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Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Cellulose-based fabrics are widely used in the preservation and storage of historic tapestries. Their ease of flammability is a serious concern that greatly limits their applications and requires the development of effective and safe flame-retardant treatments. In this work, we analysed linen and cotton textile samples before and after COEX® treatment, a patented green technology imparting anti-flame properties by functionalizing the cellulose molecules with phosphorus and sulphur groups. Some of the samples were also exposed to photo-induced ageing after the treatment. The resulting structural and chemical changes in both fibres were characterized by nonlinear optical imaging modalities, namely Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence microscopies (TPEF), and Raman and Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) spectroscopies. Complementary results evidenced a reduction in microfibril crystallinity, attributed mainly to the reduction of hydrogen bonding among cellulose macromolecules, with a concomitant increase in fluorescence possibly due to the introduction of ester groups into cellulose chains and to decomposition of lignin into fluorescent by-products.

Objective: To prove the feasibility of Multimodal Fiber Optic Spectroscopy (MFOS) analysis in bladder cancer (BCa) detection, grading, and staging. Materials and methods: Bladder specimens from patients underwent TURBT or TURP were recorded and analyzed with MFOS within 30 min from excision. In detail, our MFOS combined fluorescence, Raman spectroscopy, and diffuse reflectance. We used these optical techniques to collect spectra from bladder biopsies, then we compared the obtained results to gold standard pathological analysis. Finally, we developed a classification algorithm based on principal component analysis-linear discriminant analysis. Results: A total of 169 specimens were collected and analyzed from 114 patients, 40 (23.7%) healthy (from TURP), and 129 (76.3%) with BCa. BCa specimens were divided according to their grade—34 (26.4%) low grade (LG) and 95 (73.6%) high grade (HG) BCa—and stage—64 (49.6%) Ta, 45 (34.9%) T1, and 20 (15.5%) T2. MFOS-based classification algorithm correctly discriminated healthy versus BCa with 90% accuracy, HG versus LG with 83% accuracy. Furthermore, it assessed tumor stage with 75% accuracy for Ta versus T1, 85% for T1 versus T2, and 86% for Ta versus T2. Conclusions: Our preliminary results suggest that MFOS could be a reliable, fast, and label-free tool for BCa assessment, providing also grading and staging information. This technique could be applied in future for in vivo inspection as well as of ex vivo tissue biopsies. Thus, MFOS might improve urothelial cancer management. Further studies are required.

Identification of neoplastic and dysplastic brain tissues is of paramount importance for improving the outcomes of neurosurgical procedures. This study explores the combined application of fluorescence, Raman and diffuse reflectance spectroscopies for the detection and classification of brain tumor and cortical dysplasia with a label-free modality. Multivariate analysis was performed to evaluate classification accuracies of these techniques–employed both in individual and multimodal configuration–obtaining high sensitivity and specificity. In particular, the proposed multimodal approach allowed discriminating tumor/dysplastic tissues against control tissue with 91%/86% sensitivity and 100%/100% specificity, respectively, whereas tumor from dysplastic tissues were discriminated with 89% sensitivity and 86% specificity. Hence, multimodal optical spectroscopy allows reliably differentiating these pathologies using a non-invasive, label-free approach that is faster than the gold standard technique and does not require any tissue processing, offering the potential for the clinical translation of the technology.

Colorectal cancer presents one of the most elevated incidences of cancer worldwide. Colonoscopy relies on histopathology analysis of hematoxylin-eosin (H&E) images of the removed tissue. Novel techniques such as multi-photon microscopy (MPM) show promising results for performing real-time optical biopsies. However, clinicians are not used to this imaging modality and correlation between MPM and H&E information is not clear. The objective of this paper is to describe and make publicly available an extensive dataset of fully co-registered H&E and MPM images that allows the research community to analyze the relationship between MPM and H&E histopathological images and the effect of the semantic gap that prevents clinicians from correctly diagnosing MPM images. The dataset provides a fully scanned tissue images at 10x optical resolution (0.5 µm/px) from 50 samples of lesions obtained by colonoscopies and colectomies. Diagnostics capabilities of TPF and H&E images were compared. Additionally, TPF tiles were virtually stained into H&E images by means of a deep-learning model. A panel of 5 expert pathologists evaluated the different modalities into three classes (healthy, adenoma/hyperplastic, and adenocarcinoma). Results showed that the performance of the pathologists over MPM images was 65% of the H&E performance while the virtual staining method achieved 90%. MPM imaging can provide appropriate information for diagnosing colorectal cancer without the need for H&E staining. However, the existing semantic gap among modalities needs to be corrected. This work was supported by the PICCOLO project. This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 732111. The sole responsibility of this publication lies with the authors. The European Union is not responsible for any use that may be made of the information contained therein. This research has also been supported by the project ONKOTOOLS (KK2020/00069) funded by the Basque Government Industry Department under the ELKARTEK program.

Recent climate projections predict a more rapid increase of winter temperature than summer and global temperature averages in temperate and cold environments. As there is relatively little experimental knowledge on the effect of winter warming on cambium phenology and stem growth in species growing in cold environments, the setting of manipulative experiments is considered of primary importance, and they can help to decipher the effect of reduced winter chilling and increased forcing temperatures on cambium reactivation, growth and xylem traits. In this study, localized stem heating was applied to investigate the effect of warming from the rest to the growth phase on cambium phenology, intra-annual stem growth dynamics and ring wood features in Picea abies (L.) H.Karst. We hypothesized that reduced winter chilling induces a postponed cambium dormancy release and decrease of stem growth, while high temperature during cell wall lignification determines an enrichment of latewood-like cells. The heating device was designed to maintain a +5 °C temperature delta with respect to air temperature, thus allowing an authentic scenario of warming. Continuous stem heating from the rest (November) to the growing phase determined, at the beginning of radial growth, a reduction of the number of cell layers in the cambium, higher number of cell layers in the wall thickening phase and an asynchronous stem radial growth when comparing heated and ambient saplings. Nevertheless, heating did not induce changes in the number of produced cell layers at the end of the growing season. The analyses of two-photon fluorescence images showed that woody rings formed during heating were enriched with latewood-like cells. Our results showed that an increase of 5 °C of temperature applied to the stem from the rest to growth might not influence, as generally reported, onset of cambial activity, but it could affect xylem morphology of Norway spruce in mountain environments.

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 mu g/mL bacterial collagenase at 37 degrees C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm(-1). Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Significance: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. With a worldwide incidence rate of 2 to 3 per 100,000 people, it accounts for more than 60% of all brain cancers; currently, its 5-year survival rate is

We report the development of a novel, to the best of our knowledge, fiber-based system to realize coregistered simultaneous acquisition of fluorescence lifetime (FL) data and Raman spectra from the same area. FL measurements by means of time-correlated single photon counting are realized with periodic out-of-phase external illumination of the field of view, enabling acquisition of data under bright illumination of the specimen. Raman measurements in the near-infrared are realized asynchronously. We present a detailed characterization of this technique and validate its potential to report intrinsic contrast. Fiber-based FL and Raman maps report complementary structural, compositional, and molecular contrast in biological tissues with diverse compositional features.

Urothelial carcinoma (UC) is the most common type of bladder cancer, and its treatment depends from both tumour invasiveness (stage) and aggressiveness (grade). The gold standard for detecting UC is white-light cystoscopy, followed by tissue biopsy and histopathological examination; however, such process is invasive, time-consuming, operatordependent and prone to sampling errors. In this framework, optical spectroscopy techniques could be a promising solution for fast and label-free diagnosis of bladder tissues and for early detection of UC. Thus, we combined autofluorescence, diffuse reflectance and Raman spectroscopy in a compact and transportable setup based on an optical fibrebundle probe. This experimental setup was used for studying fresh biopsies of urothelial tumour (140 samples) and healthy bladder (50 samples) collected from 90 patients undergoing Transurethral Resection of Bladder Tumours (TURBT). The aim of this study was to develop an automated classification of the examined tissues based on the intrinsic spectral information provided by all three techniques. We found that healthy and diseased tissues showed significant spectral differences for each technique, resulting in high accuracy (up to 90%) from a Linear Discriminant Analysis (LDA) routine. In particular, fluorescence spectroscopy – excited either with blue or UV light – provided very good results in detecting UC. However, tumour grading and staging proved to be more challenging tasks, for which no single spectroscopic technique could provide sufficient sensitivity and specificity. Therefore, we found that a multimodal approach can improve significantly the diagnosis of UC stages and grades.

Liquid biopsies represent a minimally invasive tool for the precocious diagnosis of widespread diseases as well as for routinely patients monitoring by tracking selective biomarkers. Optical detection techniques based on surface enhanced Raman spectroscopy (SERS) are capable of providing information on the molecular content of analyzed samples thus representing one of the most promising analytical method in clinical research, as alternative to traditional bioassays. With the attempt to realize point-of-impact diagnostic devices, in the present study 3D printing and soft-lithography processes were combined with plasmonic nanoparticles (NPs) synthesis for the development of multifunctional lab-onchips (LOCs) integrating SERS sensors for liquid probing. As a matter of fact, LOCs enable to easily handle small volumes of samples as well as to perform multifunctional analyses. This is crucial for pathologies whose diagnosis relies on the ratio of more than one biomarker. To this end, being based on a 3D printing process, the overall design of the devices was rapidly prototyped to integrate channels and detection chambers aligned with optical fibers and portable Raman probes for signal delivering and collection. SERS functionality was achieved by immobilization of gold NPs whose chemistry was modified to enhance NPs deposition and stability. Finally, we are exploring direct laser writing for the integration of mechanical and optical microcomponents needed for liquids control and signal delivering and collection, respectively. The final devices collecting multiple functions and detection configurations will provide high sensitivity, speed of analysis, low sample and reagent consumption, measurement automation and standardization on a highly integrated dynamic platform that will revolutionize liquid biopsy making it costless, on-chip, handy and easy to use.

Tissue morpho-mechanics is gaining an increasing relevance in various fields, including biology, medicine, pathology, tissue engineering, and regenerative medicine, since it targets the relationship between morphological features and mechanical properties in biological tissues, which plays an important role in various biological processes including metastasis, wound healing and tissue regeneration. In particular, in every biological tissue, morphological, biochemical and mechanical properties are tightly connected and they influence each other in a correlative manner. For this reason, a correlative approach employing multiple techniques is ideal for targeting tissue morpho-mechanics with an optical approach. Here we report a correlative study performed by optical microscopies, disclosing the supramolecular collagen morphology correlated with its biomechanical and biochemical analyses. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy, demonstrating that the peculiar mechanical functionality of so-called sutural lamellae originates from their distinctive supramolecular organization. A theoretical model based on the ultrastructural symmetry of corneal lamellar domains provides the interpretation of the experimental data at the molecular scale. The proposed methodology opens the way to the non-invasive assessment of tissue morpho-mechanics and holds the potential to be applicable to a broad range of biological and synthetic materials.

The inability to efficiently separate autofluorescence signals from bright background illumination is a key issue that hinders widespread deployment of optical techniques such as time-correlated single photon counting (TCSPC) in clinical settings. We introduce a novel approach to TCSPC measurements that synchronizes the fluorescence acquisition with an externally modulated light source at 50 Hz, thus guaranteeing background-free fluorescence detection and “continuous” eye-perceived specimen illumination. A color camera synchronized with fluorescence detection provides spatial resolution to the single-point measurements, to generate autofluorescence lifetime maps in real-time. We believe this method provides a new research pathway for clinical translation of optical techniques.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumour in adults, and the survival rate of patients affected by this disease is strongly dependent on the successful resection of the tumour. In this framework, multimodal optical spectroscopy could provide a fast and label-free tool for improving tumour detection and guiding the surgical removal of diseased tissues. In this study, we used an optical fibre-probe system combining multiple spectroscopic techniques for in vivo examination of normal and GBM tissues in mouse brain. Spectroscopic measurements based on fluorescence, Raman, and diffuse reflectance spectroscopy were performed on anesthetized animals through two optical windows implanted on the head of each mouse. Then, the recorded data were analysed using Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) for obtaining an automated classification of the examined tissues based on the intrinsic spectral information provided by Raman and reflectance spectroscopy. These techniques provided 77% and 97% classification accuracy, respectively, by taking advantage of the intrinsic molecular content of the examined tissues. In particular, the high sensitivity and specificity achieved by means of reflectance spectroscopy indicate that such technique is the most suited for in vivo detection of GBM. The presented results demonstrate the potential of our method for improving the diagnosis of suspicious brain areas during surgery through a very fast spectroscopic inspection, thus helping the surgeon in removing all tumour tissues and reducing the probability of GBM recurrence.

Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is passed through a transmission grating and both spectral and temporal information are encoded in the SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial information is provided by an external camera together with a guiding beam that provides a visual reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence lifetime maps are augmented on white light images to provide feedback of the measurements in real-time. We validate and demonstrate the practicality of this technique in the reference fluorophores and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.

Tissue morpho-mechanics is gaining increasing in various fields, because it targets the relationship between morphological features and mechanical properties in biological tissues, which plays an important role in various fields including biology, medicine, pathology, tissue engineering, and regenerative medicine. The intimate connection between morphological, biochemical and mechanical properties in biological tissues requires a multimodal correlative approach for their exhaustive investigation. In this study, we used Second-Harmonic Generation in combination with Brillouin and Raman micro-spectroscopy in order to correlate collagen morphology at the ultrastructural level with its biomechanical and biochemical features. In particular, by imaging human corneal tissue samples with our multimodal approach, we demonstrated that the peculiar mechanical properties of corneal lamellae in the anterior portion of the corneal stroma are due to a different supramolecular organization, rather than to a different biochemical composition. This result opens new insights in the study and interpretation of corneal biomechanics thanks to the possibility of non-invasively correlating lamellar morphology with visco-elastic properties. The proposed method opens the way to the non-invasive assessment of corneal morpho-mechanics and holds the potential to be used for both diagnostic and follow-up purposes in pathologies that affect corneal biomechanics.

Tissue morpho-mechanics is gaining an increasing relevance in various fields, including biology, medicine, pathology, tissue engineering, and regenerative medicine, since it targets the relationship between morphological features and mechanical properties in biological tissues, which plays an important role in various biological processes including metastasis, wound healing and tissue regeneration. In particular, in every biological tissue, morphological, biochemical and mechanical properties are tightly connected and they influence each other in a correlative manner. For this reason, a correlative approach employing multiple techniques is ideal for targeting tissue morpho-mechanics with an optical approach. Here we report a correlative study performed by optical microscopies, disclosing the supramolecular collagen morphology correlated with its biomechanical and biochemical analyses. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy, demonstrating that the peculiar mechanical functionality of so-called sutural lamellae originates from their distinctive supramolecular organization. A theoretical model based on the ultrastructural symmetry of corneal lamellar domains provides the interpretation of the experimental data at the molecular scale. The proposed methodology opens the way to the non-invasive assessment of tissue morpho-mechanics and holds the potential to be applicable to a broad range of biological and synthetic materials.

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra-molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization-resolved second-harmonic generation (P-SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.

Bladder cancer (BC) is the eleventh most diagnosed cancer worldwide. The age-standardized incidence rate (per 100 000 person/years) is 9.0 for men and 2.2 for women [1]. Urothelial carcinoma (UC) represents about 90% of all bladder tumors, thus carrying an enormous social and economic burden [2]. UCs are classified in different stages and grades, depending on their invasiveness and on their degree of cytological abnormalities. The key aspect for a positive prognosis is the early and accurate diagnosis of the lesion stage, in order to identify the most aggressive disease forms and treat them promptly. It is well known that tissue metabolism constitutes a basic mechanism, which is at the base of many pathologies, especially BC. Being able to detect and characterize tissue metabolism and molecular fingerprints at the cellular level could be a key aspect in characterizing the pathology and enabling both early detection and therapy monitoring. The new European Union Horizon 2020 project called AMPLITUDE, the “Advanced Multimodal Photonics Laser Imaging Tool for Urothelial Diagnosis in Endoscopy”, starting in January 2020, proposes the development of an advanced multi-modal imaging tool exploiting new laser technologies in an approach combining confocal and non-linear imaging to fulfil unmet clinical needs in terms of the specificity and accuracy of urothelial cancer diagnosis and therapy monitoring. The project is coordinated by Tampere University (Finland) and carried out in cooperation with leading European research organizations including Aston Institute of Photonic Technologies—AIPT (UK), Consiglio Nazionale delle Ricerche—CNR (Italy), Institute of Photonic Sciences—ICFO (Spain), University of Milan-Bicocca, Modus Research and Innovation Ltd. (UK) and University of Florence (Italy), as well as industrial partners: Ampliconyx Oy (Finland), Femtonics Ltd. (Hungary), HC Photonics (Taiwan), and LEONI Fiber Optics GmbH (Germany).

Optical detection techniques based on surface enhanced Raman spectroscopy (SERS) are a powerful tool for biosensing applications. Meanwhile, due to technological advances, different approaches have been investigated to integrate SERS substrates on the tip of optical fibres for molecular probing in liquids. To further demonstrate the perspectives offered by SERS-on-fiber technology for diagnostic purposes, in this study, novel cap-shaped SERS sensors for reversible coupling with customized multimodal probes were prototyped via low-cost polymer casting of polydimethylsiloxane (PDMS) and further assembly of gold nanoparticles (Au NPs) of varied sizes and shapes. To demonstrate the feasibility of liquid sensing with cap sensors using backside illumination and detection, the spectra of rhodamine were acquired by coupling the caps with the fiber. As expected by UV-vis, the highest SERS efficiency was observed for NP-decorated substrates with plasmonic properties in resonance with the irradiation wavelength. Then, SERS biosensors for the specific detection of amyloid-β (Aβ) neurotoxic biomarkers were realized by covalent grafting of Aβ antibodies. As attested by fluorescence images and SERS measurements, the biosensors successfully exhibited enhanced Aβ affinity compared to the bare sensors without ligands. Finally, these versatile (bio)sensors are a powerful tool to transform any milli-sized fibers into functional (bio)sensing platforms with plasmonic and biochemical properties tailored for specific applications.

Lettera su risultati preliminari interazione luce blu-ferite superficiali. Studio in vivo in modello animale.

© 2020 by the authors., The non-invasive depth-resolved imaging of pictorial layers in paintings by means of linear optical techniques represents a challenge in the field of Cultural Heritage (CH). The presence of opaque and/or highly-scattering materials may obstruct the penetration of the radiation probe, thus impeding the visualization of the stratigraphy of paintings. Nonlinear Optical Microscopy (NLOM), which makes use of tightly-focused femtosecond pulsed lasers as illumination sources, is an emerging technique for the analysis of painted objects enabling micrometric three-dimensional (3D) resolution with good penetration capability in semi-transparent materials. In this work, we evaluated the potential of NLOM, specifically in the modality of Multi-Photon Excitation Fluorescence (MPEF), to probe the stratigraphy of egg-tempera mock-up paintings. A multi-analytical non-invasive approach, involving ultraviolet-visible-near infrared (UV-Vis-NIR) Fiber Optics Reflectance Spectroscopy, Vis-NIR photoluminescence, and Laser Induced Fluorescence, yielded key-information for the characterization of the constituting materials and for the interpretation of the nonlinear results. Furthermore, the use of three nonlinear optical systems allowed evaluation of the response of the analyzed paints to different excitation wavelengths and photon doses, which proved useful for the definition of the most suitable measurement conditions. The micrometric thickness of the paint layers, which was not measurable by means of Optical Coherence Tomography (OCT), was instead assessed by MPEF, thus demonstrating the effectiveness of this nonlinear modality in probing highly-scattering media, while ensuring the minimal photochemical disturbance to the examined materials., This research was funded by the European H2020 IPERION CH Project (Integrated Platform for the European Research Infrastructure ON Cultural Heritage (GA 654028, WP6); Spanish State Research Agency (AEI) and European Regional Development (FEDER) through Project CTQ2016-75880-P-AEI/FEDER, UE; and TOP Heritage-CM (S2018/NMT-4372) from the Community of Madrid and with the support of Plataforma Temática Interdisciplinar of CSIC “Patrimonio Abierto: Investigación y Sociedad” (PTI-PAIS).

9 pags., 6 figs., 2 tabs., Nonlinear optical microscopies (NLOM) are emerging techniques for the monitoring and preservation of cultural heritage, providing morphological, structural and compositional information on a large variety of artistic materials and objects. Being non-invasiveness the main requirement in artwork diagnostics, it becomes increasingly necessary to verify the absence of physicochemical alterations possibly induced by the use of high intensity radiation sources, and thus to find the exposure limits for safe applications. In this work, we propose a new methodology to set the criteria of damage evaluation for near infrared femtosecond pulsed lasers used for examining painted samples by means multi-photon excited fluorescence (MPEF), modality of NLOM. The method is based on the detection of changes in the MPEF signal upon repetitive irradiation on a series of modern acrylic painted samples, complemented by Raman spectroscopy measurements performed before and after irradiation. The obtained results allowed determining a safe average laser power range for the non-invasive determination of thickness in paint layers., The research leading to these results was funded by the EU Community's H2020 Research Infrastructure program under theIPERION CH Project (GA 654028), by the Spanish State Research Agency (AEI) and European Regional Development (FEDER) through Project CTQ2016-75880-P-AEI/FEDER,UE,by program TOP Heritage-CM (S2018/NMT-4372) from the Community of Madrid and by Plataforma Tem�tica Interdisciplinar of CSIC ?Patrimonio Abierto:Investigaci�n y Sociedad?(PTI-PAIS).MC is grateful for a Salvador deMadariaga mobility grant (PRX18/00029

Glioblastoma (GBM) is the most common and aggressive malignant brain tumour in adults. Patient survival rates are strongly dependent on the successfully resection of the tumour. In this framework, multimodal optical spectroscopy could provide a fast and label-free tool for improving tumour detection and guiding the removal of diseased tissue. In this study, we used an optical fibre-probe system combining multiple spectroscopic techniques for in vivo examination of normal and GBM tissues in mouse brain. Specifically, the probe – based on a fibre-bundle with optical fibres of various size and properties – allowed performing spectroscopic measurements based on fluorescence, Raman, and diffuse reflectance spectroscopy though two optical windows implanted on the head of each animal. Two visible laser diodes were used for fluorescence spectroscopy, a laser diode emitting in the NIR was used for Raman spectroscopy, and a fibre-coupled halogen lamp for diffuse reflectance. All spectral recordings were done when the animals were anesthetized; optical inspection required less than 4 minutes for each animal. The recorded data were analysed using Principal Component Analysis (PCA) for obtaining an automated classification of the examined tissues based on the intrinsic spectral information provided by Raman and reflectance spectroscopy. The presented method demonstrated high sensitivity and specificity in discriminating GBM from normal brain. Furthermore, we found that the multimodal approach is crucial for improving diagnostic capabilities beyond what can be achieved from individual techniques.

Optical spectroscopy is a fast, label-free and non-invasive method for analysing tissue composition and, thus, has the potential for improving standard diagnostic capabilities. This is particularly relevant for brain surgery due to the lack of contrast between diseased tissues (e.g. malformations and tumours) and the surrounding brain. In this study, we used an optical fibre-probe system combining multiple spectroscopic techniques for analysing ex vivo human brain biopsies taken from both tumour and dysplastic tissues. Specifically, the probe – based on a fibre-bundle with optical fibres of various size and properties – allowed performing spectroscopic measurements based on fluorescence, Raman, and diffuse reflectance spectroscopy. Two visible laser diodes were used for fluorescence spectroscopy, a laser diode emitting in the NIR was used for Raman spectroscopy, and a fibre-coupled halogen lamp for diffuse reflectance. All spectral recordings were done within 30 minutes from surgical resection, and optical inspection required less than 2 minutes for each sample. The recorded data were analysed using Principal Component Analysis (PCA) for obtaining an automated classification of the examined samples based on the intrinsic spectral information provided by all three techniques. The presented method demonstrated high sensitivity and specificity in discriminating different tissue types in good agreement with histopathological examination. Furthermore, we found that the multimodal approach is crucial for improving diagnostic capabilities beyond what can be achieved from individual techniques.

The most common type of bladder cancer is urothelial carcinoma (UC), whose treatment depends from both tumour extension (stage) and aggressiveness (grade). The gold standard for detecting UC is white-light cystoscopy, followed by tissue biopsy and pathological examination for determining tumour stage and grade. However, such process is invasive, time-consuming and prone to sampling errors. In this framework, optical spectroscopy techniques provide fast, label-free and non-invasive alternatives to standard histopathology. Thus, we combined auto-fluorescence, diffuse reflectance and Raman spectroscopy in a compact and transportable setup based on an optical fibre-probe. The latter was coupled to three laser diodes (emitting at 378 nm, 445 nm and 785 nm) and to a halogen lamp for exciting and collecting auto-fluorescence, Raman and reflectance spectra, respectively. This experimental setup was used for studying fresh biopsies of urothelial tumour (103 samples) and healthy bladder (34 samples) collected from 63 patients undergoing Transurethral Resection of Bladder Tumours (TURBT). All spectral recordings were done within 30 minutes from surgical resection, and optical inspection required less than 2 minutes for each sample. The recorded data were analysed using Principal Component Analysis (PCA) for obtaining an automated classification of the examined samples based on the intrinsic spectral information provided by all three techniques. We found that multimodal spectroscopy provides high-sensitivity, high-specificity discriminating capability for UC detection, grading and staging. The presented strategy generates results similar to gold standard histology, but in a fast and label-free way, offering the potential for endoscopic in vivo applications.

In every biological tissue, morphological and topological properties strongly affect its mechanical features and behaviour, so that ultrastructure, composition and mechanical parameters are intimately connected. Overall, it is their correct interplay that guarantees the tissue functionality. The development of experimental methods able to correlate these properties would open new opportunities both in the biological and the biomedical fields. Here, we report a correlative study intended to map supramolecular morphology, biochemical composition and viscoelastic parameters of collagen by all-optical microscopies. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy. The study highlights how subtle variations in supramolecular organization originate the peculiar mechanical behavior of different subtypes of corneal lamellae. The presented methodology paves the way to the non-invasive assessment of tissue morpho-mechanics in biological as well as synthetic materials., Raffaella Mercatelli et al. present a method incorporating Brillouin and Raman microscopy with Second-Harmonic-Generation microscopy. They examine human corneas, showing an unprecedented combination of viscoelastic, biochemical, and morphological properties of collagen.

Optical detection techniques based on surface enhanced Raman spectroscopy (SERS) and capable of providing relevant information on molecular and protein composition of biological samples, are gaining rising attention in clinical research as alternatives to traditional detection assays. Meanwhile, due to the technological advances in compact instrumentation as well as in nanofabrication processes, SERS probes based on portable guided-wave systems, have been implemented thus providing for easier accessibility into complex environments and enabling for real-time in situ detection of low concentrated target analytes. In the present study, low-cost fabrication processes were successfully combined with surface functionalization strategies for the fabrication of disposable SERS-active substrates, engineered to tightly fit the distal end of portable Raman instruments. Being based on a polymer casting fabrication process, the overall design of the substrates can be easily adapted to the varied geometry of the probes to be fit, thus guaranteeing high design versatility. SERS-functionality was achieved by immobilization of gold nanoparticles whose size and shape directly affect the plasmonic properties of the substrates. Moreover, SERS substrates can be further modified by covalently binding molecules acting as baits to selectively fish target biomarkers within heterogeneous samples thereby increasing the specificity of SERS signals. Finally, these sensors represent a powerful tool potentially implementable for the early diagnosis of widespread pathologies by real-time SERS analysis of liquid biopsy.

Schematic draw of the experimental setup used for fiber-probe spectroscopy. We evaluated the diagnostic capability of a multimodal spectroscopic approach for classifying normal brain tissue and epileptogenic focal cortical dysplasia in children. We employed fluorescence spectroscopy at two excitation wavelengths (378 nm and 445 nm) and Raman spectroscopy (at 785 nm excitation) for acquiring fluorescence and Raman spectra from 10 normal brains, 16 focal cortical dysplasia specimens and 1 cortical tuber tissue sites using a custom-built multimodal optical point spectroscopic system. We used principal component analysis combined with leave-one-sample-out-cross-validation for tissue classification. The study resulted in 100% sensitivity and 90% specificity using the information obtained from fluorescence at two distinct wavelengths and Raman spectroscopy for discriminating normal brain tissue and focal cortical dysplasia. Our results demonstrate that this methodology has the potential to be applied clinically for the detection of focal cortical dysplasia and can help to improve as precise as possible surgical resection of the dysplastic tissue during surgery for epilepsy.

In the current clinical scenario, surgical resection of tumors remains the front-line treatment modality for the management of abnormalities in both brain and central nervous system. The primary goal of surgery is to remove as much tumor as possible sparing the normal tissue. Needless removal of normal brain tissue could lead to detrimental neurological deficits causing cognitive dysfunction and motor impairment. Surgical biopsy and other imaging modalities have the disadvantages of being invasive and time-consuming, and brain shift is a major problem when using preoperative magnetic resonance images. Alternately, in the recent years, optical spectroscopy has emerged as a diagnostic modality for the detection of abnormalities in the brain. The resulting spectral features from tissues can provide information related to the biochemistry, morphology, and physiological conditions of the tissue. This technique can probe tissues in vivo or minimally invasive, and real time. In this chapter, after having covered current diagnostic modalities used in pediatric neuro-surgery, a brief outline on the fundamentals of different spectroscopic techniques is explained in detail. In the following section, a brief description of fiber optic probes and different fiber probe configurations implemented in an optical spectroscopic system is presented. Additionally, details related to different methods for spectral analysis are also discussed. Next, we review the different optical point spectroscopic modalities for the demarcation of abnormalities in the pediatric brain. We finally conclude with a discussion on promises, hopes, and opportunities of this emerging technology in the neuro-oncological application.

Although deep learning pathology diagnostic algorithms are proving comparable results with human experts in a wide variety of tasks, they still require a huge amount of well annotated data for training. Generating such extensive and well labelled datasets is time consuming and is not feasible for certain tasks and so, most of the medical datasets available are scarce in images and therefore, not enough for training. In this work we validate that the use of few shot learning techniques can transfer knowledge from a well defined source domain from Colon tissue into a more generic domain composed by Colon, Lung and Breast tissue by using very few training images. Our results show that our few-shot approach is able to obtain a balanced accuracy (BAC) of 90% with just 60 training images, even for the Lung and Breast tissues that were not present on the training set. This outperforms the finetune transfer learning approach that obtains 73% BAC with 60 images and requires 600 images to get up to 81% BAC. This study has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 732111 (PICCOLO project).