Three-dimensional and dual-color fluorescence microscopy on a chip

In this work we present two microscopes 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 devices are 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

Integrated fast optical switch fabricated by femtosecond laser micromachining

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

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. (C) 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreemen

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)

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 [...

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

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

Femtosecond laser microfabrication of a PMMA lab on a chip for high throughput size-based inertial sorting

Inertial microfluidic particles sorting represents a critical task in many areas of biology, biotechnology, and medicine, including the isolation from blood of rare target cell populations, like e.g. circulating tumor cells (CTCs) and circulating fetal cells (CFCs). Usually, cell sorter microfluidic devices are fabricated by PDMS soft lithography, which is the most widespread micromanufacturing platform enabling to cost-effectively produce Lab-on-a-Chip with resolution in the nanometer scale. However, this technology presents some drawbacks: (i) due to PDMS softness, especially for high-pressure flows, the microfluidic behavior may change along the devices, leading to ambiguous results; (ii) soft-lithography allows to pattern structures on just one side of the chip thus limiting the affordable geometries to enhance the throughput of target particles. In this work, we develop a PMMA continuous size-based inertial microfluidic sorter by femtosecond laser microfabrication (FLM). The device design includes contracting and expanding channels (microchambers) provided with siphoning outlets on the backside of the chip. Since FLM technology is in principle applicable to any type of polymer, we chose PMMA, which is a biocompatible and transparent thermoplastic polymer much stiffer than PDMS. FLM allows machining the microfluidic network on both sides of the chip, making it possible the parallelization of the sorting process. In addition, thanks to the FLM flexibility, we easily varied the chambers number and the collecting strategy (at different flow rates) in order to define a device layout maximizing the trapping efficiency and throughput