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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.

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.

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.

Aim of our observational and retrospective study is to compare efficacy and indications of endoscopic full-thickness resection device (FTRD) with the over-the-scope (OVESCO) clip closure for en bloc resection of colorectal lesions (including adenomas, early carcinomas, inflammatory polyps and neuroendocrine tumors).This article collected 36 cases of colorectal neoplasms from a single Italian referral center per colorectal disease treatment. Primary endpoints included en bloc resection, R0 resection and an early discharge of the patient. Secondary endpoints included procedure-related adverse events.Mean procedure time± standard deviation (SD) was 19.6±22.1 minutes and mean hospital stay (± SD) was 2.2±1.1 days. Overall, an en bloc resection was achieved in 34 cases (94.4%), with an R0 resection rate of 91.6%. Among the three not R0 patients, further additional treatments were needed.Along the same line of other already published articles, the main current indications of EFTR by FTRD-OVESCO are limited to superficial or low-risk malignancy lesions (eg, adenomas, early cancers or subepithelial tumors), not suitable to conventional endoscopic resection or in patients with a severe surgical risk. Both en bloc resection rate and complication rate are aligned with other authors' data.EFTR by FTRD system represents an effective and safe options whenever a recurrent lesion in a challenging environment occurres (eg, recent scar, low rectum or beyond a large colonic bend). Procedure-related adverse events are potentially severe, so that this novel technique should be performed by "expert hands".Difficult polypectomies, Early carcinomas, Endoscopic Full-Thickness Resection (EFTR), Full-Thickness Resection Device (FTRD) by Over-The-Scope (OVESCO) clip closure, Literature overview, Single center experience.La resezione di lesioni sessili del colon-retto, specialmente in siti considerati complessi (per esempio retto inferiore, a livello di austre coliche o su cicatrici di pregresse mucosectomie) costituisce un sfida per ciascun endoscopista. Considerando il constante incremento di incidenza delle neoplasie del colon retto nell’ultimo decennio, nuove metodiche di polipectomia sono state frutto della continua ricerca medica, fra cui, a partire dal 2015, la tecnica di Full-Thickness Resection Device (FTRD) basata sulla clip Over-The-Scope (OVESCO), nata da una brillante idea di Schurr MO 1,2,9,10. Pecularità di questo sistema innovativo è la capacità di realizzare, in un unico tempo (ossia “one-shot”) sia la resezione a tutto spessore della lesione target (garantendo pertanto un’alta percentuale di pazienti R0) sia la chiusura a tutto spessore della parete colica (o rettale) 9,11,15,17. Il kit FTRD consiste della specifica clip OVESCO modificata (al fine di poter afferrare a tutto spessore la parete colica o rettale), di una pinza con la funzione di sollevare la lesione target all’interno del cappuccio e, infine, di un’ansa annessa al sistema (che decorre esternamente lungo il tubo dell’endoscopio, avvolta da una guaina). Una volta che lesione è stata marcata circonferenzialmente e raggiunta con il sistema FTRD, la pinza viene fatta avanzare attraverso il canale operativo per poter afferrare le lesione e, congiuntamente ad una modesta aspirazione dell’endoscopio, includerla all’interno del cappuccio apposito (con dimensioni di 21 mm × 23 mm). Una volta ottenuta una valida duplicazione della parete colica (o rettale) la clip viene rilasciata mediante l’apposito mulinello (analogo a quella della OVESCO classica); immediatamente dopo, l’assistente provvede a chiudere l’ansa, così da poter resecare la lesione mediante un sistema di corrente monopolare (Video 1, Figs. 1, 2) 9,11. La casistica del nostro centro comprende 36 casi di lesioni colorettali, raccolte in 5 anni. La maggior parte delle lesioni sono adenomi rettali recidivi (dopo mucosectomie endoscopiche). I risultati ottenuti, in termini di resezione en bloc e di R0, sono molto incoraggianti, peraltro in perfetta linea e coerenza con quelli dei più importanti lavori europei pubblicati sinora su questo tema. L’analisi statistica dei dati raccolti ha fatto emergere percentuali di resezione en bloc ed R0 del 94.4% e 91.6%, rispettivamente, confermando l’efficacia e sicurezza di questa innovative tecnica per la resezione di lesioni complesse (“challenging”) del colon-retto, specie in pazienti ad alto rischio chirurgico. Anche le percentuali relative alle complicanze post procedurali sono confortanti, in linea, ancora una volta, con le maggiori pubblicazioni. In conclusione, alla luce del confronto fra la nostra casistica e la letteratura attuale, la resezione full-thickness basata sul sistema OVESCO, costituisce un’ottima alternativa alla resezione chirurgica di lesion polipoidi “difficili”, ovviamente se affidata a mani esperte. Dall’analisi della casistica, sottoposta a studio statistico con analisi univariata, non è emerso alcun valore di p con rilevanza statistica: questo dato, che potrebbe presentare varie chiavi di lettura, riteniamo sia fondamentalmente dovuto al numero piuttosto ristretto di casi ed al bassissimo numero di recidive (indice comunque dell’efficacia anche a lungo termine del trattamento con sistema FTRD).

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.

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.

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.

We combined Second Harmonic Generation, Two-Photon Fluorescence and Fluorescence Lifetime Imaging Microscopy for studying human carotid ex vivo tissue sections affected by atherosclerosis, resulting in the discrimination of different arterial regions within the plaques.

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.

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)

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.

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.

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.

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.

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse's hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.