Structured Illumination microscopy is a super-resolution imaging technique based on sample fluorescence excitation with a spatially modulated light pattern. The pattern properties as well as the capability to shift it over sample determine the quality of the final images. At the current state of the art, pattern generation and translation require bulky and non-trivial optical setups. Here we propose an integrated optical device for the versatile generation and translation of the light pattern. This device can be used as light source for a standard microscope, upgrading it to a super-resolution system.
Heterogeneity plays an important role in medicine and biology, which can be investigated by exploiting single cell analysis (SCA). Among SCA methods, imaging cytometry allows the analysis of individual 2D and 3D spatial features. Here we present a femtosecond laser fabricated optofluidic automated platform encompassing a thermo-optic phase shifter, cylindrical lenses and a microfluidic network to generate and shift a dual-color patterned light sheet within a microchannel where the samples of interest flow. The device can be used as add-on and can provide an acquisition rate of about 1 cell/second, or subnuclear resolution at the single cell level.
Integrated optical switches and modulators allow performing reconfigurability in integrated circuits, resulting as fundamental components in different fields ranging from optical communications to sensing and metrology. Among different methods, the thermo-optic effect has been successfully used to fabricate optical modulators by femtosecond laser micromachining (FLM) in glass substrates, proving high stability, no losses dependance but long switching time. In this work, we present an integrated optical switch realized by FLM with a switching time of less than 1 ms: which is about 1 order of magnitude faster than the other devices present in literature. This result has been achieved by carefully optimizing the geometry and the position of resistors and trenches near the waveguides through simulation and experimental validation. In addition, by means of an optimization of the applied voltage signal, we have demonstrated a further significant temporal improvement, measuring a switching time of less than 100 μs.
In this work we present a microscope on chip based on Light Sheet Fluorescence Microscopy, capable to automatically perform 3D and dual-color imaging of specimens diluted in a liquid suspension. A microfluidic channel is used for automatic sample delivery, while integrated optical components such as optical waveguides and lenses are used to illuminate the sample flowing in the channel. The device is fabricated by femtosecond laser micromachining in a glass substrate. Benefiting from the versatility of the fabrication technique we present two prototypes that have been optimized for different samples such as single cells and Drosophila embryos.
Femtosecond laser irradiation followed by chemical etching (FLICE), combined with femtosecond-laser waveguide writing, has allowed to demonstrate, in the past years, a variety of compact optofluidic devices in fused silica substrate. Applications are diverse and range from biochemical sensing to single-cell manipulation. On the other hand, femtosecond-laser written circuits with high complexity, and waveguides with very low insertion loss, have been reported in Eagle alumino-borosilicate glass (Corning). Here we show that FLICE can be applied to produce buried microchannels also in EagleXG substrate. Our results will enable quick fabrication of microfluidic devices, directly interfaced with complex and low-loss photonic circuits.
Lab on a Chip devices are compact and portable chips mainly constituted by a network of microfluidic channels. They aim at substituting bulk laboratory instrumentations, with the advantages of increasing the automation and the sensitivity of the analysis, reducing the costs and opening the possibility of performing measurements at the Point of Care. Among different Lab on Chips, optofluidic ones have the advantages of optical investigation, but the integration of optical and microfluidic components in a single substrate is very challenging from a technological point of view. A recent fabrication technique, known as femtosecond laser micromachining (FLM), has proven to be ideal for the realization of these devices, allowing the fabrication of the whole device in a single irradiation step. Here, we will present a platelet counter and a microscope on chip, that fully take advantage from the versatility of FLM. To succeed in these works a fundamental aspect to address is the capability to control the sample positioning in the microfluidic channel. A single particle per time should pass in the detection region to avoid the overlooking of specimen. Moreover, a precise control of the sample orientation and position in the channel cross section is needed for imaging. The 3D capabilities of FLM have been fundamental in the realization of advanced fluidic layouts capable of sample manipulation with no need of any additional external field. We have successfully proven red blood cells and platelets counting, as well as single cells, cellular spheroids and drosophila embryos 3D imaging.
Drosophila Melanogaster is a sample of high biological interest that is being widely used as biological model, due to the relatively short life cycle, short genome and ease in culturing. In this work we present a microscope on chip capable of processing Drosophila embryos to obtain three dimensional fluorescent images at high throughput. This device, based on light sheet microscopy, uses a plane of light intercepting the sample channel to optically and noninvasively section the embryos while flowing. This permits to automatically acquire for each sample the stack of images necessary for the subsequent 3D reconstruction with no need of any manual sample positioning and alignment. The whole chip is fabricated in a glass substrate by femtosecond laser micromachining. The device has been optimized for the specific morphology of the sample. Indeed, the highly elliptical shape of the embryos (about 100 x 500 μm2) might affect the image quality degrading both the vertical and the axial resolution of the system. To overcome this issue, we have first optimized the layout of the fluidic channel to precisely control the sample orientation by means of hydrodynamic forces. Thereafter, we have optimized the properties of the optical circuit, to realize two opposite light sheets impinging on the sample, perfectly overlapped, with a high signal to noise ratio. With these actions, we have been able to obtain high quality Drosophila reconstruction.
Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system.
We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids.
This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.
Particle focusing is an important functionality useful in a wide set of biological applications. Nevertheless, it is still challenging to realize it in microfluidics, especially in a low pressure system, because of the intrinsic 2D nature of standard microfluidic devices; long channels or complicated device geometries with several lateral channels are usually needed to avoid this limitation. In this work we present the fabrication and optimization of a compact microfluidic chip, which is capable to perform 3D particle focusing at high flow rates, thanks to the superposition of inertial focusing and intense Dean flow in tightly curving 3D channels. The device layout comprises alternating helices and straight channel sections permitting particle focusing with driving pressures < 1 bar due to the compactness of this chip. Beads characterization is performed, demonstrating the possibility of the chip to effectively focus 15 μm size sample using a single inlet and with no need of additional lateral channels that could complicate the sample processing procedure. Femtosecond laser micromachining followed by chemical etching is used to fabricate the device. This technique is a two-step process that permits fabrication of 3D structures in fused silica substrates and it is a fundamental tool to obtain 3D helices in the substrate. A surface fabrication approach has been used to avoid tapered channels. We envisage the use of this chip for high speed flow cytometer applications.
Microfluidic lenses are a powerful tool for many lab on a chip applications ranging from sensing to detection and also to
imaging purpose, with the great advantage to increase the degree of integration and compactness of these micro devices.
In this work we present the realization of such a compact microfluidic lens with reconfigurable optical properties.
The technique used to realize the device we present is femtosecond laser micromachining followed by chemical etching,
which allows to easily fabricate 3D microfluidic devices with an arbitrary shape. Thanks to that it has been possible to
easily fabricate different lens made up by cylindrical microchannel in fused silica glasses filled with liquids with a proper
refractive index. The optical properties of these devices are tested and shown to be in a good agreement with the
theoretical model previously implemented. Furthermore we have also optimized the design of these microlenses in order
to reduce the effects of spherical aberrations in the focal region, thus allowing us to obtain a set of different acylindrical
microfluidic lenses, whose validation is also reported.
In this work the lens adaptability can be achieved by replacing the liquid inside the microchannel, so that we can easily
tune the feature of the focused beam. Thus increasing the possible range of applications of these micro optical elements,
as an example we report on the validation of the device as a fast integrated optofluidic shutter.
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.
Hydrodynamic focusing is a powerful technique frequently used in microfluidics that presents a wide range of applications since it allows focusing the sample flowing in the device to a narrow region in the center of the microchannel. In fact thanks to the laminarity of the fluxes in microchannels it is possible to confine the sample solution with a low flow rate by using a sheath flow with a higher flow rate. This in turn allows the flowing of one sample element at a time in the detection region, thus enabling analysis on single particles. Femtosecond laser micromachining is ideally suited to fabricate device integrating full hydrodynamic focusing functionalities thanks to the intrinsic 3D nature of this technique, especially if compared to expensive and complicated lithographic multi-step fabrication processes. Furthermore, because of the possibility to fabricate optical waveguides with the same technology, it is possible to obtain compact optofluidic devices to perform optical analysis of the sample even at the single cell level, as is the case for optical cell stretchers and sorters. In this work we show the fabrication and the fluidic characterization of extremely compact devices having only two inlets for 2D (both in vertical and horizontal planes) as well as full 3D symmetric hydrodynamic focusing. In addition we prove one of the possible application of the hydrodynamic focusing module, by fabricating and validating (both with polystyrene beads and erythrocytes) a monolithic cell counter obtained by integrating optical waveguides in the 3D hydrodynamic focusing device.
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.
The objective of this study was to examine the in vitro effect of a single or a multiple doses of low-level
laser irradiation (LLLI) on proliferation of the human osteosarcoma cell line, SAOS-2. SAOS-2 cells were
divided in five groups and exposed to LLLI (659 nm diode laser; 11 mW power output): group I as a
control (dark), group II exposed to a single laser dose of 1 J/cm2, group III irradiated with a single dose of
3 J/cm2, and group IV and V exposed for three consecutive days to 1 or 3 J/cm², respectively. Cellular
proliferation was assessed daily up to 7 days of culturing. The obtained results showed an increase in
proliferative capacity of SAOS-2 cells during the first 96 h of culturing time in once-irradiated cells, as
compared to control cells. Furthermore, a significantly higher proliferation in the group IV and V was
detected if compared to a single dose or to control group after 96 h and 7 days. In conclusion, the effect of
the single dose on cell proliferation was transitory and repeated irradiations were necessary to observe a
strong enhancement of SAOS-2 growth. As a future perspective, we would like to determine the potential
of LLLI as a new approach for promoting bone regeneration onto biomaterials.
We present design and optimization of an optofluidic monolithic chip, able to provide optical trapping and controlled
stretching of single cells. The chip is fabricated in a fused silica glass substrate by femtosecond laser micromachining,
which can produce both optical waveguides and microfluidic channels with great accuracy.
Versatility and three-dimensional capabilities of this fabrication technology provide the possibility to fabricate circular
cross-section channels with enlarged access holes for an easy connection with an external fluidic circuit. Moreover, a
new fabrication procedure adopted allows the demonstration of microchannels with a square cross-section, thus
guaranteeing an improved quality of the trapped cell images.
Optical trapping and stretching of single red blood cells are demonstrated, thus proving the effectiveness of the proposed
device as a monolithic optical stretcher.
We believe that femtosecond laser micromachining represents a promising technique for the development of
multifunctional integrated biophotonic devices that can be easily coupled to a microscope platform, thus enabling a
complete characterization of the cells under test.
Staphylococci are important causes of nosocomial and
medical-device-related infections. Their virulence is attributed to the elaboration of biofilms that protect the organisms from immune system clearance and to increased resistance to phagocytosis and antibiotics. Photodynamic treatment (PDT) has been proposed as an alternative approach for the inactivation of bacteria in biofilms. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against
Staphylococcus epidermidis biofilms. We evaluated the effect of the combined photodynamic action of MC 540 and 532 nm laser on the viability and structure of biofilms of two Staphylococcus epidermidis strains. Significant inactivation of cells was observed in the biofilms treated with MC-540 and then exposed to laser radiation. Furthermore we found that the PDT effect, on both types of cells, was significantly dependent on both the light-dose and on the impinging lightintensity.
Disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).
Monolithic Semiconductor Ring Lasers (SRLs) are promising devices for all-optical memory and all-optical switching
applications, as they can operate in a directional bistable regime where only one directional mode (clockwise or anti-clockwise)
is active at one time. The unidirectional bistable regime can be naturally associated to a binary logic, and the
SRL represents an elementary digital memory cell that can be written all-optically, realising the function of an all-optical
flip-flop. In fact, the direction of operation can be switched by injecting an external optical signal pulse into the SRL
through one of the 4 input/output ports.
Directional switching of the SRL-based all-optical flip-flop has been demonstrated by injecting optical pulses with 5 ps
duration into one of the four input/output ports. The required switching energy is around 100 fJ, and the swiching time is
between 100 and 200 ps. The same function has been demonstrated by injecting 400 ps pulses as optical trigger.
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