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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.

Modern photonic technologies are emerging, allowing the acquisition of in-vivo endoscopic tissue imaging at a microscopic scale, with characteristics comparable to traditional histological slides, and with a label-free modality. This raises the possibility of an 'optical biopsy' to aid clinical decision making. This approach faces barriers for being incorporated into clinical practice, including the lack of existing images for training, unfamiliarity of clinicians with the novel image domains and the uncertainty of trusting 'black-box' machine learned image analysis, where the decision making remains inscrutable. In this paper, we propose a new method to transform images from novel photonics techniques (e.g. autofluorescence microscopy) into already established domains such as Hematoxilyn-Eosin (H-E) microscopy through virtual reconstruction and staining. We introduce three main innovations: 1) we propose a transformation method based on a Siamese structure that simultaneously learns the direct and inverse transformation ensuring domain back-transformation quality of the transformed data. 2) We also introduced an embedding loss term that ensures similarity not only at pixel level, but also at the image embedding description level. This drastically reduces the perception distortion trade-off problem existing in common domain transfer based on generative adversarial networks. These virtually stained images can serve as reference standard images for comparison with the already known H-E images. 3) We also incorporate an uncertainty margin concept that allows the network to measure its own confidence, and demonstrate that these reconstructed and virtually stained images can be used on previously-studied classification models of H-E images that have been computationally degraded and de-stained. The three proposed methods can be seamlessly incorporated on any existing architectures. We obtained balanced accuracies of 0.95 and negative predictive values of 1.00 over the reconstructed and virtually stained image-set on the detection of color-rectal tumoral tissue. This is of great importance as we reduce the need for extensive labeled datasets for training, which are normally not available on the early studies of a new imaging technology This work was supported in part by the European Union's Horizon 2020 Research and Innovation Programme under Grant 732111 (PICCOLO project), and in part by the Basque Government's Industry Department through the ELKARTEK Program's Project 3KIA under Grant KK-2020/00049. The work of Andoni Elola was supported by his pre-doctoral research from the Basque Government under Grant PRE_2019_2_0100 and Grant IT1229-19

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

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.

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.

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.

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).

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).

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.

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.

Background: Colorectal cancer has a high incidence rate worldwide, with over 1.8 million new cases and 880,792 deaths in 2018. Fortunately, its early detection significantly increases the survival rate, reaching a cure rate of 90% when diagnosed at a localized stage. Colonoscopy is the gold standard technique for detection and removal of colorectal lesions with potential to evolve into cancer. When polyps are found in a patient, the current procedure is their complete removal. However, in this process, gastroenterologists cannot assure complete resection and clean margins which are given by the histopathology analysis of the removed tissue, which is performed at laboratory. Aims: In this paper, we demonstrate the capabilities of multiphoton microscopy (MPM) technology to provide imaging biomarkers that can be extracted by deep learning techniques to identify malignant neoplastic colon lesions and distinguish them from healthy, hyperplastic, or benign neoplastic tissue, without the need for histopathological staining. Materials and Methods: To this end, we present a novel MPM public dataset containing 14,712 images obtained from 42 patients and grouped into 2 classes. A convolutional neural network is trained on this dataset and a spatially coherent predictions scheme is applied for performance improvement. Results: We obtained a sensitivity of 0.8228 ± 0.1575 and a specificity of 0.9114 ± 0.0814 on detecting malignant neoplastic lesions. We also validated this approach to estimate the self-confidence of the network on its own predictions, obtaining a mean sensitivity of 0.8697 and a mean specificity of 0.9524 with the 18.67% of the images classified as uncertain. Conclusions: This work lays the foundations for performing in vivo optical colon biopsies by combining this novel imaging technology together with deep learning algorithms, hence avoiding unnecessary polyp resection and allowing in situ diagnosis assessment. This work was supported by the PICCOLO project. This project has received funding from the European Union's Horizon2020 Research and Innovation Programme under grant agreement No. 732111. This research has also been supported by the project ONKOTOOLS (KK-2020/00069) funded by the Basque Government Industry Department under the ELKARTEK program

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.

Urothelial carcinoma (UC) is the most common bladder tumour. Proper treatment requires tumour resection for diagnosing its grade (aggressiveness) and stage (invasiveness). White-light cystoscopy and histopathological examination are the gold standard procedures for clinical and histopathological diagnostics, respectively. However, cystoscopy is limited in terms of specificity, histology requires long tissue processing, both procedures rely on operator's experience. Multimodal optical spectroscopy can provide a powerful tool for detecting, staging and grading bladder tumours in a fast, reliable and label-free modality. In this study, we collected fluorescence, Raman and reflectance spectra from 50 biopsies obtained from 32 patients undergoing transurethral resection of bladder tumour using a multimodal fibre-probe. Principal component analysis allowed distinguishing normal from pathological tissues, as well as discriminating tumour stages and grades. Each individual spectroscopic technique provided high specificity and sensitivity in classifying all tissues; however, a multimodal approach resulted in a considerable increase in diagnostic accuracy (>= 95%), which is of paramount importance for tumour grading and staging. The presented method offers the potential for being applied in cystoscopy and for providing an automated diagnosis of UC at the clinical level, with an improvement with respect to current state-of-the-art procedures.

Urothelial carcinoma (UC) is the most common type of bladder cancer. The gold standard for detecting UC is white-light cystoscopy, which is followed by tissue biopsy and pathological examination. However, such process is invasive, timeconsuming and prone to sampling errors. In this framework, optical spectroscopy techniques provide fast, label-free and non-invasive alternatives to standard histopathology. Thus, the aim of this study is to discriminate normal bladder tissues from urothelial tumours, and to identify the different stages of the disease, by means of combined auto-fluorescence, diffuse reflectance and Raman spectroscopy. In fact, these techniques were implemented in a compact and transportable setup based on two optical fibre probes: one coupled to fluorescence and reflectance excitation sources, while the other one to the 785 nm laser. Raman, fluorescence and reflected light signals were collected though the same probe used for excitation and sent to a spectrograph. We used this experimental setup for studying fresh biopsies of urothelial tumour and healthy bladder collected from 32 patients undergoing Transurethral Resection of Bladder Tumours (TURBT). Scoring methods based on ratiometric approach and Principal Component Analysis (PCA) allowed not only to discriminate healthy biopsies from tumour ones, but also to recognize three tumour stages.

Coronary heart disease is the most common type of heart disease caused by atherosclerosis. In fact, an arterial wall lesion centered on the accumulation of cholesterol-rich lipids and the accompanying inflammatory response generates a plaque, whose rupture may result in a thrombus with fatal consequences. Plaque characterization for assessing the severity of atherosclerosis is generally performed through standard histopathological examination based on hematoxylin/eosin staining, which is operator-dependent and requires relatively long procedures. In this framework, nonlinear optical microscopy is a valid, label-free alternative to standard diagnostic methods. We combined second-harmonic generation (SHG), two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy in a multimodal scheme for obtaining morphological and molecular information on human carotid ex vivo specimens affected by atherosclerosis. In this study, discrimination between different tissues within the atherosclerotic plaque was achieved based on both lifetime, TPEF-to-SHG ratio, and image pattern analysis. The presented methodology aims to be a starting point for future fully automated and fast characterization of atherosclerotic biopsies; moreover, it could be extended to the study of other tissues and pathologies. Combined TPEF/SHG mapping of a carotid specimen affected by atherosclerosis.

Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view, especially for what is concerning collagen content and organization because collagen plays a crucial role in plaque vulnerability. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Non-linear microscopy techniques offer the potential for providing morpho-functional information on the examined tissues in a label-free way. In this study, we employed combined SHG and FLIM microscopy for characterizing collagen organization in both normal arterial wall and within atherosclerotic plaques. Image pattern analysis of SHG images allowed characterizing collagen organization in different tissue regions. In addition, the analysis of collagen fluorescence decay contributed to the characterization of the samples on the basis of collagen fluorescence lifetime. Different values of collagen fiber mean size, collagen distribution, collagen anisotropy and collagen fluorescence lifetime were found in normal arterial wall and within plaque depositions, prospectively allowing for automated classification of atherosclerotic lesions and plaque vulnerability. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. Sutural lamellae are located in the first 30 ?m of the stromal layer below Bowman's membrane; they affect the mechanical properties of the cornea and are responsible of the overall corneal stiffness. This stromal layer is particularly interesting for diagnosing and following-up keratoconus, an eye disorder that features a reduced stiffness of the cornea and its consequent pathological deformation. The methodology was tested by acquiring image stacks of the central portion of the cornea in ex vivo samples of healthy corneas, keratoconic corneas and keratoconic corneas treated with cross-linking (CXL), a treatment aimed at hindering the progression of keratoconus. The samples were imaged and characterized based on forward/backward SHG ratio, finding a different ratio in keratoconus with respect to both the healthy cornea and cross-linked keratoconus. The inclination of corneal sutural lamellae was then characterized by means of a three-dimensional correlation analysis on SHG images, acquired using a backward detection geometry. Such method provided good discrimination capabilities, demonstrating that this approach can be used not only for diagnosing keratoconus in a very early stage, but also for performing treatment follow-up on cross-linked corneas.

We combined Second Harmonic Generation and Two-Photon Fluorescence for imaging ex vivo tissue sections of human bladder affected by urothelial carcinoma. We studied different grades of the tumor, and compared them to healthy bladder mucosa.

Every optical imaging technique is limited in its penetration depth by scattering occurring in biological tissues. Possible solutions to overcome this problem consist of limiting the detrimental effects of scattering by reducing optical inhomogeneities within the sample. This can be achieved either by using physical methods (such as refractive index matching solutions) or by chemical methods (such as the removal of scatterers), based on tissue transformation protocols. This review provides an overview of the current state-of-the-art methods used for both ex-vivo and in-vivo optical clearing of biological tissues. We start with a brief history of the development of the most widespread clearing methods across the new millennium, then we describe the working principles of both physical and chemical methods. Clearing methods are then reviewed, pointing the attention of the reader on both physical and chemical methods, classified based on the tissue size and type for each specific application. A small section is reserved for methods that have already found in-vivo applications at the research level. Finally, a detailed discussion highlighting both the most relevant results achieved and the new ongoing developments in this field is reported in the last part, together with future perspectives for the clearing methodology. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Non-linear optical microscopy is becoming popular as a non-invasive in vivo imaging modality in dermatology. In this study, combined TPF and SHG microscopy were used to monitor collagen remodelling in vivo after micro-ablative fractional laser resurfacing. Papillary dermis of living subjects, covering a wide age range, was imaged immediately before and forty days after treatment. A qualitative visual examination of acquired images demonstrated an age-dependent remodelling effect on collagen. Additional quantitative analysis of new collagen production was performed by means of two image analysis methods. A higher increase in SHG to TPF ratio, corresponding to a stronger treatment effectiveness, was found in older subjects, whereas the effect was found to be negligible in young, and minimal in middle age subjects. Analysis of collagen images also showed a dependence of the treatment effectiveness with age but with controversial results. While the diagnostic potential of in vivo multiphoton microscopy has already been demonstrated for skin cancer and other skin diseases, here we first successfully explore its potential use for a non-invasive follow-up of a laser-based treatment.

Atherosclerosis is characterized by the accumulation of lipids within the arterial wall and is commonly diagnosed using standard histology. Non-linear microscopy represents a possible label-free alternative to standard diagnostic methods for imaging various tissue components. Here we employ SHG and CARS microscopy for imaging thin cross-sections of atherosclerotic arterial tissue, demonstrating that both cholesterol deposition in the lumen and collagen in the normal arterial wall can be imaged and discriminated using SHG and CARS microscopy. A simultaneous detection of both forward and backward scattered SHG signals allows distinguishing collagen fibres from cholesterol. Further analysis, based on image pattern evaluation algorithms, is used to characterize collagen organization in the healthy arterial wall against collagen found within plaques. Different values of fibre mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue.

Two-photon spectral resolved imaging was used to image fresh human biopsies of colon tissue and to characterize healthy colon mucosa, adenomatous polyp and adenocarcinoma by means of a morpho-functional analysis. Morphological examination, performed using endogenous tissue fluorescence, discriminated adenomatous and adenocarcinoma tissues from normal mucosa in terms of cellular asymmetry and nucleus-to-cytoplasm ratio. Good agreement was found between multiphoton images and histological examination performed on the same samples. Further characterization, performed by means of spectral-resolved analysis of NADH and FAD fluorescence, demonstrated an altered metabolic activity in both adenomatous and adenocarcinoma tissues compared to healthy mucosa. This morpho-functional approach may represent a powerful method to be used in combination with endoscopy for in vivo optical diagnosis of colon cancer and may be extended to other tissues. (C) 2013 Optical Society of America

Focal cortical dysplasia (FCD) is an abnormality in the cerebral cortex that is caused by malformations during cortical development. Currently, magnetic resonance imaging (MRI) and electro-corticography (ECoG) are used for detecting FCD. On the downside, MRI is very much insensitive to small malformations in the brain, while ECoG is an invasive and time consuming procedure. Recently, optical techniques were widely exploited as a minimally invasive and quantitative approaches for disease diagnosis. These techniques include fluorescence and Raman spectroscopy. The aim of this investigation is to study the diagnostic performances of optical spectroscopy incorporating fluorescence (at 378 nm and 445 nm excitation wavelengths) and Raman spectroscopy (at 785 nm excitation) for the discrimination of FCD from normal brain in pediatric subjects. The study included 10 normal and 17 FCD tissue sites from 3 normal and 7 FCD samples. The emission spectra of FCD at 378 nm excitation wavelength presented a blue-shifted peak with respect to normal tissue. Prominent spectral differences between normal and FCD tissue were observed at 1298 cm -1 , 1302 cm -1 , 1445 cm -1 and 1660 cm -1 using Raman spectroscopy. Tissue classification models were developed using a multivariate statistical method, principal component analysis. This study demonstrates that a combined spectroscopic approach can provide a better diagnostic capability for classifying normal and FCD tissues. Further, the implementation of the technology within a fiber probe could open the way for in vivo diagnostics and intra-operative surgical guidance.

A faster healing process was observed in superficial skin wounds after irradiation with a blue LED (EmoLED) photocoagulator. EmoLED is a compact handheld device, used to induce a thermal effect and thus coagulation in superficial abrasions. We present the results of an in vivo study, conducted in a mouse model, to analyze the induced wound healing. Two superficial abrasions were produced on the back of the mice: one area was treated with EmoLED (1.4 W/cm2, 30 s treatment time), while the other one was left naturally recovering. During the treatment, a temperature around 40-45°C was induced on the abrasion surface. Mice back healthy skin was used as a control. The animals underwent a follow up study and were sacrificed at 0, 1, 3, 6, 9, 12, 18, 21, 24 hours p.o. and 6 days p.o.. Samples from the two abraded areas were harvested and examined by histopathological and immunofluorescence analysis, SHG imaging and confocal microscopy. The aim of the study was to investigate the inflammatory infiltrate, mastocyte population, macrophage subpopulation, fibroblasts and myofibroblasts. Our results show that soon after the treatment, both the inflammatory infiltrate and the M1 macrophage subpopulation appear earlier in the treated, compared to a delayed appearance in the untreated samples. There was no alteration in collagen morphology in the recovered wound. This study confirms the preliminary results obtained in a previous study on a rat model: the selective photothermal effect we used for inducing immediate coagulation in superficial wounds seems to be associated to a faster and improved healing process.

A faster healing process was observed in superficial skin wounds after irradiation with a blue LED (EmoLED) photocoagulator. EmoLED is a compact handheld device, used to induce a thermal effect and thus coagulation in superficial abrasions. We present the results of an in vivo study, conducted in different mouse model, to analyze the induced wound healing. Two superficial abrasions were produced on the back of the mice: one area was treated with EmoLED (1.4 W/cm2, 30 s treatment time), while the other one was left naturally recovering. During the treatment, a temperature around 40-45°C was induced on the abrasion surface. Mice back healthy skin was used as a control. We compared the treatment in black mice, healthy albino mice, diabetic albino mice and albino mice with coagulation problem. The animals underwent a follow up study and were sacrificed at 0, 3, 6, 9, 18, 24 hours p.o.. Samples from the two abraded areas were harvested and examined by histopathological and immunofluorescence analysis, SHG imaging and confocal microscopy. The aim of the study was to compare the effects in the different target groups and to investigate the early phase of the wound healing process. Our results show that the effects are comparable in all the treated groups and that the healing process appears to be faster in respect to the naturally recovered wounds. This study confirms the previous results obtained in a study on a rat model an in a study on healthy albino mice: the selective photothermal effect we used for inducing immediate coagulation in superficial wounds seems to be associated to a faster and improved healing process.

Keratoconus is an eye disorder that features a reduced stiffness of the cornea and its consequent pathological deformation. Cross-Linking (CXL) treatment has proven useful in hindering the progression of keratoconus, offering a minimally-invasive alternative to corneal surgical transplantation. In this study, the biomechanical characteristics of a human keratoconic cornea were clinically examined in vivo soon before keratoplasty, and the morphological alterations of the collagen scaffold in the same cornea were examined ex vivo by means of Second-Harmonic Generation (SHG) microscopy. A healthy cornea and a CXL-treated keratoconus were compared. In particular, the lamellar organization in the three corneal samples was characterized in different stromal layers by detecting both forward- and backwardscattered SHG signal and then considering the forward/backward (F/B) ratio as parameter. The F/B ratio was used to characterize the morphological organization of collagen lamellae within different stromal layers, finding an increased disorder at the level of Bowman's membrane, opposed to a more regular organization within deeper stromal layers in all the examined samples. The organization of collagen lamellae in CXL-treated keratoconic samples was similar to that one found in healthy corneas, demonstrating that the CXL is able to rearrange the collagen scaffold and partially recover the properties of a healthy condition. The obtained results are in agreement with previous results obtained in studies aimed at monitoring the organization of fibrillar collagen using F/B SHG ratio. In conclusion, the proposed method might be useful for both diagnosing keratoconus as well as for monitoring the effects of the CXL treatment.

SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. In particular, the attention was focused on the organization and orientation of corneal collagen lamellae in the first 30 mu m of the stromal layer below Bowman's membrane. In fact, it is thought that the irregular orientation of these specific lamellae, an in particular the existence of so-called sutural lamellae, affects the mechanical properties of the cornea and is responsible of the overall corneal stiffness. The methodology was tested by acquiring image stacks of the central portion of the cornea in ex vivo samples of healthy corneas, keratoconic corneas and keratoconic corneas treated with cross-linking. These samples represent a good benchmark for testing the methodology, considering that keratoconus is an ophthalmic disease in which the cornea loses its stiffness and acquires an abnormal conical shape because of a different organization of sutural lamellae. The samples were first imaged and characterized on the basis of forward/backward SHG ratio, finding a different ratio in keratoconus with respect to both the healthy cornea and cross-linked keratoconus. The inclination of corneal sutural lamellae was then characterized by means of a three-dimensional correlation analysis on SHG images, acquired using a backward detection geometry. Such method provided good discrimination capabilities, demonstrating that this approach can be used not only for diagnosing keratoconus in a very early stage, but also for performing treatment follow-up on cross-linked corneas.

Two optical fibre-based probes for spectroscopic measurements on human tissues were designed and developed. The two probes combine fluorescence and Raman spectroscopy in a multimodal approach. The fluorescence excitation was provided by two laser diodes emitting in the UV (378 nm) and in the visible (445 nm) range, while a third source in the NIR (785 nm) was used for Raman. The device was tested on freshly excised human skin biopsies clinically diagnosed as malignant melanoma, melanocytic nevus, or healthy skin. Discrimination of lesions based on their fluorescence and Raman spectra showed good correlation with the subsequent histological examination. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

SHG image acquired with sagittal optical sectioning (A) of a healthy cornea and (B) of a keratoconic cornea. Scale bars: 30 μm. Keratoconus is an eye disorder that causes the cornea to take an abnormal conical shape, thus impairing its refractive functions and causing blindness. The late diagnosis of keratoconus is among the principal reasons for corneal surgical transplantation. This pathology is characterized by a reduced corneal stiffness in the region immediately below Bowman's membrane, probably due to a different lamellar organization, as suggested by previous studies. Here, the lamellar organization in this corneal region is characterized in three dimensions by means of second-harmonic generation (SHG) microscopy. In particular, a method based on a three-dimensional correlation analysis allows to probe the orientation of sutural lamellae close to the Bowman's membrane, finding statistical differences between healthy and keratoconic samples. This method is demonstrated also in combination with an epi-detection scheme, paving the way for a potential clinical ophthalmic application of SHG microscopy for the early diagnosis of keratoconus.

Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. The rupture of an atherosclerotic plaque, resulting in a thrombus, is one of the leading causes of death in the Western World. Preventive assessment of plaque vulnerability is therefore extremely important and can be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Instead, a label-free and non-invasive alternative is provided by nonlinear microscopy. In this study, we combined SHG and FLIM microscopy in order to characterize collagen organization and lipids in human carotid ex vivo tissues affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were processed through image pattern analysis methods (FFT, GLCM). The resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterization of carotid samples and discrimination of different tissue regions. The presented method can be applied for automated classification of atherosclerotic lesions and plaque vulnerability. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in clinical setting.

Focal cortical dysplasia (FCD) is one of most common causes of intractable epilepsy in pediatric population and these are often insensitive to anti-epileptic drugs. FCD is characterized by a disarray in localized regions of the cerebral cortex and abnormal neurons which results them to misfire with incorrect signals. Resective neurosurgery to remove or disconnect the affected parts from the rest of the brain seems to be a viable option to treat FCD. Before neurosurgery the subject could undergo imaging studies including magnetic resonance imaging (MRI) or computed tomography (CT) scans. On the downside FCD could be elusive in MRI images and may be practically invisible in CT scans. Furthermore, unnecessary removal of normal tissues is to be taken into consideration as this could lead to neurological defects. In this context, optical spectroscopy have been widely investigated as an alternative technique for the detection of abnormal tissues in different organ sites. Disease progression is accompanied by a number of architectural, biochemical and morphological changes. These variations are reflected in the spectral intensity and line shape. Here, in this proof of concept study we propose to investigate the application of tissue optical spectroscopy based on fluorescence excitation at two wavelength 378 and 445 nm coupled along with Raman spectroscopy for the detection of FCD on formalin fixed tissue specimens from pediatric subjects. For fluorescence at both the excitation wavelengths FCD showed a decreased intensity at longer wavelength when compared to normal tissues. Also, differences exist in the Raman spectral profiles of normal and FCD.

Keratoconus is an ophthalmic disease in which the cornea acquires an abnormal conical shape that prevents the correct focusing on the retina, causing visual impairment. The late diagnosis of keratoconus is among the principal causes of corneal transplantation surgery. In this study, we characterize the morphology of keratoconic corneas by means of the correlation of SHG images, finding that keratoconus can be diagnosed by analyzing the inclination of lamellae below Bowman’s membrane. In addition, imaging performed with both sagittal and “en face” geometry demonstrated that this morphological features can be highlighted both ex vivo and in vivo.

Nonlinear optical microscopy has the potential of being used in vivo as a noninvasive high-resolution imaging modality for skin examination at both epidermal and dermal levels. In particular, two-photon fluorescence microscopy can be used to detect morphological and functional changes in the epidermis via the high signal generated by the reduced form of mitochondrial nicotinamide adenine dinucleotide (NADH) [49]. This microscopic modality allows differentiation of epidermal layers with different structural and cytological features. Further, the combination of two-photon fluorescence with second harmonic generation microscopy can be successfully employed for dermal imaging. In particular, the two mentioned imaging techniques are able to selectively image elastic fibers and collagen, hence providing an exhaustive characterization of the skin dermis. In conclusion, nonlinear imaging techniques, especially if used with a multimodal approach, could find a stable place in a clinical dermatological setting for both diagnosis and follow-up purposes.

Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. It is one of the leading causes of death in the Western World, as the rupture of an atherosclerotic plaque may result in a thrombus with fatal consequences. Preventive assessment of plaque vulnerability is therefore extremely important and it could be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Nonlinear microscopy is a labelfree and non-invasive tool for achieving such goal, as an alternative to the histopathological examination. In this study, we used Second Harmonic Generation (SHG) microscopy, Two-Photon Fluorescence (TPF) microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) with a combined approach in order to characterize collagen organization and lipids in human carotid ex vivo tissue sections affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were used to measure the corresponding TPF/SHG ratios. Moreover, such images were processed through image pattern analysis methods such as Fast Fourier Transform (FFT) and Grey-Level Co-occurrence Matrix (GLCM). Finally, the resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterizing carotid samples and discriminating different tissue regions. The presented method offers the potential to be applied for automated classification of atherosclerotic lesions and plaque vulnerability. In addition, the methodology could be extended to the diagnostics of other tissues and pathologies. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in a clinical setting.

Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view, especially for what is concerning collagen content and organization because collagen plays a crucial role in plaque vulnerability. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Non-linear microscopy techniques offer the potential for providing morpho-functional information on the examined tissues in a label-free way. In this study, we employed combined SHG and FLIM microscopy for characterizing collagen organization in both normal arterial wall and within atherosclerotic plaques. Image pattern analysis of SHG images allowed characterizing collagen organization in different tissue regions. In addition, the analysis of collagen fluorescence decay contributed to the characterization of the samples based on collagen fluorescence lifetime. Different values of collagen fiber mean size, collagen distribution, and collagen anisotropy and collagen fluorescence lifetime were found in normal arterial wall and within plaque depositions, prospectively allowing for automated classification of atherosclerotic lesions and plaque vulnerability. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

The healing process of superficial skin wounds treated with a blue-LED haemostatic device is studied. Four mechanical abrasions are produced on the back of 10 Sprague Dawley rats: two are treated with the blue-LED device, while the other two are left to naturally recover. Visual observations, non-linear microscopic imaging, as well as histology and immunofluorescence analyses are performed 8 days after the treatment, demonstrating no adverse reactions neither thermal damages in both abraded areas and surrounding tissue. A faster healing process and a better-recovered skin morphology are observed: the treated wounds show a reduced inflammatory response and a higher collagen content. Blue LED induced photothermal effect on superficial abrasions.

The standard clinical practice for detection of bladder tumours is standard white light cystoscopy. Major concerns related to cystoscopy include its invasive, causes discomfort to the patient and also it misses flat tumors (carcinoma in situ). Recently, considerable progress has been made in the development of optical spectroscopy and imaging based on fluorescence, reflectance and Raman for disease diagnosis. The basic objective behind optical technologies to is skip the time consuming tissue pathology, to provide real time results and moreover to reduce the emotional strain on the patients waiting for the results. In this context, we propose to implement diffuse reflectance spectroscopy for staging and grading urothelial carcinoma by estimating the tissue optical properties such as absorption and reduced scattering co-efficients. Tissue optical properties emulates the underlying physiological and morphological properties of the tissue. In the visible region of spectrum light absorption is dominated by hemoglobin. While light scattering is primarily due to the changes in nuclear size and density as well as from the collagen fibres. Tumour tissues exhibit considerable changes in these properties which could be quantified using reflectance spectroscopic measurements. Our study indicates a changes in the reflectance spectral profile between normal and different grades of urothelial carcinoma. Also, at wavelengths greater than 640 nm (where scattering is dominant) we found an increased intensity when compared to the normal tissues.

A faster healing process was observed in superficial skin wounds after irradiation with the EMOLED photocoagulator. The instrument consists of a compact handheld photocoagulation device, useful for inducing coagulation in superficial abrasions. In this work we present the results of an in vivo study, in a murine model. Two superficial wounds were produced on the back of 12 mice: one area was left untreated, the other one was treated with EMOLED. Healthy skin was used as a control. The animals were sacrificed 3 hours, 12 hours, 1 day, 6 day after treatment. The treatment effects on back skin was monitored by visual observations, histopathological analysis, immuno-histochemical analysis, and non-linear microscopic imaging performed at each follow up time, finding no adverse reactions and no thermal damage in both treated areas and surrounding tissues. In addition, a faster healing process, a reduced inflammatory response, a higher collagen content, and a better-recovered skin morphology was evidenced in the treated tissue with respect to the untreated tissue. These morphological features were characterized by means of immuno-histochemical analysis, aimed at imaging fibroblasts and myofibroblasts, and by SHG microscopy, aimed at characterizing collagen organization, demonstrating a fully recovered aspect of dermis as well as a faster neocollagenesis in the treated regions. This study demonstrates that the selective photothermal effect we used for inducing immediate coagulation in superficial wounds is associated to a minimal inflammatory response, which provides reduced recovery times and improved healing process.

Detection of pre-malignant lesions in skin could help in reducing the 5 year patient mortality rates and greatly advancing the quality of life. Current gold standard for the detection of skin pathologies is a tissue biopsy and followed by a series of steps before it is examined under a light microscope by a pathologist. The disadvantage with this method is its invasiveness. Light based biomedical point spectroscopic techniques offers an adjunct technique to invasive tissue pathology. In this context, we have implemented a simple multiplexed ratiometric approach (F470/F560 and F510/F580) based on fluorescence at two excitation wavelengths 378 nm and 445 nm respectively. The emission profile at these excitation wavelengths showed a shift towards the longer wavelengths for melanoma when compared with normal and nevus. At both excitation wavelengths, we observed an increased intensity ratios for normal, followed by nevus and melanoma. This intensity ratios provide a good diagnostic capability in differentiating normal, nevus and melanocytic skin lesions. This method could be applied in vivo because of the simplicity involved in discriminating normal and pathological skin tissues.