Dysplasia and tumor discrimination in brain tissues by combined fluorescence, Raman, and diffuse reflectance spectroscopies.

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.

Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques.

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.

Autofluorescence enhancement for label-free imaging of myelinated fibers in mammalian brains.

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.

The feasibility of multimodal fiber optic spectroscopy analysis in bladder cancer detection, grading, and staging.

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.

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques.

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 µg/mL bacterial collagenase at 37 °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.

Supervised learning methods for the recognition of melanoma cell lines through the analysis of their Raman spectra.

Malignant melanoma is an aggressive form of skin cancer, which develops from the genetic mutations of melanocytes - the most frequent involving BRAF and NRAS genes. The choice and the effectiveness of the therapeutic approach depend on tumour mutation; therefore, its assessment is of paramount importance. Current methods for mutation analysis are destructive and take a long time; instead, Raman spectroscopy could provide a fast, label-free and non-destructive alternative. In this study, confocal Raman microscopy has been used for examining three in vitro melanoma cell lines, harbouring different molecular profiles and, in particular, specific BRAF and NRAS driver mutations. The molecular information obtained from Raman spectra has served for developing two alternative classification algorithms based on linear discriminant analysis and artificial neural network. Both methods provide high accuracy (≥90%) in discriminating all cell types, suggesting that Raman spectroscopy may be an effective tool for detecting molecular differences between melanoma mutations.

Endoscopic Full-thickness Resection with the Full-Thickness Resection Device (FTRD) for "difficult to resect" colonic lesions. A single-center experience.

INTRODUCTION: 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). MATERIAL AND METHODS: 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. RESULTS: 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. DISCUSSION: 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. CONCLUSIONS: 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". KEY WORDS: 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.

In vivo detection of murine glioblastoma through Raman and reflectance fiber-probe spectroscopies.

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 < 5 % . GBM treatment relies mainly on surgical resection. In this framework, multimodal optical spectroscopy could provide a fast and label-free tool for improving tumor detection and guiding the removal of diseased tissues. Aim: Discriminating healthy brain from GBM tissues in an animal model through the combination of Raman and reflectance spectroscopies. Approach: EGFP-GL261 cells were injected into the brains of eight laboratory mice for inducing murine GBM in these animals. A multimodal optical fiber probe combining fluorescence, Raman, and reflectance spectroscopy was used to localize in vivo healthy and tumor brain areas and to collect their spectral information. Results: Tumor areas were localized through the detection of EGFP fluorescence emission. Then, Raman and reflectance spectra were collected from healthy and tumor tissues, and later analyzed through principal component analysis and linear discriminant analysis in order to develop a classification algorithm. Raman and reflectance spectra resulted in 92% and 93% classification accuracy, respectively. Combining together these techniques allowed improving the discrimination between healthy and tumor tissues up to 97%. Conclusions: These preliminary results demonstrate the potential of multimodal fiber-probe spectroscopy for in vivo label-free detection and delineation of brain tumors, and thus represent an additional, encouraging step toward clinical translation and deployment of fiber-probe spectroscopy.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Label-free grading and staging of urothelial carcinoma through multimodal fibre-probe spectroscopy.

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.

Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples.

Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications.

Improved label-free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy.

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.

Non-linear imaging and characterization of atherosclerotic arterial tissue using combined SHG and FLIM microscopy.

Atherosclerosis is one of the leading causes of death in the Western World and its characterization is extremely interesting from the diagnostic point of view. Here, we employed combined SHG-FLIM microscopy to characterize arterial tissue with atherosclerosis. The shorter mean fluorescence lifetime measured within plaque depositions (1260 ± 80 ps) with respect to normal arterial wall (1480 ± 100 ps) allowed discriminating collagen from lipids. SHG measurements and image analysis demonstrated that the normal arterial wall has a more anisotropic Aspect Ratio (0.37 ± 0.02) with respect to plaque depositions (0.61 ± 0.02) and that the correlation length can be used for discriminating collagen fibre bundles (2.0 ± 0.6 µm) from cholesterol depositions (4.1 ± 0.6 µm). The presented method has the potential to find place in a clinical setting as well as to be applied in vivo in the near future. Graphic composition of SHG and FLIM images representing normal arterial wall and plaque depositions.

Combined fluorescence-Raman spectroscopic setup for the diagnosis of melanocytic lesions.

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.