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.
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.
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.
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.
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.
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 immunehistochemical 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.