Integrated optical device for Structured Illumination Microscopy.

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.

Yield stress "in a flash": investigation of nonlinearity and yielding in soft materials with an optofluidic microrheometer.

Yield stress materials deform as elastic solids or flow as viscous liquids, depending on the applied stress, which also allows them to trap particles below a certain size or density threshold. To investigate the conditions for such a transition at the microscale, we use an optofluidic microrheometer, based on the scattering of an infrared beam onto a microbead, which reaches forces in the nN scale. We perform creep experiments on a model soft material composed of swollen microgels, determining the limits of linear response and yield stress values, and observe quantitative agreement with bulk measurements. However, the motion of the microbead, both below and above yielding, reflects distinctive microscale features of the surrounding material, whose plastic rearrangements were investigated by us using small, passive tracers.

Microfluidic Based Optical Microscopes on Chip.

Last decade's advancements in optofluidics allowed obtaining an ever increasing integration of different functionalities in lab on chip devices to culture, analyze, and manipulate single cells and entire biological specimens. Despite the importance of optical imaging for biological sample monitoring in microfluidics, imaging is traditionally achieved by placing microfluidics channels in standard bench-top optical microscopes. Recently, the development of either integrated optical elements or lensless imaging methods allowed optical imaging techniques to be implemented in lab on chip systems, thus increasing their automation, compactness, and portability. In this review, we discuss known solutions to implement microscopes on chip that exploit different optical methods such as bright-field, phase contrast, holographic, and fluorescence microscopy.

Optofluidic light modulator integrated in lab-on-a-chip.

Microfluidic lenses are relevant optical components for sensing application in lab-on-a-chip devices, guaranteeing a robust alignment of the elements, a high level of compactness and tunable optical properties. In this work we describe an innovative integrated in-plane microfluidic lens. The device shows both an optimized shape capable of reducing spherical aberrations and periodically tunable optical properties. Indeed through the combination of the lens with a droplet generator module, we have been able to obtain an integrated optofluidic modulator capable of both on-demand on/off switching and periodic modulation of light. The device possesses a simple 3D geometry, which has been realized by exploiting the 3D capability of the femtosecond laser micromachining fabrication technique.

Structured-light-sheet imaging in an integrated optofluidic platform.

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.

Editorial for the Special Issue on New Trends and Applications in Femtosecond Laser Micromachining.

Femtosecond laser micromachining is becoming an established fabrication technique for transparent material processing in three dimensions [...].

Strategies for improved temporal response of glass-based optical switches.

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.

On the robustness of machine learning algorithms toward microfluidic distortions for cell classification via on-chip fluorescence microscopy.

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).

Effects of Thermal Annealing on Femtosecond Laser Micromachined Glass Surfaces.

Femtosecond laser micromachining (FLM) of fused silica allows for the realization of three-dimensional embedded optical elements and microchannels with micrometric feature size. The performances of these components are strongly affected by the machined surface quality and residual roughness. The polishing of 3D buried structures in glass was demonstrated using different thermal annealing processes, but precise control of the residual roughness obtained with this technique is still missing. In this work, we investigate how the FLM irradiation parameters affect surface roughness and we characterize the improvement of surface quality after thermal annealing. As a result, we achieved a strong roughness reduction, from an average value of 49 nm down to 19 nm. As a proof of concept, we studied the imaging performances of embedded mirrors before and after thermal polishing, showing the capacity to preserve a minimum feature size of the reflected image lower than µ5µm. These results allow for us to push forward the capabilities of this enabling fabrication technology, and they can be used as a starting point to improve the performances of more complex optical elements, such as hollow waveguides or micro-lenses.

Automatic imaging of Drosophila embryos with light sheet fluorescence microscopy on chip.

We present a microscope on chip for automated imaging of Drosophila embryos by light sheet fluorescence microscopy. This integrated device, constituted by both optical and microfluidic components, allows the automatic acquisition of a 3D stack of images for specimens diluted in a liquid suspension. The device has been fully optimized to address the challenges related to the specimens under investigation. Indeed, the thickness and the high ellipticity of Drosophila embryos can degrade the image quality. In this regard, optical and fluidic optimization has been carried out to implement dual-sided illumination and automatic sample orientation. In addition, we highlight the dual color investigation capabilities of this device, by processing two sample populations encoding different fluorescent proteins. This work was made possible by the versatility of the used fabrication technique, femtosecond laser micromachining, which allows straightforward fabrication of both optical and fluidic components in glass substrates.

High-throughput 3D imaging of single cells with light-sheet fluorescence microscopy on chip.

Single-cell analysis techniques are fundamental to study the heterogeneity of cellular populations, which is the basis to understand several biomedical mechanisms. Light-sheet fluorescence microscopy is a powerful technique for obtaining high-resolution imaging of individual cells, but the complexity of the setup and the sample mounting procedures limit its overall throughput. In our work, we present an optofluidic microscope-on-chip with integrated microlenses for light-sheet shaping and with a fluidic microchannel that allows the automatic and continuous delivery of samples of a few tens of microns in size. The device is used to perform dual-color fluorescence analysis and 3D reconstruction of xenograft-derived mouse breast cancer cells.

Particle Manipulation by Optical Forces in Microfluidic Devices.

Since the pioneering work of Ashkin and coworkers, back in 1970, optical manipulation gained an increasing interest among the scientific community. Indeed, the advantages and the possibilities of this technique are unsubtle, allowing for the manipulation of small particles with a broad spectrum of dimensions (nanometers to micrometers size), with no physical contact and without affecting the sample viability. Thus, optical manipulation rapidly found a large set of applications in different fields, such as cell biology, biophysics, and genetics. Moreover, large benefits followed the combination of optical manipulation and microfluidic channels, adding to optical manipulation the advantages of microfluidics, such as a continuous sample replacement and therefore high throughput and automatic sample processing. In this work, we will discuss the state of the art of these optofluidic devices, where optical manipulation is used in combination with microfluidic devices. We will distinguish on the optical method implemented and three main categories will be presented and explored: (i) a single highly focused beam used to manipulate the sample, (ii) one or more diverging beams imping on the sample, or (iii) evanescent wave based manipulation.

Particle focusing by 3D inertial microfluidics.

Three-dimensional (3D) particle focusing in microfluidics is a fundamental capability with a wide range of applications, such as on-chip flow cytometry, where high-throughput analysis at the single-cell level is performed. Currently, 3D focusing is achieved mainly in devices with complex layouts, additional sheath fluids, and complex pumping systems. In this work, we present a compact microfluidic device capable of 3D particle focusing at high flow rates and with a small footprint, without the requirement of external fields or lateral sheath flows, but using only a single-inlet, single-outlet microfluidic sequence of straight channels and tightly curving vertical loops. This device exploits inertial fluidic effects that occur in a laminar regime at sufficiently high flow rates, manipulating the particle positions by the combination of inertial lift forces and Dean drag forces. The device is fabricated by femtosecond laser irradiation followed by chemical etching, which is a simple two-step process enabling the creation of 3D microfluidic networks in fused silica glass substrates. The use of tightly curving three-dimensional microfluidic loops produces strong Dean drag forces along the whole loop but also induces an asymmetric Dean flow decay in the subsequent straight channel, thus producing rapid cross-sectional mixing flows that assist with 3D particle focusing. The use of out-of-plane loops favors a compact parallelization of multiple focusing channels, allowing one to process large amounts of samples. In addition, the low fluidic resistance of the channel network is compatible with vacuum driven flows. The resulting device is quite interesting for high-throughput on-chip flow cytometry.

Selective plane illumination microscopy on a chip.

Selective plane illumination microscopy can image biological samples at a high spatiotemporal resolution. Complex sample preparation and system alignment normally limit the throughput of the method. Using femtosecond laser micromachining, we created an integrated optofluidic device that allows obtaining continuous flow imaging, three-dimensional reconstruction and high-throughput analysis of large multicellular spheroids at a subcellular resolution.

Research highlights: surface-based microfluidic control.

Microfluidic systems are often dominated by their surfaces because of the high surface area to volume ratios in microchannel flows or drop-based systems. Here we highlight recent work on engineering and exploiting surface effects to control the formation and motion of microdrops. We highlight work using precisely microstructured wetting surfaces to repel all manner of liquids even when the liquid-air surface tension is low. In a second paper, selective capillary filling and draining is used to pattern liquid and cell-laden gels for 3D culture. A final paper making use of vapor-driven surface tension effects to drive the motion of drop ensembles is also examined, exploring a new mechanism for drop control - including motion and merging. Surface-driven motion and patterning has been a widely successful area in microfluidics (e.g. electrowetting or patterned self-assembled monolayers) and recent work is extending into new directions that, once well-understood, should enable new applications.

Straightforward 3D hydrodynamic focusing in femtosecond laser fabricated microfluidic channels.

We report on the use of femtosecond laser irradiation followed by chemical etching as a microfabrication tool for innovative microfluidic networks that implement hydrodynamic focusing. The capability of our microfabrication technology to interconnect microchannels in three dimensions was exploited to demonstrate 2D hydrodynamic focusing, either in the horizontal or in the vertical plane, and full 3D hydrodynamic focusing. In all cases only two inlets were required, one for the sample and one for the sheath flows. Fluidic characterization of all devices was provided. In addition, taking advantage of the possibility to write optical waveguides using the same technology, a monolithic cell counter based on 3D hydrodynamic focusing and integrated optical detection was validated. Counting rates up to 5000 cells s(-1) were achieved in this very compact device, where focusing and counting operations were implemented in less than 1 mm(3). Integration of this hydrodynamic focusing module into several devices fabricated by the same technology as optical cell stretchers and cell sorters is envisaged.