A Multimodal Ensemble Driven by Multiobjective Optimisation to Predict Overall Survival in Non-Small-Cell Lung Cancer.

Lung cancer accounts for more deaths worldwide than any other cancer disease. In order to provide patients with the most effective treatment for these aggressive tumours, multimodal learning is emerging as a new and promising field of research that aims to extract complementary information from the data of different modalities for prognostic and predictive purposes. This knowledge could be used to optimise current treatments and maximise their effectiveness. To predict overall survival, in this work, we investigate the use of multimodal learning on the CLARO dataset, which includes CT images and clinical data collected from a cohort of non-small-cell lung cancer patients. Our method allows the identification of the optimal set of classifiers to be included in the ensemble in a late fusion approach. Specifically, after training unimodal models on each modality, it selects the best ensemble by solving a multiobjective optimisation problem that maximises both the recognition performance and the diversity of the predictions. In the ensemble, the labels of each sample are assigned using the majority voting rule. As further validation, we show that the proposed ensemble outperforms the models learning a single modality, obtaining state-of-the-art results on the task at hand.

CNN-Based Approaches with Different Tumor Bounding Options for Lymph Node Status Prediction in Breast DCE-MRI.

BACKGROUND: The axillary lymph node status (ALNS) is one of the most important prognostic factors in breast cancer (BC) patients, and it is currently evaluated by invasive procedures. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), highlights the physiological and morphological characteristics of primary tumor tissue. Deep learning approaches (DL), such as convolutional neural networks (CNNs), are able to autonomously learn the set of features directly from images for a specific task. MATERIALS AND METHODS: A total of 155 malignant BC lesions evaluated via DCE-MRI were included in the study. For each patient's clinical data, the tumor histological and MRI characteristics and axillary lymph node status (ALNS) were assessed. LNS was considered to be the final label and dichotomized (LN+ (27 patients) vs. LN- (128 patients)). Based on the concept that peritumoral tissue contains valuable information about tumor aggressiveness, in this work, we analyze the contributions of six different tumor bounding options to predict the LNS using a CNN. These bounding boxes include a single fixed-size box (SFB), a single variable-size box (SVB), a single isotropic-size box (SIB), a single lesion variable-size box (SLVB), a single lesion isotropic-size box (SLIB), and a two-dimensional slice (2DS) option. According to the characteristics of the volumes considered as inputs, three different CNNs were investigated: the SFB-NET (for the SFB), the VB-NET (for the SVB, SIB, SLVB, and SLIB), and the 2DS-NET (for the 2DS). All the experiments were run in 10-fold cross-validation. The performance of each CNN was evaluated in terms of accuracy, sensitivity, specificity, the area under the ROC curve (AUC), and Cohen's kappa coefficient (K). RESULTS: The best accuracy and AUC are obtained by the 2DS-NET (78.63% and 77.86%, respectively). The 2DS-NET also showed the highest specificity, whilst the highest sensibility was attained by the VB-NET based on the SVB and SIB as bounding options. CONCLUSION: We have demonstrated that a selective inclusion of the DCE-MRI's peritumoral tissue increases accuracy in the lymph node status prediction in BC patients using CNNs as a DL approach.

Artificial Intelligence in Bone Metastases: An MRI and CT Imaging Review.

(1) Background: The purpose of this review is to study the role of radiomics as a supporting tool in predicting bone disease status, differentiating benign from malignant bone lesions, and characterizing malignant bone lesions. (2) Methods: Two reviewers conducted the literature search independently. Thirteen articles on radiomics as a decision support tool for bone lesions were selected. The quality of the methodology was evaluated according to the radiomics quality score (RQS). (3) Results: All studies were published between 2018 and 2021 and were retrospective in design. Eleven (85%) studies were MRI-based, and two (15%) were CT-based. The sample size was <200 patients for all studies. There is significant heterogeneity in the literature, as evidenced by the relatively low RQS value (average score = 22.6%). There is not a homogeneous protocol used for MRI sequences among the different studies, although the highest predictive ability was always obtained in T2W-FS. Six articles (46%) reported on the potential application of the model in a clinical setting with a decision curve analysis (DCA). (4) Conclusions: Despite the variability in the radiomics method application, the similarity of results and conclusions observed is encouraging. Substantial limits were found; prospective and multicentric studies are needed to affirm the role of radiomics as a supporting tool.

3T-MRI Artificial Intelligence in Patients with Invasive Breast Cancer to Predict Distant Metastasis Status: A Pilot Study.

BACKGROUND: The incidence of breast cancer metastasis has decreased over the years. However, 20-30% of patients with early breast cancer still die from metastases. The purpose of this study is to evaluate the performance of a Deep Learning Convolutional Neural Networks (CNN) model to predict the risk of distant metastasis using 3T-MRI DCE sequences (Dynamic Contrast-Enhanced). METHODS: A total of 157 breast cancer patients who underwent staging 3T-MRI examinations from January 2011 to July 2022 were retrospectively examined. Patient data, tumor histological and MRI characteristics, and clinical and imaging follow-up examinations of up to 7 years were collected. Of the 157 MRI examinations, 39/157 patients (40 lesions) had distant metastases, while 118/157 patients (120 lesions) were negative for distant metastases (control group). We analyzed the role of the Deep Learning technique using a single variable size bounding box (SVB) option and employed a Voxel Based (VB) NET CNN model. The CNN performance was evaluated in terms of accuracy, sensitivity, specificity, and area under the ROC curve (AUC). RESULTS: The VB-NET model obtained a sensitivity, specificity, accuracy, and AUC of 52.50%, 80.51%, 73.42%, and 68.56%, respectively. A significant correlation was found between the risk of distant metastasis and tumor size, and the expression of PgR and HER2. CONCLUSIONS: We demonstrated a currently insufficient ability of the Deep Learning approach in predicting a distant metastasis status in patients with BC using CNNs.

On the Additional Information Provided by 3T-MRI ADC in Predicting Tumor Cellularity and Microscopic Behavior.

BACKGROUND: to evaluate whether Apparent Diffusion Coefficient (ADC) values of invasive breast cancer, provided by 3T Diffusion Weighted-Images (DWI), may represent a non-invasive predictor of pathophysiologic tumor aggressiveness. METHODS: 100 Patients with histologically proven invasive breast cancers who underwent a 3T-MRI examination were included in the study. All MRI examinations included dynamic contrast-enhanced and DWI/ADC sequences. ADC value were calculated for each lesion. Tumor grade was determined according to the Nottingham Grading System, and immuno-histochemical analysis was performed to assess molecular receptors, cellularity rate, on both biopsy and surgical specimens, and proliferation rate (Ki-67 index). Spearman's Rho test was used to correlate ADC values with histological (grading, Ki-67 index and cellularity) and MRI features. ADC values were compared among the different grading (G1, G2, G3), Ki-67 (<20% and >20%) and cellularity groups (<50%, 50-70% and >70%), using Mann-Whitney and Kruskal-Wallis tests. ROC curves were performed to demonstrate the accuracy of the ADC values in predicting the grading, Ki-67 index and cellularity groups. RESULTS: ADC values correlated significantly with grading, ER receptor status, Ki-67 index and cellularity rates. ADC values were significantly higher for G1 compared with G2 and for G1 compared with G3 and for Ki-67 < 20% than Ki-67 > 20%. The Kruskal-Wallis test showed that ADC values were significantly different among the three grading groups, the three biopsy cellularity groups and the three surgical cellularity groups. The best ROC curves were obtained for the G3 group (AUC of 0.720), for G2 + G3 (AUC of 0.835), for Ki-67 > 20% (AUC of 0.679) and for surgical cellularity rate > 70% (AUC of 0.805). CONCLUSIONS: 3T-DWI ADC is a direct predictor of cellular aggressiveness and proliferation in invasive breast carcinoma, and can be used as a supporting non-invasive factor to characterize macroscopic lesion behavior especially before surgery.

The Impact of Tumor Edema on T2-Weighted 3T-MRI Invasive Breast Cancer Histological Characterization: A Pilot Radiomics Study.

BACKGROUND: to evaluate the contribution of edema associated with histological features to the prediction of breast cancer (BC) prognosis using T2-weighted MRI radiomics. METHODS: 160 patients who underwent staging 3T-MRI from January 2015 to January 2019, with 164 histologically proven invasive BC lesions, were retrospectively reviewed. Patient data (age, menopausal status, family history, hormone therapy), tumor MRI-features (location, margins, enhancement) and histological features (histological type, grading, ER, PgR, HER2, Ki-67 index) were collected. Of the 160 MRI exams, 120 were considered eligible, corresponding to 127 lesions. T2-MRI were used to identify edema, which was classified in four groups: peritumoral, pre-pectoral, subcutaneous, or diffuse. A semi-automatic segmentation of the edema was performed for each lesion, using 3D Slicer open-source software. Main radiomics features were extracted and selected using a wrapper selection method. A Random Forest type classifier was trained to measure the performance of predicting histological factors using semantic features (patient data and MRI features) alone and semantic features associated with edema radiomics features. RESULTS: edema was absent in 37 lesions and present in 127 (62 peritumoral, 26 pre-pectoral, 16 subcutaneous, 23 diffuse). The AUC-classifier obtained by associating edema radiomics with semantic features was always higher compared to the AUC-classifier obtained from semantic features alone, for all five histological classes prediction (0.645 vs. 0.520 for histological type, 0.789 vs. 0.590 for grading, 0.487 vs. 0.466 for ER, 0.659 vs. 0.546 for PgR, and 0.62 vs. 0.573 for Ki67). CONCLUSIONS: radiomic features extracted from tumor edema contribute significantly to predicting tumor histology, increasing the accuracy obtained from the combination of patient clinical characteristics and breast imaging data.

AIforCOVID: Predicting the clinical outcomes in patients with COVID-19 applying AI to chest-X-rays. An Italian multicentre study.

Recent epidemiological data report that worldwide more than 53 million people have been infected by SARS-CoV-2, resulting in 1.3 million deaths. The disease has been spreading very rapidly and few months after the identification of the first infected, shortage of hospital resources quickly became a problem. In this work we investigate whether artificial intelligence working with chest X-ray (CXR) scans and clinical data can be used as a possible tool for the early identification of patients at risk of severe outcome, like intensive care or death. Indeed, further to induce lower radiation dose than computed tomography (CT), CXR is a simpler and faster radiological technique, being also more widespread. In this respect, we present three approaches that use features extracted from CXR images, either handcrafted or automatically learnt by convolutional neuronal networks, which are then integrated with the clinical data. As a further contribution, this work introduces a repository that collects data from 820 patients enrolled in six Italian hospitals in spring 2020 during the first COVID-19 emergency. The dataset includes CXR images, several clinical attributes and clinical outcomes. Exhaustive evaluation shows promising performance both in 10-fold and leave-one-centre-out cross-validation, suggesting that clinical data and images have the potential to provide useful information for the management of patients and hospital resources.

Deep Reinforcement Learning for Fractionated Radiotherapy in Non-Small Cell Lung Carcinoma.

Lung cancer is by far the leading cause of cancer death among both men and women. Radiation therapy is one of the main approaches to lung cancer treatment, and its planning is crucial for the therapy outcome. However, the current practice that uniformly delivers the dose does not take into account the patient-specific tumour features that may affect treatment success. Since radiation therapy is by its very nature a sequential procedure, Deep Reinforcement Learning (DRL) is a well-suited methodology to overcome this limitation. In this respect, in this work we present a DRL controller optimizing the daily dose fraction delivered to the patient on the basis of CT scans collected over time during the therapy, offering a personalized treatment not only for volume adaptation, as currently intended, but also for daily fractionation. Furthermore, this contribution introduces a virtual radiotherapy environment based on a set of ordinary differential equations modelling the tissue radiosensitivity by combining both the effect of the radiotherapy treatment and cell growth. Their parameters are estimated from CT scans routinely collected using the Particle Swarm Optimization algorithm. This permits the DRL to learn the optimal behaviour through an iterative trial and error process with the environment. We performed several experiments considering three rewards functions modelling treatment strategies with different tissue aggressiveness and two exploration strategies for the exploration-exploitation dilemma. The results show that our DRL approach can adapt to radiation therapy treatment, optimizing its behaviour according to the different reward functions and outperforming the current clinical practice.

3T MRI-Radiomic Approach to Predict for Lymph Node Status in Breast Cancer Patients.

BACKGROUND: axillary lymph node (LN) status is one of the main breast cancer prognostic factors and it is currently defined by invasive procedures. The aim of this study is to predict LN metastasis combining MRI radiomics features with primary breast tumor histological features and patients' clinical data. METHODS: 99 lesions on pre-treatment contrasted 3T-MRI (DCE). All patients had a histologically proven invasive breast cancer and defined LN status. Patients' clinical data and tumor histological analysis were previously collected. For each tumor lesion, a semi-automatic segmentation was performed, using the second phase of DCE-MRI. Each segmentation was optimized using a convex-hull algorithm. In addition to the 14 semantics features and a feature ROI volume/convex-hull volume, 242 other quantitative features were extracted. A wrapper selection method selected the 15 most prognostic features (14 quantitative, 1 semantic), used to train the final learning model. The classifier used was the Random Forest. RESULTS: the AUC-classifier was 0.856 (label = positive or negative). The contribution of each feature group was lower performance than the full signature. CONCLUSIONS: the combination of patient clinical, histological and radiomics features of primary breast cancer can accurately predict LN status in a non-invasive way.

Visual4DTracker: a tool to interact with 3D + t image stacks.

BACKGROUND: Biological phenomena usually evolves over time and recent advances in high-throughput microscopy have made possible to collect multiple 3D images over time, generating [Formula: see text] (or 4D) datasets. To extract useful information there is the need to extract spatial and temporal data on the particles that are in the images, but particle tracking and feature extraction need some kind of assistance. RESULTS: This manuscript introduces our new freely downloadable toolbox, the Visual4DTracker. It is a MATLAB package implementing several useful functionalities to navigate, analyse and proof-read the track of each particle detected in any [Formula: see text] stack. Furthermore, it allows users to proof-read and to evaluate the traces with respect to a given gold standard. The Visual4DTracker toolbox permits the users to visualize and save all the generated results through a user-friendly graphical user interface. This tool has been successfully used in three applicative examples. The first processes synthetic data to show all the software functionalities. The second shows how to process a 4D image stack showing the time-lapse growth of Drosophila cells in an embryo. The third example presents the quantitative analysis of insulin granules in living beta-cells, showing that such particles have two main dynamics that coexist inside the cells. CONCLUSIONS: Visual4DTracker is a software package for MATLAB to visualize, handle and manually track [Formula: see text] stacks of microscopy images containing objects such cells, granules, etc.. With its unique set of functions, it remarkably permits the user to analyze and proof-read 4D data in a friendly 3D fashion. The tool is freely available at https://drive.google.com/drive/folders/19AEn0TqP-2B8Z10kOavEAopTUxsKUV73?usp=sharing.

Positive tissue transglutaminase antibodies with negative endomysial antibodies: Unresolved issues in diagnosing celiac disease.

BACKGROUND: The serological screening for celiac disease (CD) is currently based on the detection of anti-transglutaminase (tTG) IgA antibodies, subsequently confirmed by positive endomysial antibodies (EMA). When an anti-tTG IgA positive/EMA IgA negative result occurs, it can be due either to the lower sensitivity of the EMA test or to the lower specificity of the anti-tTG test. This study aimed at verifying how variation in analytical specificity among different anti-tTG methods could account for this discrepancy. METHODS: A total of 130 consecutive anti-tTG IgA positive/EMA negative samples were collected from the local screening routine and tested using five anti-tTG IgA commercial assays: two chemiluminescence methods, one fluoroimmunoenzymatic method, one immunoenzymatic method and one multiplex flow immunoassay method. RESULTS: Twenty three/130 (17.7%) patients were diagnosed with CD. In the other 107 cases a diagnosis of CD was not confirmed. The overall agreement among the five anti-tTG methods ranged from 28.5% to 77.7%. CD condition was more likely linked to the positivity of more than one anti-tTG IgA assay (monopositive = 2.5%, positive with ≥ three methods = 29.5%; p = 0.0004), but it was not related to anti-tTG IgA antibody levels (either positive or borderline; p = 0.5). CONCLUSIONS: Patients with positive anti-tTG/negative EMA have a low probability of being affected by CD. Given the high variability among methods to measure anti-tTG IgA antibodies, anti-tTG-positive/EMA-negative result must be considered with extreme caution. It is advisable that the laboratory report comments on any discordant results, suggesting to consider the data in the proper clinical context and to refer the patient to a CD reference center for prolonged follow up.

Exploiting Multi-Level Parallelism for Stitching Very Large Microscopy Images.

Due to the limited field of view of the microscopes, acquisitions of macroscopic specimens require many parallel image stacks to cover the whole volume of interest. Overlapping regions are introduced among stacks in order to make it possible automatic alignment by means of a 3D stitching tool. Since state-of-the-art microscopes coupled with chemical clearing procedures can generate 3D images whose size exceeds the Terabyte, parallelization is required to keep stitching time within acceptable limits. In the present paper we discuss how multi-level parallelization reduces the execution times of TeraStitcher, a tool designed to deal with very large images. Two algorithms performing dataset partition for efficient parallelization in a transparent way are presented together with experimental results proving the effectiveness of the approach that achieves a speedup close to 300×, when both coarse- and fine-grained parallelism are exploited. Multi-level parallelization of TeraStitcher led to a significant reduction of processing times with no changes in the user interface, and with no additional effort required for the maintenance of code.

A radiomic approach for adaptive radiotherapy in non-small cell lung cancer patients.

The primary goal of precision medicine is to minimize side effects and optimize efficacy of treatments. Recent advances in medical imaging technology allow the use of more advanced image analysis methods beyond simple measurements of tumor size or radiotracer uptake metrics. The extraction of quantitative features from medical images to characterize tumor pathology or heterogeneity is an interesting process to investigate, in order to provide information that may be useful to guide the therapies and predict survival. This paper discusses the rationale supporting the concept of radiomics and the feasibility of its application to Non-Small Cell Lung Cancer in the field of radiation oncology research. We studied 91 stage III patients treated with concurrent chemoradiation and adaptive approach in case of tumor reduction during treatment. We considered 12 statistics features and 230 textural features extracted from the CT images. In our study, we used an ensemble learning method to classify patients' data into either the adaptive or non-adaptive group during chemoradiation on the basis of the starting CT simulation. Our data supports the hypothesis that a specific signature can be identified (AUC 0.82). In our experience, a radiomic signature mixing semantic and image-based features has shown promising results for personalized adaptive radiotherapy in non-small cell lung cancer.

Phase separation of the plasma membrane in human red blood cells as a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus.

Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins can alter lipid density, packing and interactions, and are considered an important factor that affects fluidity variation in membranes. Red blood cells (RBC) membrane physical state, showing pronounced alterations in Type 1 diabetes mellitus (T1DM), could be the ideal candidate for monitoring the disease progression and the effects of therapies. On these grounds, the measurement of RBC membrane fluidity alterations can furnish a more sensitive index in T1DM diagnosis and disease progression than Glycosylated hemoglobin (HbA1c), which reflects only the information related to glycosylation processes. Here, through a functional two-photon microscopy approach we retrieved fluidity maps at submicrometric scale in RBC of T1DM patients with and without complications, detecting an altered membrane equilibrium. We found that a phase separation between fluid and rigid domains occurs, triggered by systemic effects on membranes fluidity of glycation and oxidation. The phase separation patterns are different among healthy, T1DM and T1DM with complications patients. Blood cholesterol and LDL content are positively correlated with the extent of the phase separation patterns. To quantify this extent a machine learning approach is employed to develop a Decision-Support-System (DSS) able to recognize different fluidity patterns in RBC. Preliminary analysis shows significant differences(p<0.001) among healthy, T1DM and T1DM with complications patients. The development of an assay based on Phase separation of the plasma membrane of the Red Blood cells is a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus, and could allow customization and the selection of medical treatments in T1DM in clinical settings, and enable the early detection of complications.

Age-related changes of cortical excitability and connectivity in healthy humans: non-invasive evaluation of sensorimotor network by means of TMS-EEG.

The sensorimotor cortical system undergoes structural and functional changes across its lifespan. Some of these changes are physiological and parallel the normal aging process, while others might represent pathophysiological mechanisms underlying neurodegenerative disorders. In the last years, the study of possible age-related modifications in brain sensorimotor functional characteristics has been the focus of several research projects. Here we have used the transcranial magnetic stimulation (TMS)-electroencephalography (EEG) navigated co-registration to investigate the influence of physiological aging on the excitability and connectivity of the human sensorimotor cortical system. To this end, we compared the TMS-evoked EEG potentials (TEPs) collected after stimulating the dominant primary motor cortex (M1) in healthy young subjects (mean age 24.5years) with those collected in healthy older adults (mean age 67.6years). We have shown that, after stimulation of the left motor cortex, TEPs are significantly affected by physiological aging. This phenomenon has a clear spatio-temporal specificity and we speculate that normal aging per se leads to some changes in the excitability of specific cortical neural assemblies whereas other alterations could reflect compensatory mechanisms to such changes.

Age related differences in functional synchronization of EEG activity as evaluated by means of TMS-EEG coregistrations.

It was recently demonstrated that the characteristics of EEG rhythms preceding a transcranial magnetic stimulation (TMS) of the motor cortex (M1) influence the motor-evoked potential (MEP) amplitude with a peculiar pattern, thus reflecting the M1 functional state. As physiological aging is related to a decrease in motor performance and changes in excitability and connectivity strength within cerebral sensorimotor circuits, we aimed to explore whether aging affects EEG-MEP interactions. Using MRI-navigated TMS and multichannel EEG, we compared the EEG-MEP interactions observed in healthy aged subjects with those observed in young volunteers. We divided the MEPs amplitude into two different subgroups consisting of "high" and "low" MEPs, based on the 50th percentile of their amplitude distribution. Then we analysed the characteristics of the pre-stimulus EEG from M1 and correlated areas separately for the "high" and "low" MEPs, comparing the two conditions. In both young and old subjects, significantly larger MEPs were evoked when the stimulated M1 was coupled in the beta-2 band with the homolateral prefrontal cortex. Conversely, only in young participants was the MEP size modulated when the M1 and homolateral parieto-occipital cortices were coupled in the delta band. The elderly didn't show this kind of pattern. Importantly, this coupling was significantly higher in elderly brains than in young brains, both for high and low MEPs. Our results suggest an age-related significant influence of time-varying coupling of spatially patterned EEG rhythms on motor cortex excitability in response to TMS.

Automated Neuron Tracing Methods: An Updated Account.

The reconstruction of neuron morphology allows to investigate how the brain works, which is one of the foremost challenges in neuroscience. This process aims at extracting the neuronal structures from microscopic imaging data. The great advances in microscopic technologies have made a huge amount of data available at the micro-, or even lower, resolution where manual inspection is time consuming, prone to error and utterly impractical. This has motivated the development of methods to automatically trace the neuronal structures, a task also known as neuron tracing. This paper surveys the latest neuron tracing methods available in the scientific literature as well as a selection of significant older papers to better place these proposals into context. They are categorized into global processing, local processing and meta-algorithm approaches. Furthermore, we point out the algorithmic components used to design each method and we report information on the datasets and the performance metrics used.

Sensorimotor cortex excitability and connectivity in Alzheimer's disease: A TMS-EEG Co-registration study.

Several studies have shown that, in spite of the fact that motor symptoms manifest late in the course of Alzheimer's disease (AD), neuropathological progression in the motor cortex parallels that in other brain areas generally considered more specific targets of the neurodegenerative process. It has been suggested that motor cortex excitability is enhanced in AD from the early stages, and that this is related to disease's severity and progression. To investigate the neurophysiological hallmarks of motor cortex functionality in early AD we combined transcranial magnetic stimulation (TMS) with electroencephalography (EEG). We demonstrated that in mild AD the sensorimotor system is hyperexcitable, despite the lack of clinically evident motor manifestations. This phenomenon causes a stronger response to stimulation in a specific time window, possibly due to locally acting reinforcing circuits, while network activity and connectivity is reduced. These changes could be interpreted as a compensatory mechanism allowing for the preservation of sensorimotor programming and execution over a long period of time, regardless of the disease's progression. Hum Brain Mapp 37:2083-2096, 2016. © 2016 Wiley Periodicals, Inc.

Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging.

In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated.