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.
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.
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.
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.
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.
We demonstrate a way of light harvesting in integrated microfluidic chips fabricated by femtosecond laser micromachining. The architecture consists of waveguide arrays fabricated in the vicinity of the microchannel filled with a fluorescent organic solution (e.g., polyfluorene solution). Amplified spontaneous emission from the microchannel is efficiently coupled by the waveguides to the outside of the chip.