, spectral resolution and the effect of errors in the initialization of the algorithm. Then, we compare the performance of our method to that of traditional spectral unmixing algorithms using novel multispectral flow and image cytometry systems. In all cases, we show that our blind unmixing algorithm performs robust unmixing of highly spatially and spectrally mixed data with an unprecedently low computational cost. In summary, we present the first use of a blind unmixing method in multispectral flow and image cytometry, opening the door to the widespread use of our method to efficiently pre-process multiplex immunostaining samples without the need of experimental controls. AVAILABILITY AND IMPLEMENTATION: https://github.com/djimenezsanchez/Blind_Unmixing_NMF_RI/ contains the source code and all datasets used in this manuscript. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Eye tracking technology in low resolution scenarios is not a completely solved issue to date. The possibility of using eye tracking in a mobile gadget is a challenging objective that would permit to spread this technology to non-explored fields. In this paper, a knowledge based approach is presented to solve gaze estimation in low resolution settings. The understanding of the high resolution paradigm permits to propose alternative models to solve gaze estimation. In this manner, three models are presented: a geometrical model, an interpolation model and a compound model, as solutions for gaze estimation for remote low resolution systems. Since this work considers head position essential to improve gaze accuracy, a method for head pose estimation is also proposed. The methods are validated in an optimal framework, I2Head database, which combines head and gaze data. The experimental validation of the models demonstrates their sensitivity to image processing inaccuracies, critical in the case of the geometrical model. Static and extreme movement scenarios are analyzed showing the higher robustness of compound and geometrical models in the presence of user's displacement. Accuracy values of about 3° have been obtained, increasing to values close to 5° in extreme displacement settings, results fully comparable with the state-of-the-art.

Head pose estimation (HPE) is currently a growing research field, mainly because of the proliferation of human–computer interfaces (HCI) in the last decade. It offers a wide variety of applications, including human behavior analysis, driver assistance systems or gaze estimation systems. This article aims to contribute to the development of robust and accurate HPE methods based on 2D tracking of the face, enhancing performance of both 2D point tracking and 3D pose estimation. We start with a baseline method for pose estimation based on POSIT algorithm. A novel weighted variant of POSIT is then proposed, together with a methodology to estimate weights for the 2D–3D point correspondences. Further, outlier detection and correction methods are also proposed in order to enhance both point tracking and pose estimation. With the aim of achieving a wider impact, the problem is addressed using a global approach: all the methods proposed are generalizable to any kind of object for which an approximate 3D model is available. These methods have been evaluated for the specific task of HPE using two different head pose video databases; a recently published one that reflects the expected performance of the system in current technological conditions, and an older one that allows an extensive comparison with state-of-the-art HPE methods. Results show that the proposed enhancements improve the accuracy of both 2D facial point tracking and 3D HPE, with respect to the implemented baseline method, by over 15% in normal tracking conditions and over 30% in noisy tracking conditions. Moreover, the proposed HPE system outperforms the state of the art on the two databases.

We present a dynamic atlas composed of neuromelanin-enhanced magnetic resonance brain images of 40 healthy subjects. The performance of this atlas is evaluated on the fully automated segmentation of two paired neuromelanin-rich brainstem healthy structures: the substantia nigra pars compacta and the locus coeruleus. We show that our dynamic atlas requires in average 60% less images and, therefore, 60% less computation time than a static multi-image atlas while achieving a similar segmentation performance. Then, we show that by applying our dynamic atlas, composed of healthy subjects, to the segmentation and neuromelanin quantification of a set of brain images of 39 Parkinson disease patients, we are able to find significant quantitative differences in the level of neuromelanin between healthy subjects and Parkinson disease patients, thus opening the door to the use of these structures as image biomarkers in future computer aided diagnosis systems for the diagnosis of Parkinson disease.

A multistage procedure to detect eye features is presented. Multiresolution and topographic classification are used to detect the iris center. The eye corner is calculated combining valley detection and eyelid curve extraction. The algorithm is tested in the BioID database and in a proprietary database containing more than 1200 images. The results show that the suggested algorithm is robust and accurate. Regarding the iris center our method obtains the best average behavior for the BioID database compared to other available algorithms. Additional contributions are that our algorithm functions in real time and does not require complex post processing stages.