KEYWORDS: In vivo imaging, Tissues, Windows, Animal model studies, Nonlinear optics, Microlens, Optical microscopy, Histopathology, Tissue optics, Signal generators
Tissue histopathology, reliant on costly and time-consuming hematoxylin and eosin (H&E) staining of thin tissue slices, faces limitations. Label-free non-linear optical microscopy in vivo presents a solution, allowing work on fresh samples. Implantable microstructures prove effective for systematic longitudinal in vivo studies of immunological responses to biomaterials using label-free non-linear optical microscopy. Employing two-photon laser polymerization, we implanted a matrix of 3D lattices in the chorioallantoic membrane of chicken embryos, establishing a 3D reference frame for cell counting. H&E analysis is compared to label-free in vivo non-linear excitation imaging for cell quantification and identifying granulocytes, collagen, and microvessels. Preliminary results in higher animal models demonstrate the transformative potential of this approach, offering an alternative to conventional histopathology for validating biomaterials in in vivo longitudinal studies.
Femtosecond Laser Irradiation followed by Chemical Etching is exploited to create microfluidic devices for High-order Harmonic Generation (HHG) in noble gases. A finetuning of the channels’ diameter and length permits the production of high-order harmonics in completely different regimes, going from the hollow waveguiding regime to the sub-mm interaction regime. We envisage that the high adaptability of our microfluidic approach will allow us to integrate more functionalities in the same integrated device thus paving the way to palm-top HHG solutions.
Deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. In this framework we propose a miniaturized in-vivo imaging window composed of high dioptric power microlenses coupled to micro-scaffold, which were fabricated by two-photon polymerization (2PP) in the biocompatible photoresist SZ2080. We adopt a single-irradiation strategy for the fabrication of the whole structure: We first irradiate the micro-scaffold on the bottom side of the chip and afterwards the microlenses on the top. For the microlenses we adopted a hybrid approach by combining the 2PP of the micro lenses surface with a subsequent UV crosslinking of the inner volume. We explored different lenses profiles (plano-convex and parabolic) with variable parameters like diameter and focal lengths, to optimize the imaging characteristics. We envisage that these imaging windows will open the way to direct and continuous optical inspection of biological processes in vivo.
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.
Femtosecond laser irradiation followed by chemical etching in NaOH is a material processing technique capable of creating hollow channels with submicrometric cross-sections, extending up to a millimeter in length. In this study, we unveil the fabrication conditions leading to an effective nano-structuring of bulk fused silica, overcoming the limitations of traditional machining techniques in terms of both minimum achievable dimensions and high aspect ratios.
We report new methods of two-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.
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.
Since the first observation of High-order Harmonic Generation (HHG) in gas twenty years ago, the combination of intense research together with technological developments, has led to impressive progress in the field of extreme ultraviolet spectroscopy and attosecond science. Beam lines based on HHG extend over several meters and are based on instrumentation that requires careful alignment and even active stabilization systems. Therefore, a miniaturization of HHG beams will reduce the cost of these light sources and pave the way to their application in numerous new fields.
Femtosecond laser micromachining followed by chemical etching (FLICE) has already demonstrated its high potential in the fabrication of fused silica lab-on-a-chip devices; it can directly produce microfluidic networks in a 3D geometry directly buried in the glass substrate. Until now, they have been extensively used for the manipulation of fluids but they are perfectly suitable for the manipulation of gas as well.
In this work, we will demonstrate HHG in a gas filled microchannel network fabricated by the FLICE technique. The device structure will be based on hollow waveguides: several inlets will deliver the gas into a central hollow waveguide where the ultrafast laser will be coupled and HHG will take place. The high versatility of the FLICE technique will allow us to fabricate devices with modulated gas concentration and waveguide profile to achieve quasi-phase-matching conditions. Moreover, we will also demonstrate an integrated filtering chip that will allow to geometrically separate the main laser radiation from the XUV generated beam.
In this century of continuous exponential growth of communications worldwide, traditional electrical interconnection is finding increasingly difficult to respond to the bandwidth pressure, and photonic interconnection will most likely be the future standard.
Planar lightwave circuit (PLC) technology is capable of high-throughput fabrication of low loss waveguides, but is in general limited to its 2D geometry.
On the other hand femtosecond direct writing (FDW) provides a solid tool for the fabrication of optical circuits with great flexibility, exploiting its truly 3D properties, but suffers from higher losses and lower throughput.
By combining with PLC technology, FDW could aid in the bridging of different layers of optical circuits, exponentially decreasing their footprint. We report in this work the fabrication of such optical vias.
The fabrication of vertical waveguides in fused silica, using a IR femtosecond fiber laser, with parameters optimised to induce the previously reported micro-explosions mechanism inside fused silica. By using a
long working distance water immersion objective, we reduced spherical aberrations due to a better phase matching with the glass. A helix path was applied to create a cone of damaged material, leaving a stress-induced central waveguide, with propagation losses lower than 1 dB/mm.
Finally, we analyse the possibility of tilting these waveguides and its effect on their optical properties. This feature adds to the flexibility of this method, that could for example accommodate input/output angles of common coupling strategies used with PLC technologies.
Within the European Project TERABOARD, a photonic integration platforms including electronic-photonic integration is developed to demonstrate high bandwidth high-density modules and to demonstrate cost and energy cost target objectives. Large count high bandwidth density EO interfaces for onboard and intra-data center interconnection are reported. For onboard large count interconnections a novel concept based on optical-TSV interconnection platform with no intersections and no WDM multiplexing is reported. All input/output coupler arrays based on a pluggable silica platform are reported as well.
We present a cost-effective and highly-portable plastic prototype that can be interfaced with a cell phone to implement an optofluidic imaging cytometry platform. It is based on a PMMA microfluidic chip that fits inside an opto-mechanical platform fabricated by a 3D printer. The fluorescence excitation and imaging is performed using the LED and the CMOS from the cell phone increasing the compactness of the system. A custom developed application is used to analyze the images and provide a value of particle concentration.
Microfluidic optical stretchers are valuable optofluidic devices for studying single cell mechanical properties. These usually consist of a single microfluidic channel where cells, with dimensions ranging from 5 to 20 μm are trapped and manipulated through optical forces induced by two counter-propagating laser beams. Recently, monolithic optical stretchers have been directly fabricated in fused silica by femtosecond laser micromachining (FLM). Such a technology allows writing in a single step in the substrate volume both the microfluidic channel and the optical waveguides with a high degree of precision and flexibility. However, this method is very slow and cannot be applied to cheaper materials like polymers. Therefore, novel technological platforms are needed to boost the production of such devices on a mass scale.
In this work, we propose integration of FLM with micro-injection moulding (μIM) as a novel route towards the cost-effective and flexible manufacturing of polymeric Lab-on-a-Chip (LOC) devices. In particular, we have fabricated and assembled a polymethylmethacrylate (PMMA) microfluidic optical stretcher by exploiting firstly FLM to manufacture a metallic mould prototype with reconfigurable inserts. Afterwards, such mould was employed for the production, through μIM, of the two PMMA thin plates composing the device. The microchannel with reservoirs and lodgings for the optical fibers delivering the laser radiation for cell trapping were reproduced on one plate, while the other included access holes to the channel. The device was assembled by direct fs-laser welding, ensuring sealing of the channel and avoiding thermal deformation and/or contamination.
We report the fabrication and validation of a microfluidic chip for fluorescence detection, which incorporates in the same glass substrate the microfluidic network, the excitation, the filtering, and the collection elements. The device is fabricated in a hybrid approach combining different technologies, such as femtosecond laser micromachining and RF sputtering, to increase their individual capabilities. The validation of the chip demonstrates a good wavelength selective light filtering and a limit of detection of a 600-nM concentration of Oxazine 720 perchlorate dye.
Femtosecond-pulsed laser welding of transparent materials on a micrometer scale is a versatile tool for the fabrication and assembly of electronic, electromechanical, and especially biomedical micro-devices. In this paper, we report on microwelding of two transparent layers of polymethyl methacrylate (PMMA) with femtosecond laser pulses at 1030 nm in the MHz regime. We aim at exploiting localized heat accumulation to weld the two layers without any preprocessing of the sample and any intermediate absorbing media, by focusing fs-laser pulses at the interface.
The modifications produced by the focused laser beam into the bulk material have been firstly investigated depending on the laser process parameters aiming to produce continuous melting. Results have been evaluated based on heat accumulation models. Finally, fs-laser welding of PMMA samples have been successfully demonstrated and tested by leakage tests for application in direct laser assembly of microfluidic devices.
Optical stretching is a powerful technique for the mechanical phenotyping of single suspended cells that exploits cell deformability as an inherent functional marker. Dual-beam optical trapping and stretching of cells is a recognized tool to investigate their viscoelastic properties. The optical stretcher has the ability to deform cells through optical forces without physical contact or bead attachment. In addition, it is the only method that can be combined with microfluidic delivery, allowing for the serial, high-throughput measurement of the optical deformability and the selective sorting of single specific cells. Femtosecond laser micromachining can fabricate in the same chip both the microfluidic channel and the optical waveguides, producing a monolithic device with a very precise alignment between the components and very low sensitivity to external perturbations. Femtosecond laser irradiation in a fused silica chip followed by chemical etching in hydrofluoric acid has been used to fabricate the microfluidic channels where the cells move by pressure-driven flow. With the same femtosecond laser source two optical waveguides, orthogonal to the microfluidic channel and opposing each other, have been written inside the chip. Here we present an optimized writing process that provides improved wall roughness of the micro-channels allowing high-quality imaging. In addition, we will show results on cell sorting on the basis of mechanical properties in the same device: the different deformability exhibited by metastatic and tumorigenic cells has been exploited to obtain a metastasis-cells enriched sample. The enrichment is verified by exploiting, after cells collection, fluorescence microscopy.
Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.
We report the fabrication of micro-Fresnel lenses by femtosecond laser surface ablation on one-dimensional (1-D) polymer photonic crystals. This device is designed to focus the transmitted wavelength (520 nm) of the photonic crystal and filter the wavelengths corresponding to the photonic band-gap region (centered at 630 nm, ranging from 530 to 700 nm). Integration of such devices in a wavelength selective light harvesting and filtering microchip is envisaged.
We have fabricated entirely by femtosecond micromachining a plastic optofluidic chip with integrated microfluidics and
optical excitation/detection. First a microfluidic channel and two fiber grooves were ablated on one surface of the
PMMA substrate. In order to collect and focus the fluorescence signal onto a detector, two binary Fresnel lenses were
micromachined on the back surface of the substrate. The operatio of the integrated optofluidic chip was demonstrated by
filling the channel with different Rhodamine 6G solution, and a limit of detection of 50 nM was achieved.
We report on the use of femtosecond laser pulses to fabricate photonic devices (waveguides and interferometers) inside
commercial CE chips without affecting the manufacturing procedure of the microfluidic part of the device. The
fabrication of single waveguides intersecting the channels allows one to perform absorption or Laser Induced
Fluorescence (LIF) sensing of the molecules separated inside the microchannels. Microfluidic channels, with access
holes, are fabricated using femtosecond laser irradiation followed by chemical etching. Mach-Zehnder interferometers
are used for label-free sensing of the samples flowing in the microfluidic channels by means of refractive index changes
detection.
A lab-on-a-chip (LOC) is a device that incorporates in a single substrate the functionalities of a biological laboratory, i.e.
a network of fluidic channels, reservoirs, valves, pumps and sensors, all with micrometer dimensions. Its main
advantages are the possibility of working with small samples quantities (from nano- to picoliters), high sensitivity, speed
of analysis and the possibility of measurement automation and standardization. They are becoming the most powerful
tools of analytical chemistry with a broad application in life sciences, biotechnology and drug development. The next
technological challenge of LOCs is direct on-chip integration of photonic functionalities for sensing of biomolecules
flowing in the microchannels. Ultrafast laser processing of the bulk of a dielectric material is a very flexible and simple
method to produce photonic devices inside microfluidic chips for capillary electrophoresis (CE) or chemical
microreactors. By taking advantage of the unique three-dimensional capabilities of this fabrication technique, more
complex functionalities, such as splitters or Mach-Zehnder interferometers, can be implemented. In this work we report
on the use of femtosecond laser pulses to fabricate photonic devices (as waveguides, splitters and interferometers) inside
commercial CE chips, without affecting the manufacturing procedure of the microfluidic part of the device. The
fabrication of single waveguides intersecting the channels allows one to perform absorption or Laser Induced
Fluorescence (LIF) sensing of the molecules separated inside the microchannels. Waveguide splitters are used for
multipoint excitation of the microfluidic channel for parallel or higher sensitivity measurements. Finally, Mach-Zehnder
interferometers are used for label-free sensing of the samples flowing in the microfluidic channels by means of refractive
index changes detection.
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