Alessandro Cosci

Time- and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ.

Combined non-linear imaging techniques were used to deeply image human ex-vivo fresh biopsies of bladder as well as to discriminate between healthy bladder mucosa and carcinoma in situ. Morphological examination by two-photon excited fluorescence and second-harmonic generation has shown a good agreement with corresponding common routine histology performed on the same samples. Tumor cells appeared slightly different in shape and with a smaller cellular-to-nuclear dimension ratio with respect to corresponding normal cells. Further differences between the two tissue types were found in both spectral emission and fluorescence lifetime distribution by performing temporal- and spectral- resolved analysis of fluorescence. This method may represent a promising tool to be used in a multi-photon endoscope, in a confocal endoscope or in a spectroscopic probe for in-vivo optical diagnosis of bladder cancer.

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.

Confocal reflectance microscopy for determination of microbubble resonator thickness

Optical Micro Bubble Resonators (OMBR) are emerging as new type of sensors characterized by high Q-factor and embedded micro-fluidic. Sensitivity is related to cavity field penetration and, therefore, to the resonator thickness. At the state of the art, methods for OMBRs wall thickness evaluation rely only on a theoretical approach. The purpose of this study is to create a non-destructive method for measuring the shell thickness of a microbubble using reflectance confocal microscopy. The method was validated through measurements on etched capillaries with different thickness and finally it was applied on microbubble resonators.

Transparent Oxyfluoride Nano-Glass Ceramics Doped with Pr3+ and Pr3+–Yb3+ for NIR Emission

Pr3+-Yb3+ co-doped oxyfluoride glasses and glass-ceramics (GC) containing LaF3 nanocrystals have been prepared to obtain NIR emission of Yb3+ ions upon Pr3+ excitation in the blue region of the visible spectrum. Two different compositions have been tested 0.1-0.5 Pr-Yb and 0.5-1 Pr-Yb, in addition to Pr3+ singly doped samples. The crystallization mechanism of the nano-glass-ceramics was studied by DTA revealing that it occurs from a constant number of nuclei, the crystal growth being limited by diffusion. HR-TEM demonstrated that phase separation acts as precursor for LaF3 crystallization and a detailed analysis of the chemical composition (EDXS) revealed the enrichment in RE3+ ions inside the initial phase separated droplets, from which the LaF3 crystals are formed. The RE3+ ions incorporation inside LaF3 crystals was also proved by photoluminescence measurements showing Stark splitting of the RE3+ ions energy levels in the glass-ceramic samples. Lifetimes measurements showed the existence of a better energy transfer process between Pr3+ and Yb3+ ions in the glass-ceramics compared to the as made glass, and the highest value of energy transfer efficiency is 59% and the highest theoretical quantum efficiency is 159%, obtained for glass-ceramics GC0.1-0.5 Pr-Yb treated at 620 oC-40 h.

In vivo multiphoton imaging of collagen remodeling after microablative fractional rejuvenation

The potential of multiphoton microscopy in providing in-vivo early diagnosis of skin lesions has already been demonstrated, while its capability in therapy follow-up has not been deeply explored so far. Two-photon excited fluorescence and second-harmonic generation microscopy were used in combination to follow-up collagen remodeling after laser micro-ablative rejuvenation. Treated regions of volunteers were imaged with multiphoton microscopy before and after treatment, and we found a strong age-dependence of the treatment effectiveness. In particular, the photorejuvenating effect was negligible in young subjects (< 30 years), whereas a significant production of new collagen was observed in aged subjects (> 70 years). Quantification of the amount of newly produced collagen and its organization were performed by means of visual examination of two-photon images. The obtained results demonstrate the performance of laser fractional micro-ablative rejuvenation without the need of an invasive biopsy as well as the wide applicability range of applications for multiphoton microscopy in clinical dermatology.

Time- And Spectral-Resolved Multiphoton Imaging Of Fresh Bladder Biopsies

Human tissues intrinsically contains many fluorophores, as such NADH, elastin, collagen, and flavins, that can be excited and imaged using multiphoton microscopy, up to 150 microns depth. In this work we used combined two photon intrinsic fluorescence (TPE), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopic analyses allowed to characterize both healthy and pathological tissue samples in a good agreement with common routine histology. In particular, we examined tissue samples from bladder normal mucosa, and bladder carcinoma in-situ (CIS), finding both morphological and spectroscopic differences. From the morphological point of view, cancer cells appeared more elongated with respect to corresponding normal cells; they also exhibited a different nucleus to cytoplasm ratio. From the spectroscopic point of view, we found differences between the two tissue types in both spectral emission and fluorescence lifetime distribution. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or cystoscope to be used in in-vivo imaging applications.

Multispectral multiphoton lifetime analysis of human bladder tissue

Human tissues intrinsically contain many fluorophores, as such NADH, elastin, collagen, and flavins, that can be excited and imaged using multiphoton microscopy, up to 150 μm depth. In this work we have used combined two-photon excited fluorescence (TPE), fluorescence lifetime imaging microscopy (FLIM), and multispectral two photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopic analyses have allowed to characterize both healthy and pathological tissue samples. In particular, we have examined tissue samples from healthy bladder mucosa, and bladder carcinoma in-situ (CIS), finding both morphological and spectroscopic differences. From the morphological point of view, cancer cells appeared more elongated with respect to corresponding normal cells; they also exhibited a different nucleus to cytoplasm ratio. From the spectroscopic point of view, we have found differences between the two tissue types in both spectral emission and fluorescence lifetime distribution. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or cystoscope to be used in in-vivo imaging applications.

THz Pyro-Optical Detector Based on LiNbO3 Whispering Gallery Mode Microdisc Resonator

This study analyzes the capabilities of a LiNbO3 whispering gallery mode microdisc resonator as a potential bolometer detector in the THz range. The resonator is theoretically characterized in the stationary regime by its thermo-optic and thermal coefficients. Considering a Q-factor of 107, a minimum detectable power of 20 μW was evaluated, three orders of magnitude above its noise equivalent power. This value opens up the feasibility of exploiting LiNbO3 disc resonators as sensitive room-temperature detectors in the THz range.

Localized immunoassay in flow-through optical microbubble resonator (Conference Presentation)

The integration of the Whispering Gallery Modes (WGMs) resonators in a microfluidics platform represents an important feature towards the realization of a compact high performance label-free biosensor. These hollow resonant microstructures present the advantage to combine the WGM resonator properties with the intrinsic capability of integrated microfluidics. In this sense, optical microbubble resonators (OMBRs), intended as a hollow core spherical bulge realized in a glass microcapillary by a suitable fabrication process, with their high Q factors (< 107 in air) well satisfy this requirement. Their operation is based on the fact that, given a small enough wall thickness of the bubble, the WGM optical field extends on both sides of the wall, so that it is possible to couple light into the resonator from an outer waveguide, and at the same time to have interaction of the WGM field with the inner fluid and analyte. The biosensing mechanism of these devices is based on the WGMs morphological dependence: any change on the OMBR inner surface, due to some chemical and/or biochemical binding, causes a shift of the resonance position and reduces the Q factor of the OMBR. By measuring these changes, important information about the sensing capability of the device can be obtained. In order to develop an OMBR based biosensor and optimize its performance, a crucial step is represented by its chemical/biochemical functionalization. Here we present a novel technique able to guarantee that the chemical interaction occurs in the OMBR inner wall, leaving the other microfluidic parts completely inert from a biochemical point of view. The method is based on UV photoactivation, which allows to localize the biolayers only in correspondence of the OMBR inner wall. As a proof of concept, an immunoassay based on rabbit IgG/anti rabbit-IgG interaction was performed and. The anti rabbit-IgG antibody was labelled with Alexa Fluor 488 to verify, by a fluorescence characterization, the goodness of this procedure. Moreover, an anti mouse-IgG, labelled with the same fluorophore (Alexa Fluor 488) was used for specificity-tests of the IgG/anti-IgG interaction. The immunoassay based on fluorescence was characterized using an optical microscope (Zeiss AXIO inverted fluorescence microscope) working at the wavelengths of 470 nm for excitation of Alexa Fluor 488. The real time measurement of the resonance broadening after each functionalization step together with the high Q factor (< 105) measured after the IgG/anti-IgG interaction in water, gives a further proof for the method validity.

Fluorescence ratiometric classifier for the detection of skin pathologies

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.