58 results on '"Osellame R"'
Search Results
2. Non-linear excitation microscopy of live cells by using an implantable microscope objective-on-a-chip
- Author
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Tarnok, A, Houston, JP, Nardini, A, Vázquez, R, Conci, C, Grassi, M, Marini, M, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Raimondi, M, Chirico, G, Nardini A., Vázquez R. M., Conci C., Grassi M., Marini M., Bouzin M., Collini M., Osellame R., Cerullo G. N., Kariman B. S., Raimondi M. T., Chirico G., Tarnok, A, Houston, JP, Nardini, A, Vázquez, R, Conci, C, Grassi, M, Marini, M, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Raimondi, M, Chirico, G, Nardini A., Vázquez R. M., Conci C., Grassi M., Marini M., Bouzin M., Collini M., Osellame R., Cerullo G. N., Kariman B. S., Raimondi M. T., and Chirico G.
- Abstract
In the context of biomaterials, small-molecules and drugs testing, intravital microscopy allows to quantify in-vivo the immune reaction, reducing the number of laboratory animals required to statistically validate the product. However, fluorescence microscopy is affected by limited tissue penetration due to light scattering and by optical aberrations, induced on focused beams, by the animal tissue surrounding the implant. In this framework, we developed a system of microlenses coupled to microscaffolds, both incorporated in a miniaturized imaging window. The chip is designed to act as an in-situ microscope objective with the aim to overcome the restrictions of in-vivo imaging (i.e. spherical aberrations) and to allow multiple biological observations in the same animal (by including fluorescent beacons). The device is fabricated by two-photon polymerizing a biocompatible photoresist called SZ2080. The microlenses are manufactured by the concentric polar scanning of the laser beam to realize their outer surface, followed by the UV bulk polymerization of their inner SZ2080. We preliminarily characterized the imaging capabilities of our implantable system on live cells cultured on flat substrates and 3D microscaffolds by coupling it to low magnification objectives. The microlenses optical quality is sufficient to induce non-linear excitation and collect two-photon excitation images with the same level of laser intensity and signal-to-noise ratio. Remarkably, they allow to efficiently excite the fluorescence of labelled human fibroblasts collecting high resolution magnified images. These results will open the way to the application of implanted micro-optics for the real-time and continuous in-vivo observation of complex biological processes.
- Published
- 2024
3. A flexible beamline combining XUV attosecond pulses with few-femtosecond UV and near-infrared pulses for time-resolved experiments.
- Author
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Wanie, V., Ryabchuk, S., Colaizzi, L., Galli, M., Månsson, E. P., Trabattoni, A., Wahid, A. B., Hahne, J., Cartella, A., Saraswathula, K., Frassetto, F., Lopes, D. P., Martínez Vázquez, R., Osellame, R., Poletto, L., Légaré, F., Nisoli, M., and Calegari, F.
- Subjects
ATTOSECOND pulses ,THIRD harmonic generation ,LIGHT absorption ,ULTRAVIOLET radiation ,BINOCULARS - Abstract
We describe a beamline where few-femtosecond ultraviolet (UV) pulses are generated and synchronized to few-cycle near-infrared (NIR) and extreme ultraviolet (XUV) attosecond pulses. The UV light is obtained via third-harmonic generation in argon or neon gas when focusing a phase-stabilized NIR driving field inside a glass cell that was designed to support high pressures for enhanced conversion efficiency. A recirculation system allows reducing the large gas consumption required for the nonlinear process. Isolated attosecond pulses are generated using the polarization gating technique, and the photon spectrometer employed to characterize the XUV radiation consists of a new design based on the combination of a spherical varied-line-space grating and a cylindrical mirror. This design allows for compactness while providing a long entrance arm for integrating different experimental chambers. The entire interferometer is built under vacuum to prevent both absorption of the XUV light and dispersion of the UV pulses, and it is actively stabilized to ensure an attosecond delay stability during experiments. This table-top source has been realized with the aim of investigating UV-induced electron dynamics in neutral states of bio-relevant molecules, but it also offers the possibility to implement a manifold of novel time-resolved experiments based on photo-ionization/excitation of gaseous and liquid targets by ultraviolet radiation. UV pump–XUV probe measurements in ethyl-iodide showcase the capabilities of the attosecond beamline. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
4. In vivo label-free tissue histology through a microstructured imaging window
- Author
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Conci, C, Sironi, L, Jacchetti, E, Panzeri, D, Inverso, D, Martínez Vázquez, R, Osellame, R, Collini, M, Cerullo, G, Chirico, G, Teresa Raimondi, M, Claudio Conci, Laura Sironi, Emanuela Jacchetti, Davide Panzeri, Donato Inverso, Rebeca Martínez Vázquez, Roberto Osellame, Maddalena Collini, Giulio Cerullo, Giuseppe Chirico, Manuela Teresa Raimondi, Conci, C, Sironi, L, Jacchetti, E, Panzeri, D, Inverso, D, Martínez Vázquez, R, Osellame, R, Collini, M, Cerullo, G, Chirico, G, Teresa Raimondi, M, Claudio Conci, Laura Sironi, Emanuela Jacchetti, Davide Panzeri, Donato Inverso, Rebeca Martínez Vázquez, Roberto Osellame, Maddalena Collini, Giulio Cerullo, Giuseppe Chirico, and Manuela Teresa Raimondi
- Abstract
Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of
- Published
- 2024
5. Laser microfabrication for optical sensing and imaging in vivo
- Author
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Bianchini, P, Diaspro, A, Sheppard, CJR, Bouzin, M, Sironi, L, Marini, M, D'Alfonso, L, Collini, M, Chirico, G, Conci, C, Jacchetti, E, Raimondi, M, Nardini, A, Cerullo, G, Martinez Vazquez, R, Osellame, R, Sironi L., Marini M., Bouzin M., D'Alfonso L., Collini M., Chirico G., Conci C., Jacchetti E., Raimondi M. T., Nardini A., Cerullo G., Martinez Vazquez R., Osellame R., Bianchini, P, Diaspro, A, Sheppard, CJR, Bouzin, M, Sironi, L, Marini, M, D'Alfonso, L, Collini, M, Chirico, G, Conci, C, Jacchetti, E, Raimondi, M, Nardini, A, Cerullo, G, Martinez Vazquez, R, Osellame, R, Sironi L., Marini M., Bouzin M., D'Alfonso L., Collini M., Chirico G., Conci C., Jacchetti E., Raimondi M. T., Nardini A., Cerullo G., Martinez Vazquez R., and Osellame R.
- Abstract
The possibility to fabricate microstructures to be used in the medical field is a reality. Further steps in this direction consist in the fabrication of active microstructures for implants and/or for cellular treatments. Two major areas of interest will be briefly treated here. The first is the one of stimulus-responsive optical polymers, especially hydrogels, that can be shaped by means of laser 3D printing and ablation. These can be used for thermal stimulation, energy transduction and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. A second field of interest is the microfabrication of rigid microstructures that can stand the tissue-induced stresses in implants. The increasing demand of microdevices for nanomedicine and personalized medicine has fostered the quest for an efficient combination of composite and hybrid photo-responsive materials and digital micro/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. The aim of this report is to provide a short overview of the basics of photo-polymerization induced by two-photon excitation and to discuss two case studies. In the first one, we discuss the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photo-thermal activity, chemical swelling and chemical sensing. In the second case, we outline the fabrication steps and the first tests of a novel chip which aims at enabling longitudinal studies of the reaction to the biomater
- Published
- 2023
6. Two-Photon Polymerization of an In-Vivo Multiphoton Imaging Window
- Author
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Vazquez, R, Nardini, A, Marini, M, Conci, C, Kariman, B, Bouzin, M, Collini, M, Chirico, G, Raimondi, M, Osellame, R, Cerullo, G, Vazquez R. M., Nardini A., Marini M., Conci C., Kariman B. S., Bouzin M., Collini M., Chirico G., Raimondi M. T., Osellame R., Cerullo G., Vazquez, R, Nardini, A, Marini, M, Conci, C, Kariman, B, Bouzin, M, Collini, M, Chirico, G, Raimondi, M, Osellame, R, Cerullo, G, Vazquez R. M., Nardini A., Marini M., Conci C., Kariman B. S., Bouzin M., Collini M., Chirico G., Raimondi M. T., Osellame R., and Cerullo G.
- Abstract
Intravital microscopy windows for in vivo observation of tissue are desirable in many biomedical fields, from immunology to the assessment of the foreign body reaction to biomaterials. Moreover, they allow one to reduce the number of animals required to statistically validate biomaterials if compared with traditional histopathological analyses of animal biopsies [1,2].
- Published
- 2023
7. Micro-Fabricated Optics for Multiphoton Microscopy
- Author
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Jaworski, M, Marciniak, M, Vazquez, R, Nardini, A, Conci, C, Jacchetti, E, Marini, M, Panzeri, D, Sironi, L, Bouzin, M, Collini, M, Inverso, D, Kariman, B, Kabouraki, E, Farsari, M, Osellame, R, Cerullo, G, Raimondi, M, Chirico, G, Vazquez R. M., Nardini A., Conci C., Jacchetti E., Marini M., Panzeri D., Sironi L., Bouzin M., Collini M., Inverso D., Kariman B. S., Kabouraki E., Farsari M., Osellame R., Cerullo G., Raimondi M. T., Chirico G., Jaworski, M, Marciniak, M, Vazquez, R, Nardini, A, Conci, C, Jacchetti, E, Marini, M, Panzeri, D, Sironi, L, Bouzin, M, Collini, M, Inverso, D, Kariman, B, Kabouraki, E, Farsari, M, Osellame, R, Cerullo, G, Raimondi, M, Chirico, G, Vazquez R. M., Nardini A., Conci C., Jacchetti E., Marini M., Panzeri D., Sironi L., Bouzin M., Collini M., Inverso D., Kariman B. S., Kabouraki E., Farsari M., Osellame R., Cerullo G., Raimondi M. T., and Chirico G.
- Abstract
We report new methods of 2 photon polymerization of microlenses with high numerical aperture, large diameter and good optical quality. We characterize the aberrations of these lenses that, coupled to raster scanning optical microscopes, allow two-photon excitation imaging of cells. In-vivo non-linear imaging will be also discussed, opening the possibility to use these micro-lenses in implants for the continuous inspection of biological dynamics in vivo.
- Published
- 2023
8. Microlenses Fabricated by Two‐Photon Laser Polymerization for Cell Imaging with Non‐Linear Excitation Microscopy
- Author
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Marini, M, Nardini, A, Martínez Vázquez, R, Conci, C, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Farsari, M, Kabouraki, E, Raimondi, M, Chirico, G, Marini, M., Nardini, A., Martínez Vázquez, R., Conci, C., Bouzin, M., Collini, M., Osellame, R., Cerullo, G., Kariman, B. S., Farsari, M., Kabouraki, E., Raimondi, M. T., Chirico, G., Marini, M, Nardini, A, Martínez Vázquez, R, Conci, C, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Farsari, M, Kabouraki, E, Raimondi, M, Chirico, G, Marini, M., Nardini, A., Martínez Vázquez, R., Conci, C., Bouzin, M., Collini, M., Osellame, R., Cerullo, G., Kariman, B. S., Farsari, M., Kabouraki, E., Raimondi, M. T., and Chirico, G.
- Abstract
Non-linear excitation microscopy offers several advantages for in-vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in-vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, Fibroblast cell culture plano-convex microlenses to be used for non-linear imaging of biological tissue are developed and tested. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. Magnified fluorescence images can be retrieved through the microlenses coupled to commercial confocal and two-photon excitation scanning microscopes. The signal-to-noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro-optics for optical in-vivo inspection of biological processes.
- Published
- 2023
9. Two-photon laser polymerized microlenses for non-linear excitation microscopy of biological samples
- Author
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Ferraro, P, Psaltis, D, Grilli, S, Nardini, A, Vazquez, R, Marini, M, Conci, C, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Farsari, M, Kabouraki, E, Chirico, G, Raimondi, M, Nardini A., Vazquez R. M., Marini M., Conci C., Bouzin M., Collini M., Osellame R., Cerullo G., Kariman B. S., Farsari M., Kabouraki E., Chirico G., Raimondi M. T., Ferraro, P, Psaltis, D, Grilli, S, Nardini, A, Vazquez, R, Marini, M, Conci, C, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Farsari, M, Kabouraki, E, Chirico, G, Raimondi, M, Nardini A., Vazquez R. M., Marini M., Conci C., Bouzin M., Collini M., Osellame R., Cerullo G., Kariman B. S., Farsari M., Kabouraki E., Chirico G., and Raimondi M. T.
- Abstract
We report new methods of 2 photon polymerization of microlenses with high numerical aperture, large diameter and good optical quality. We characterize the aberrations of these lenses that, coupled to raster scanning optical microscopes, allow two-photon excitation imaging of cells. In-vivo non-linear imaging will be also discussed, opening the possibility to use these micro-lenses in implants for the continuous inspection of biological dynamics in vivo.
- Published
- 2023
10. Micro-Fabricated Optics for Multiphoton Microscopy
- Author
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Vázquez, R. Martínez, primary, Nardini, A., additional, Conci, C., additional, Jacchetti, E., additional, Marini, M., additional, Panzeri, D., additional, Sironi, L., additional, Bouzin, M., additional, Collini, M., additional, Inverso, D., additional, Kariman, B.S., additional, Kabouraki, E., additional, Farsari, M., additional, Osellame, R., additional, Cerullo, G., additional, Raimondi, M.T., additional, and Chirico, G., additional
- Published
- 2023
- Full Text
- View/download PDF
11. A Miniaturized Imaging Window to Quantify Intravital Tissue Regeneration within a 3D Microscaffold in Longitudinal Studies
- Author
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Conci, C, Jacchetti, E, Sironi, L, Gentili, L, Cerullo, G, Osellame, R, Chirico, G, Raimondi, M, Conci C., Jacchetti E., Sironi L., Gentili L., Cerullo G., Osellame R., Chirico G., Raimondi M. T., Conci, C, Jacchetti, E, Sironi, L, Gentili, L, Cerullo, G, Osellame, R, Chirico, G, Raimondi, M, Conci C., Jacchetti E., Sironi L., Gentili L., Cerullo G., Osellame R., Chirico G., and Raimondi M. T.
- Abstract
The biocompatibility assessment of biomaterials or the dynamic response of implanted constructs entails inflammatory events primary reflected in cell behavior at the microcirculatory system. Current protocols are based on histopathology which are over 40 years old and require the sacrifice of a huge number of laboratory animal with an unsustainable ethical burden of animal research. Intravital microscopy techniques are actually used to study implantation outcomes in real time. However, no device providing a specific tracking geometry to reposition the field of view of the microscope, for repeated analyses, exists yet. The synthetic photoresist SZ2080 is characterized here, allowing the development and in vivo validation of a miniaturized imaging window, the Microatlas, that, fabricated via two-photon polymerization, is implanted in living chicken embryos and imaged by fluorescence microscopy 3 and 4 days after the implant. The characterization of their elastomechanical and fluorescence properties highlights planar raster spacing as the most important parameter in tuning the mechanical and spectroscopic features of the structures. The quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration and as beacon for 3D repositioning of the microscope field of view and correction of optical aberrations.
- Published
- 2022
12. A miniaturized chip for 3D optical imaging of tissue regeneration in vivo
- Author
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Popp, J, Gergely, C, Conci, C, Jacchetti, E, Sironi, L, Gentili, L, Cerullo, G, Martinez, R, Osellame, R, Marini, M, Bouzin, M, Collini, M, D'Alfonso, L, Kabouraki, E, Farsari, M, Ranella, A, Kehagias, N, Chirico, G, Raimondi, M, Conci C., Jacchetti E., Sironi L., Gentili L., Cerullo G., Martinez R., Osellame R., Marini M., Bouzin M., Collini M., D'Alfonso L., Kabouraki E., Farsari M., Ranella A., Kehagias N., Chirico G., Raimondi M. T., Popp, J, Gergely, C, Conci, C, Jacchetti, E, Sironi, L, Gentili, L, Cerullo, G, Martinez, R, Osellame, R, Marini, M, Bouzin, M, Collini, M, D'Alfonso, L, Kabouraki, E, Farsari, M, Ranella, A, Kehagias, N, Chirico, G, Raimondi, M, Conci C., Jacchetti E., Sironi L., Gentili L., Cerullo G., Martinez R., Osellame R., Marini M., Bouzin M., Collini M., D'Alfonso L., Kabouraki E., Farsari M., Ranella A., Kehagias N., Chirico G., and Raimondi M. T.
- Abstract
The current protocols for biocompatibility assessment of biomaterials, based on histopathology, require the sacrifice of a huge number of laboratory animals with an unsustainable ethical burden and remarkable cost. Intravital microscopy techniques can be used to study implantation outcomes in real time though with limited capabilities of quantification in longitudinal studies, mainly restricted by the light penetration and the spatial resolution in deep tissues. We present the outline and first tests of a novel chip which aims to enable longitudinal studies of the reaction to the biomaterial implant. The chip is composed of a regular reference microstructure fabricated via two-photon polymerization in the SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in-vitro characterization and in vivo validation of the Microatlas is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration.
- Published
- 2022
13. Microlenses Fabricated by Two‐Photon Laser Polymerization for Cell Imaging with Non‐Linear Excitation Microscopy
- Author
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Marini, M., primary, Nardini, A., additional, Martínez Vázquez, R., additional, Conci, C., additional, Bouzin, M., additional, Collini, M., additional, Osellame, R., additional, Cerullo, G., additional, Kariman, B. S., additional, Farsari, M., additional, Kabouraki, E., additional, Raimondi, M. T., additional, and Chirico, G., additional
- Published
- 2023
- Full Text
- View/download PDF
14. Generation of four-photon GHZ states on a laser written integrated platform
- Author
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Corrielli, G., primary, Pont, M., additional, Fyrillas, A., additional, Agresti, I., additional, Carvacho, G., additional, Maring, N., additional, Emeriau, P. E., additional, Ceccarelli, F., additional, Albiero, R., additional, Ferreira, P.-H. Dias, additional, Somaschi, N., additional, Senellart, J., additional, Morassi, M., additional, Lemaitre, A., additional, Sagnes, I., additional, Senellart, P., additional, Sciarrino, F., additional, Liscidini, M., additional, Belabas, N., additional, and Osellame, R., additional
- Published
- 2023
- Full Text
- View/download PDF
15. Microlenses fabricated by two-photon laser polymerization for cell imaging with non-linear excitation microscopy
- Author
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Marini, M., primary, Nardini, A., additional, Vázquez, R. Martínez, additional, Conci, C., additional, Bouzin, M., additional, Collini, M., additional, Osellame, R., additional, Cerullo, G., additional, Kariman, B.S., additional, Farsari, M., additional, Kabouraki, E., additional, Raimondi, M.T., additional, and Chirico, G., additional
- Published
- 2022
- Full Text
- View/download PDF
16. Microfluidic devices for quasi-phase-matching in high-order harmonic generation
- Author
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Ciriolo, A. G., primary, Martínez Vázquez, R., additional, Crippa, G., additional, Devetta, M., additional, Faccialà, D., additional, Barbato, P., additional, Frassetto, F., additional, Negro, M., additional, Bariselli, F., additional, Poletto, L., additional, Tosa, V., additional, Frezzotti, A., additional, Vozzi, C., additional, Osellame, R., additional, and Stagira, S., additional
- Published
- 2022
- Full Text
- View/download PDF
17. Low-power programmable integrated photonic circuits fabricated by femtosecond laser micromachining
- Author
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Pentangelo, C, Ceccarelli, F, Piacentini, S, Atzeni, S, Crespi, A, and Osellame, R
- Published
- 2022
18. High-fidelity and polarization-insensitive universal photonic processors fabricated by femtosecond laser writing
- Author
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Pentangelo Ciro, Di Giano Niki, Piacentini Simone, Arpe Riccardo, Ceccarelli Francesco, Crespi Andrea, and Osellame Roberto
- Subjects
femtosecond laser writing ,universal photonic processor ,integrated photonics ,Physics ,QC1-999 - Abstract
Universal photonic processors (UPPs) are fully programmable photonic integrated circuits that are key components in quantum photonics. With this work, we present a novel platform for the realization of low-loss, low-power, and high-fidelity UPPs based on femtosecond laser writing (FLW) and compatible with a large wavelength spectrum. In fact, we demonstrate different UPPs, tailored for operation at 785 nm and 1550 nm, providing similar high-level performances. Moreover, we show that standard calibration techniques applied to FLW-UPPs result in Haar random polarization-insensitive photonic transformations implemented with average amplitude fidelity as high as 0.9979 at 785 nm (0.9970 at 1550 nm), with the possibility of increasing the fidelity over 0.9990 thanks to novel optimization algorithms. Besides being the first demonstrations of polarization-insensitive UPPs, these devices show the highest level of control and reconfigurability ever reported for a FLW circuit. These qualities will be greatly beneficial to applications in quantum information processing.
- Published
- 2024
- Full Text
- View/download PDF
19. Universal photonic processors fabricated by femtosecond laser writing
- Author
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García-Blanco, Sonia M., Cheben, Pavel, Pentangelo, C., Ceccarelli, F., Piacentini, S., Albiero, R., Urbinati, E., Di Giano, N., Atzeni, S., Crespi, A., and Osellame, R.
- Published
- 2022
- Full Text
- View/download PDF
20. Non-linear excitation microscopy of live cells by using an implantable microscope objective-on-a-chip
- Author
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Tarnok, Attila, Houston, Jessica P., Nardini, A., Martínez Vázquez, R., Conci, C., Grassi, M., Marini, M., Bouzin, M., Collini, M., Osellame, R., Cerullo, G. N., Kariman, B. S., Raimondi, M. T., and Chirico, G.
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- 2024
- Full Text
- View/download PDF
21. Two-photon laser polymerized microlenses for non-linear excitation microscopy of biological samples.
- Author
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Nardini, A., Martínez Vázquez, R., Marini, M., Conci, C., Bouzin, M., Collini, M., Osellame, R., Cerullo, G., Kariman, B. S., Farsari, M., Kabouraki, E., Chirico, G., and Raimondi, M. T.
- Published
- 2023
- Full Text
- View/download PDF
22. Femtosecond-laser written universal quantum photonic processors.
- Author
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Pentangelo, C., Ceccarelli, F., Di Giano, N., Arpe, R., Piacentini, S., Crespi, A., and Osellame, R.
- Published
- 2023
- Full Text
- View/download PDF
23. Femtosecond laser micromachining for integrated quantum photonics
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Corrielli Giacomo, Crespi Andrea, and Osellame Roberto
- Subjects
femtosecond laser micromachining ,integrated optics ,integrated quantum photonics ,photonic circuits ,quantum technologies ,Physics ,QC1-999 - Abstract
Integrated quantum photonics, i.e. the generation, manipulation, and detection of quantum states of light in integrated photonic chips, is revolutionizing the field of quantum information in all applications, from communications to computing. Although many different platforms are being currently developed, from silicon photonics to lithium niobate photonic circuits, none of them has shown the versatility of femtosecond laser micromachining (FLM) in producing all the components of a complete quantum system, encompassing quantum sources, reconfigurable state manipulation, quantum memories, and detection. It is in fact evident that FLM has been a key enabling tool in the first-time demonstration of many quantum devices and functionalities. Although FLM cannot achieve the same level of miniaturization of other platforms, it still has many unique advantages for integrated quantum photonics. In particular, in the last five years, FLM has greatly expanded its range of quantum applications with several scientific breakthroughs achieved. For these reasons, we believe that a review article on this topic is very timely and could further promote the development of this field by convincing end-users of the great potentials of this technological platform and by stimulating more research groups in FLM to direct their efforts to the exciting field of quantum technologies.
- Published
- 2021
- Full Text
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24. Two-photon laser polymerized microlenses for non-linear excitation microscopy of biological samples
- Author
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Ferraro, Pietro, Psaltis, Demetri, Grilli, Simonetta, Nardini, A., Martínez Vázquez, R., Marini, M., Conci, C., Bouzin, M., Collini, M., Osellame, R., Cerullo, G., Kariman, B. S., Farsari, M., Kabouraki, E., Chirico, G., and Raimondi, M. T.
- Published
- 2023
- Full Text
- View/download PDF
25. Femtosecond laser rapid prototyping and characterization of microfluidic device for particles sorting
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Volpe Annalisa, Petruzzellis Isabella, Mezzapesa Francesco P., Gaudiuso Caterina, Osellame Roberto, Ancona Antonio, and Martínez Vázquez Rebeca
- Subjects
Physics ,QC1-999 - Abstract
Rapid prototyping methods for the fabrication of polymeric labs-on-a-chip (LoC) are on the rise, as they allow high degrees of precision and flexibility. In this contest, the flexibility of ultrafast laser technology enables the rapid prototyping and high-precision micromachining of 3D LoC devices with complex microfluidic channel networks. In this paper, we describe the realization process of a microfluidic tool for fully inertial particles sorting. The microfluidic network was realized in polymethyl methacrylate (PMMA), exploiting femtosecond laser technology. The multilayer device was assembled through a facile and low-cost solvent-assisted method. In particular, we studied the particle focusing in curved inertial microfluidic channel with trapezoidal cross section. A particles focusing along the walls of the device, sensitive to particle size and flow rate, was observed based on the principle of Dean-coupled inertial migration in spiral microchannel.
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- 2023
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26. Fabrication of Plano-convex Microlenses using Two-Photon Polymerization for Bioimaging with Non-Linear Excitation Microscopy
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Kariman Behjat S., Nardini Alessandra, Grassi Marco, Marini Mario, Conci Claudio, Bouzin Margaux, Collini Maddalena, Raimondi Manuela T., Chirico Giuseppe, Osellame Roberto, Cerullo Giulio, and Martínez Vázquez Rebeca
- Subjects
Physics ,QC1-999 - Abstract
A recent challenge in bioimaging is the observation and imaging of vital, thick, and complex tissues in real time and in non-invasive mode. In the last decade, non-linear excitation microscopy showed several advantages for in-vivo imaging compared to conventional confocal techniques. Nevertheless, deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. A low numerical aperture objective lens coupled to high dioptric power microlenses, implanted in the tissue, can be beneficial for the reduction of optical aberrations. In this context, we fabricated a system of plano-convex microlenses and microscaffolds on a single chip by means of two-photon polymerization), to be used for non-linear imaging of biological specimens.
- Published
- 2023
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27. Femtosecond-laser written universal quantum photonic processors
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Hemmer, Philip R., Migdall, Alan L., Pentangelo, C., Ceccarelli, F., Di Giano, N., Arpe, R., Piacentini, S., Crespi, A., and Osellame, R.
- Published
- 2023
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28. Universal photonic processors fabricated by femtosecond laser writing.
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Pentangelo, C., Ceccarelli, F., Piacentini, S., Albiero, R., Urbinati, E., Di Giano, N., Atzeni, S., Crespi, A., and Osellame, R.
- Published
- 2021
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29. One-dimensional photonic crystal for polarization-sensitive surface-enhanced spectroscopy
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Mogni Erika, Pellegrini Giovanni, Gil-Rostra Jorge, Yubero Francisco, Simone Giuseppina, Fossati Stefan, Dostálek Jakub, Martinez-Vazquez Rebeca, Osellame Roberto, Celebrano Michele, Finazzi Marco, and Biagioni Paolo
- Subjects
Physics ,QC1-999 - Abstract
We realize and experimentally characterize a novel platform for surface-enhanced sensing through Bloch Surface Waves (BSWs). We test a one-dimensional photonic crystal, with a high index inclusion in the top layer, that sustains surfaces modes with, in principle, arbitrary polarization. This is achieved through the coherent superposition of TE and TM dispersion relations of BSWs, which can also provide superchiral fields over a wide spectral range (down to the UV). The resulting platform paves the way to the implementation of polarization-resolved surface-enhanced techniques.
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- 2022
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30. Microlenses fabricated by two-photon laser polymerization for cell imaging with non-linear excitation microscopy
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M. Marini, A. Nardini, R. Martínez Vázquez, C. Conci, M. Bouzin, M. Collini, R. Osellame, G. Cerullo, B. S. Kariman, M. Farsari, E. Kabouraki, M. T. Raimondi, G. Chirico, Marini, M, Nardini, A, Martínez Vázquez, R, Conci, C, Bouzin, M, Collini, M, Osellame, R, Cerullo, G, Kariman, B, Farsari, M, Kabouraki, E, Raimondi, M, and Chirico, G
- Subjects
3D micro scaffolds, confocal microscopy, microlenses, SZ2080, twophoton imaging, two-photon polymerization ,SZ2080 ,FIS/07 - FISICA APPLICATA (A BENI CULTURALI, AMBIENTALI, BIOLOGIA E MEDICINA) ,twophoton imaging ,3D micro scaffold ,Condensed Matter Physics ,confocal microscopy ,3D micro scaffolds ,Electronic, Optical and Magnetic Materials ,Biomaterials ,two-photon polymerization ,microlenses ,microlense ,Electrochemistry ,two-photon imaging - Abstract
Non-linear excitation microscopy offers several advantages for in-vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in-vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, we develop and test on fibroblast cell culture plano-convex microlenses to be used for non-linear imaging of biological tissue. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. We could retrieve magnified fluorescence images through the microlenses coupled to commercial confocal and two-photon excitation scanning microscopes. The signal-to-noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro-optics for optical in-vivo inspection of biological processes.
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- 2023
31. A miniaturized chip for 3D optical imaging of tissue regeneration in vivo
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Claudio Conci, Emanuela Jacchetti, Laura Sironi, Lorenzo Gentili, Giulio Cerullo, Rebeca Martínez Vázquez, Roberto Osellame, Mario Marini, Margaux Bouzin, Maddalena Collini, Laura D'Alfonso, Elmina Kabouraki, Maria Farsari, Anthi Ranella, Nikos Kehagias, Giuseppe Chirico, Manuela Teresa Raimondi, Popp, J, Gergely, C, Conci, C, Jacchetti, E, Sironi, L, Gentili, L, Cerullo, G, Martinez, R, Osellame, R, Marini, M, Bouzin, M, Collini, M, D'Alfonso, L, Kabouraki, E, Farsari, M, Ranella, A, Kehagias, N, Chirico, G, and Raimondi, M
- Subjects
two-photon polymerization ,3D-microstructured scaffold ,elasto-mechanic ,in vivo implant ,ex ovo implant ,intravital imaging window ,confocal microscopy ,two-photon imaging - Abstract
The current protocols for biocompatibility assessment of biomaterials, based on histopathology, require the sacrifice of a huge number of laboratory animals with an unsustainable ethical burden and remarkable cost. Intravital microscopy techniques can be used to study implantation outcomes in real time though with limited capabilities of quantification in longitudinal studies, mainly restricted by the light penetration and the spatial resolution in deep tissues. We present the outline and first tests of a novel chip which aims to enable longitudinal studies of the reaction to the biomaterial implant. The chip is composed of a regular reference microstructure fabricated via two-photon polymerization in the SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in-vitro characterization and in vivo validation of the Microatlas is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration.
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- 2022
32. A Miniaturized Imaging Window to Quantify Intravital Tissue Regeneration within a 3D Microscaffold in Longitudinal Studies
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Claudio Conci, Emanuela Jacchetti, Laura Sironi, Lorenzo Gentili, Giulio Cerullo, Roberto Osellame, Giuseppe Chirico, Manuela Teresa Raimondi, Conci, C, Jacchetti, E, Sironi, L, Gentili, L, Cerullo, G, Osellame, R, Chirico, G, and Raimondi, M
- Subjects
two-photon polymerization ,3D-microstructured scaffold ,elastomechanic ,in vivo implant ,ex ovo implant ,intravital imaging window ,confocal microscopy ,two-photon imaging ,Atomic and Molecular Physics, and Optics ,Electronic, Optical and Magnetic Materials - Abstract
The biocompatibility assessment of biomaterials or the dynamic response of implanted constructs entails inflammatory events primary reflected in cell behavior at the microcirculatory system. Current protocols are based on histopathology which are over 40 years old and require the sacrifice of a huge number of laboratory animal with an unsustainable ethical burden of animal research. Intravital microscopy techniques are actually used to study implantation outcomes in real time. However, no device providing a specific tracking geometry to reposition the field of view of the microscope, for repeated analyses, exists yet. The synthetic photoresist SZ2080 is characterized here, allowing the development and in vivo validation of a miniaturized imaging window, the Microatlas, that, fabricated via two-photon polymerization, is implanted in living chicken embryos and imaged by fluorescence microscopy 3 and 4 days after the implant. The characterization of their elastomechanical and fluorescence properties highlights planar raster spacing as the most important parameter in tuning the mechanical and spectroscopic features of the structures. The quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration and as beacon for 3D repositioning of the microscope field of view and correction of optical aberrations.
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- 2022
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33. Nanochannels in Fused Silica through NaOH Etching Assisted by Femtosecond Laser Irradiation.
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Barbato P, Osellame R, and Martínez Vázquez R
- Abstract
Sodium hydroxide (NaOH) is increasingly drawing attention as a highly selective etchant for femtosecond laser-modified fused silica. Unprecedented etching contrasts between the irradiated and pristine areas have enabled the fabrication of hollow, high-aspect-ratio structures in the bulk of the material, overcoming the micrometer threshold as the minimum feature size. In this work, we systematically study the effect of NaOH solutions under different etching conditions (etchant concentration, temperature, and etching time) on the tracks created by tightly focused femtosecond laser pulses to assess the best practices for the fabrication of hollow nanostructures in bulk fused silica.
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- 2024
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34. Lab-on-Chip Systems for Cell Sorting: Main Features and Advantages of Inertial Focusing in Spiral Microchannels.
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Petruzzellis I, Martínez Vázquez R, Caragnano S, Gaudiuso C, Osellame R, Ancona A, and Volpe A
- Abstract
Inertial focusing-based Lab-on-Chip systems represent a promising technology for cell sorting in various applications, thanks to their alignment with the ASSURED criteria recommended by the World Health Organization: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Delivered. Inertial focusing techniques using spiral microchannels offer a rapid, portable, and easy-to-prototype solution for cell sorting. Various microfluidic devices have been investigated in the literature to understand how hydrodynamic forces influence particle focusing in spiral microchannels. This is crucial for the effective prototyping of devices that allow for high-throughput and efficient filtration of particles of different sizes. However, a clear, comprehensive, and organized overview of current research in this area is lacking. This review aims to fill this gap by offering a thorough summary of the existing literature, thereby guiding future experimentation and facilitating the selection of spiral geometries and materials for cell sorting in microchannels. To this end, we begin with a detailed theoretical introduction to the physical mechanisms underlying particle separation in spiral microfluidic channels. We also dedicate a section to the materials and prototyping techniques most commonly used for spiral microchannels, highlighting and discussing their respective advantages and disadvantages. Subsequently, we provide a critical examination of the key details of inertial focusing across various cross-sections (rectangular, trapezoidal, triangular, hybrid) in spiral devices as reported in the literature.
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- 2024
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35. Anisotropy Parameters for Two-Color Photoionization Phases in Randomly Oriented Molecules: Theory and Experiment in Methane and Deuteromethane.
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Ertel D, Busto D, Makos I, Schmoll M, Benda J, Bragheri F, Osellame R, Lindroth E, Patchkovskii S, Mašín Z, and Sansone G
- Abstract
We present combined theoretical and experimental work investigating the angle-resolved phases of the photoionization process driven by a two-color field consisting of an attosecond pulse train and an infrared pulse in an ensemble of randomly oriented molecules. We derive a general form for the two-color photoelectron (and time-delay) angular distribution valid also in the case of chiral molecules and when relative polarizations of the photons contributing to the attosecond photoelectron interferometer differ. We show a comparison between the experimental data and theoretical predictions in an ensemble of methane and deuteromethane molecules, discussing the effect of nuclear dynamics on the photoionization phases. Finally, we demonstrate that the oscillating component and the phase of the two-color signal can be fitted by using complex asymmetry parameters, in perfect analogy to the atomic case.
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- 2024
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36. In vivo label-free tissue histology through a microstructured imaging window.
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Conci C, Sironi L, Jacchetti E, Panzeri D, Inverso D, Martínez Vázquez R, Osellame R, Collini M, Cerullo G, Chirico G, and Raimondi MT
- Abstract
Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo , largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in i n vivo longitudinal studies., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)
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- 2024
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37. Methods and Protocol for Single-Cell Motility Assays under Topological Constraints.
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Ficorella C, Sala F, Martínez Vázquez R, Osellame R, and Käs JA
- Subjects
- Humans, Cell Line, Tumor, Chemotactic Factors pharmacology, Chemotactic Factors metabolism, Single-Cell Analysis methods, Cell Movement, Cell Migration Assays methods, Cell Migration Assays instrumentation
- Abstract
The extracellular environment plays a crucial role in many physiological and pathological processes involving cell motility, such as metastatic invasion in cancer development, by heavily impacting the migration strategies adopted by the cells. The study of how mechanical constraints affect the dynamics of cell migration may be relevant to gain more insight into such processes, and it may prove to be a powerful tool in the hands of biologists. In this chapter, we describe the methods used to investigate the ability of neoplastic cells to migrate through narrowing, rigid microstructures upon chemoattractant stimulation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2024
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38. Structured-light-sheet imaging in an integrated optofluidic platform.
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Paiè P, Calisesi G, Candeo A, Comi A, Sala F, Ceccarelli F, De Luigi A, Veglianese P, Muhlberger K, Fokine M, Valentini G, Osellame R, Neil M, Bassi A, and Bragheri F
- Subjects
- Humans, Microscopy, Fluorescence methods, Flow Cytometry methods, Microfluidics, Imaging, Three-Dimensional methods
- Abstract
Heterogeneity investigation at the single-cell level reveals morphological and phenotypic characteristics in cell populations. In clinical research, heterogeneity has important implications in the correct detection and interpretation of prognostic markers and in the analysis of patient-derived material. Among single-cell analysis, imaging flow cytometry allows combining information retrieved by single cell images with the throughput of fluidic platforms. Nevertheless, these techniques might fail in a comprehensive heterogeneity evaluation because of limited image resolution and bidimensional analysis. Light sheet fluorescence microscopy opened new ways to study in 3D the complexity of cellular functionality in samples ranging from single-cells to micro-tissues, with remarkably fast acquisition and low photo-toxicity. In addition, structured illumination microscopy has been applied to single-cell studies enhancing the resolution of imaging beyond the conventional diffraction limit. The combination of these techniques in a microfluidic environment, which permits automatic sample delivery and translation, would allow exhaustive investigation of cellular heterogeneity with high throughput image acquisition at high resolution. Here we propose an integrated optofluidic platform capable of performing structured light sheet imaging flow cytometry (SLS-IFC). The system encompasses a multicolor directional coupler equipped with a thermo-optic phase shifter, cylindrical lenses and a microfluidic network to generate and shift a patterned light sheet within a microchannel. The absence of moving parts allows a stable alignment and an automated fluorescence signal acquisition during the sample flow. The platform enables 3D imaging of an entire cell in about 1 s with a resolution enhancement capable of revealing sub-cellular features and sub-diffraction limit details.
- Published
- 2023
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39. Experimental certification of contextuality, coherence, and dimension in a programmable universal photonic processor.
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Giordani T, Wagner R, Esposito C, Camillini A, Hoch F, Carvacho G, Pentangelo C, Ceccarelli F, Piacentini S, Crespi A, Spagnolo N, Osellame R, Galvão EF, and Sciarrino F
- Abstract
Quantum superposition of high-dimensional states enables both computational speed-up and security in cryptographic protocols. However, the exponential complexity of tomographic processes makes certification of these properties a challenging task. In this work, we experimentally certify coherence witnesses tailored for quantum systems of increasing dimension using pairwise overlap measurements enabled by a six-mode universal photonic processor fabricated with a femtosecond laser writing technology. In particular, we show the effectiveness of the proposed coherence and dimension witnesses for qudits of dimensions up to 5. We also demonstrate advantage in a quantum interrogation task and show it is fueled by quantum contextuality. Our experimental results testify to the efficiency of this approach for the certification of quantum properties in programmable integrated photonic platforms.
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- 2023
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40. Six-telescope integrated optics beam combiner fabricated using ultrafast laser inscription for J- and H-band astronomy.
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Dinkelaker AN, Smarzyk S, Nayak AS, Piacentini S, Corrielli G, Osellame R, Pedretti E, Roth MM, and Madhav K
- Abstract
We have built and characterized, to our knowledge, the first six-telescope discrete beam combiner (DBC) for stellar interferometry in the astronomical J-band. It is the DBC with the largest number of beam combinations and was manufactured using ultrafast laser inscription in borosilicate glass, with a throughput of ≈56 % . For calibration of the visibility-to-pixel matrix, we use a two-input Michelson interferometer and extract the complex visibility. A visibility amplitude of 1.05 and relative precision of 2.9% and 3.8% are extracted for 1328 nm and 1380 nm, respectively. Broadband (≤40 n m ) characterization is affected by dispersion but shows similar performance.
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- 2023
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41. 3D photopolymerized microstructured scaffolds influence nuclear deformation, nucleo/cytoskeletal protein organization, and gene regulation in mesenchymal stem cells.
- Author
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Donnaloja F, Raimondi MT, Messa L, Barzaghini B, Carnevali F, Colombo E, Mazza D, Martinelli C, Boeri L, Rey F, Cereda C, Osellame R, Cerullo G, Carelli S, Soncini M, and Jacchetti E
- Abstract
Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications., Competing Interests: Since December 5, 2019, M.T.R., G.C., and R.O. are co-founders of MOAB S.r.l and hold stock in the company. The other authors declare no competing financial interest., (© 2023 Author(s).)
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- 2023
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42. Whole transcriptomic analysis of mesenchymal stem cells cultured in Nichoid micro-scaffolds.
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Testa C, Oliveto S, Jacchetti E, Donnaloja F, Martinelli C, Pinoli P, Osellame R, Cerullo G, Ceri S, Biffo S, and Raimondi MT
- Abstract
Mesenchymal stem cells (MSCs) are known to be ideal candidates for clinical applications where not only regenerative potential but also immunomodulation ability is fundamental. Over the last years, increasing efforts have been put into the design and fabrication of 3D synthetic niches, conceived to emulate the native tissue microenvironment and aiming at efficiently controlling the MSC phenotype in vitro . In this panorama, our group patented an engineered microstructured scaffold, called Nichoid. It is fabricated through two-photon polymerization, a technique enabling the creation of 3D structures with control of scaffold geometry at the cell level and spatial resolution beyond the diffraction limit, down to 100 nm. The Nichoid's capacity to maintain higher levels of stemness as compared to 2D substrates, with no need for adding exogenous soluble factors, has already been demonstrated in MSCs, neural precursors, and murine embryonic stem cells. In this work, we evaluated how three-dimensionality can influence the whole gene expression profile in rat MSCs. Our results show that at only 4 days from cell seeding, gene activation is affected in a significant way, since 654 genes appear to be differentially expressed (392 upregulated and 262 downregulated) between cells cultured in 3D Nichoids and in 2D controls. The functional enrichment analysis shows that differentially expressed genes are mainly enriched in pathways related to the actin cytoskeleton, extracellular matrix (ECM), and, in particular, cell adhesion molecules (CAMs), thus confirming the important role of cell morphology and adhesions in determining the MSC phenotype. In conclusion, our results suggest that the Nichoid, thanks to its exclusive architecture and 3D cell adhesion properties, is not only a useful tool for governing cell stemness but could also be a means for controlling immune-related MSC features specifically involved in cell migration., Competing Interests: MTR, RO, and GC are co-founders of a university spin-off company, MOAB srl, and hold shares. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Testa, Oliveto, Jacchetti, Donnaloja, Martinelli, Pinoli, Osellame, Cerullo, Ceri, Biffo and Raimondi.)
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- 2023
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43. On the robustness of machine learning algorithms toward microfluidic distortions for cell classification via on-chip fluorescence microscopy.
- Author
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Ahmad A, Sala F, Paiè P, Candeo A, D'Annunzio S, Zippo A, Frindel C, Osellame R, Bragheri F, Bassi A, and Rousseau D
- Subjects
- Machine Learning, Microscopy, Fluorescence, Neural Networks, Computer, Algorithms, Microfluidics
- Abstract
Single-cell imaging and sorting are critical technologies in biology and clinical applications. The power of these technologies is increased when combined with microfluidics, fluorescence markers, and machine learning. However, this quest faces several challenges. One of these is the effect of the sample flow velocity on the classification performances. Indeed, cell flow speed affects the quality of image acquisition by increasing motion blur and decreasing the number of acquired frames per sample. We investigate how these visual distortions impact the final classification task in a real-world use-case of cancer cell screening, using a microfluidic platform in combination with light sheet fluorescence microscopy. We demonstrate, by analyzing both simulated and experimental data, that it is possible to achieve high flow speed and high accuracy in single-cell classification. We prove that it is possible to overcome the 3D slice variability of the acquired 3D volumes, by relying on their 2D sum z -projection transformation, to reach an efficient real time classification with an accuracy of 99.4% using a convolutional neural network with transfer learning from simulated data. Beyond this specific use-case, we provide a web platform to generate a synthetic dataset and to investigate the effect of flow speed on cell classification for any biological samples and a large variety of fluorescence microscopes (https://www.creatis.insa-lyon.fr/site7/en/MicroVIP).
- Published
- 2022
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44. Integrated optical device for Structured Illumination Microscopy.
- Author
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Calvarese M, Paiè P, Candeo A, Calisesi G, Ceccarelli F, Valentini G, Osellame R, Gong H, Neil M, Bragheri F, and Bassi A
- Subjects
- Microscopy, Fluorescence methods, Lighting, Optical Devices
- Abstract
Structured Illumination Microscopy (SIM) is a key technology for high resolution and super-resolution imaging of biological cells and molecules. The spread of portable and easy-to-align SIM systems requires the development of novel methods to generate a light pattern and to shift it across the field of view of the microscope. Here we show a miniaturized chip that incorporates optical waveguides, splitters, and phase shifters, to generate a 2D structured illumination pattern suitable for SIM microscopy. The chip creates three point-sources, coherent and controlled in phase, without the need for further alignment. Placed in the pupil of a microscope's objective, the three sources generate a hexagonal illumination pattern on the sample, which is spatially translated thanks to thermal phase shifters. We validate and use the chip, upgrading a commercial inverted fluorescence microscope to a SIM setup and we image biological sample slides, extending the resolution of the microscope.
- Published
- 2022
- Full Text
- View/download PDF
45. Microfluidic Lab-on-a-Chip for Studies of Cell Migration under Spatial Confinement.
- Author
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Sala F, Ficorella C, Osellame R, Käs JA, and Martínez Vázquez R
- Subjects
- Cell Movement, Microfluidics, Microtechnology, Printing, Lab-On-A-Chip Devices, Microfluidic Analytical Techniques
- Abstract
Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists' analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.
- Published
- 2022
- Full Text
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46. Toward Higher Integration Density in Femtosecond-Laser-Written Programmable Photonic Circuits.
- Author
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Albiero R, Pentangelo C, Gardina M, Atzeni S, Ceccarelli F, and Osellame R
- Abstract
Programmability in femtosecond-laser-written integrated circuits is commonly achieved with the implementation of thermal phase shifters. Recent work has shown how such phase shifters display significantly reduced power dissipation and thermal crosstalk with the implementation of thermal isolation structures. However, the aforementioned phase shifter technology is based on a single gold film, which poses severe limitations on integration density and circuit complexity due to intrinsic geometrical constraints. To increase the compactness, we propose two improvements to this technology. Firstly, we fabricated thermal phase shifters with a photolithography process based on two different metal films, namely (1) chromium for microheaters and (2) copper for contact pads and interconnections. Secondly, we developed a novel curved isolation trench design that, along with a state-of-the-art curvature radius, allows for a significant reduction in the optical length of integrated circuits. As a result, curved Cr-Cu phase shifters provide a compact footprint with low parasitic series resistance and no significant increase in power dissipation (∼38 mW) and thermal crosstalk (∼20%). These results pave the way toward the fabrication of femtosecond-laser-written photonic circuits with a steep increase in terms of layout complexity.
- Published
- 2022
- Full Text
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47. Laser-written vapor cells for chip-scale atomic sensing and spectroscopy.
- Author
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Lucivero VG, Zanoni A, Corrielli G, Osellame R, and Mitchell MW
- Abstract
We report the fabrication of alkali-metal vapor cells using femtosecond laser machining. This laser-written vapor-cell (LWVC) technology allows arbitrarily-shaped 3D interior volumes and has potential for integration with photonic structures and optical components. We use non-evaporable getters both to dispense rubidium and to absorb buffer gas. This enables us to produce cells with sub-atmospheric buffer gas pressures without vacuum apparatus. We demonstrate sub-Doppler saturated absorption spectroscopy and single beam optical magnetometry with a single LWVC. The LWVC technology may find application in miniaturized atomic quantum sensors and frequency references.
- Published
- 2022
- Full Text
- View/download PDF
48. Storage and analysis of light-matter entanglement in a fiber-integrated system.
- Author
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Rakonjac JV, Corrielli G, Lago-Rivera D, Seri A, Mazzera M, Grandi S, Osellame R, and de Riedmatten H
- Abstract
The deployment of a full-fledged quantum internet poses the challenge of finding adequate building blocks for entanglement distribution between remote quantum nodes. A practical system would combine propagation in optical fibers with quantum memories for light, leveraging on the existing communication network while featuring the scalability required to extend to network sizes. Here, we demonstrate a fiber-integrated quantum memory entangled with a photon at telecommunication wavelength. The storage device is based on a fiber-pigtailed laser-written waveguide in a rare earth-doped solid and allows an all-fiber stable addressing of the memory. The analysis of the entanglement is performed using fiber-based interferometers. Our results feature orders-of-magnitude advances in terms of storage time and efficiency for integrated storage of light-matter entanglement and constitute a substantial step forward toward quantum networks using integrated devices.
- Published
- 2022
- Full Text
- View/download PDF
49. Effect of 3D Synthetic Microscaffold Nichoid on the Morphology of Cultured Hippocampal Neurons and Astrocytes.
- Author
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Musi CA, Colnaghi L, Giani A, Priori EC, Marchini G, Tironi M, Conci C, Cerullo G, Osellame R, Raimondi MT, Remuzzi A, and Borsello T
- Subjects
- Coculture Techniques, Hippocampus, Humans, Neurons metabolism, Astrocytes, Brain Diseases metabolism
- Abstract
The human brain is the most complex organ in biology. This complexity is due to the number and the intricate connections of brain cells and has so far limited the development of in vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in vitro system based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology. We investigated whether these 3D microscaffold devices can create an environment allowing the manipulation, monitoring, and functional assessment of a mixed population of brain cells in vitro. With this aim, we set up a new model of hippocampal neurons and astrocytes co-cultured in the Nichoid microscaffold to generate brain micro-tissues of 30 μm thickness. After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold, and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that within this period the different cell types had become well-differentiated. This was further elaborated with the use of drebrin, PSD-95, and synaptophysin antibodies that labeled the majority of neurons, both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to form connections than those co-cultured in a 2D condition. Our results show that the Nichoid can be used as a 3D device to investigate the structure and morphology of neurons and astrocytes in vitro. In the future, this model can be used as a tool to study brain cell interactions in the discovery of important mechanisms governing neuronal plasticity and to determine the factors that form the basis of different human brain diseases. This system may potentially be further used for drug screening in the context of various brain diseases.
- Published
- 2022
- Full Text
- View/download PDF
50. Strategies for improved temporal response of glass-based optical switches.
- Author
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Calvarese M, Paiè P, Ceccarelli F, Sala F, Bassi A, Osellame R, and Bragheri F
- Abstract
We present an optimization of the dynamics of integrated optical switches based on thermal phase shifters. These devices have been fabricated in the volume of glass substrates by femtosecond laser micromachining and are constituted by an integrated Mach-Zehnder interferometer and a superficial heater. Simulations, surface micromachining and innovative layouts allowed us to improve the temporal response of the optical switches down to a few milliseconds. In addition, taking advantage of an electrical pulse shaping approach where an optimized voltage signal is applied to the heater, we proved a switching time as low as 78 µs, about two orders of magnitude shorter with respect to the current state of the art of thermally-actuated optical switches in glass., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
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