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

Viscoelasticity measurements by an optofluidic micro-rheometer

During the last decades, microrheology attracted a significant attention thanks to the possibility of investigating the viscoelastic properties of complex systems (e.g. cells and soft materials) at micrometer scale. The inherent low-consumption of sample offered by microrheology makes it the ideal candidate to study the rheological properties of precious/limited materials. In active microrheology, optical or magnetic forces enable trapping and manipulation of micro-probes in the fluid under test. The probe's response to external stimuli is used to derive the rheological properties of the surrounding medium. While this approach has been already reported in the scientific literature mainly using optical tweezers [1], in this document we propose a different system configuration based on a dual beam laser trap, previously exploited to realize a simple viscometer [2,3]. The here proposed device has all the features of a rheometer, also allowing to measure the elastic properties, and has the advantage of requiring a lower beam intensity while being able to apply larger forces with respect to standard optical tweezers. Additionally the system can be easily integrated in a glass substrate, requiring just an external connection to a CW-laser source and a low-magnification objective for sample observation

Spatiotemporal Amplitude and Phase Retrieval of Bessel-X pulses using a Hartmann-Shack Sensor

We propose a new experimental technique, which allows for a complete characterization of ultrashort optical pulses both in space and in time. Combining the well-known Frequency-Resolved-Optical-Gating technique for the retrieval of the temporal profile of the pulse with a measurement of the near-field made with an Hartmann-Shack sensor, we are able to retrieve the spatiotemporal amplitude and phase profile of a Bessel-X pulse. By following the pulse evolution along the propagation direction we highlight the superluminal propagation of the pulse peak

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)

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

Conical-emission and shock-front dynamics in femtosecond laser-pulse filamentation

We investigate both experimentally and numerically the space-time dynamics of an ultrashort laser pulse during self-focusing and nonlinear propagation in water by means of a time-gated angular-spectrum characterization. The results identify the formation of shock fronts on both trailing and leading edges of the wave packet that are due to the formation of subluminal and superluminal group velocity intensity peaks, sustained by conical emission

Integrated optofluidic chip for low-volume fluid viscosity measurement

In the present work, an integrated optofluidic chip for fluid viscosity measurements in the range from 1 mPas to 100 mPas is proposed. The device allows the use of small sample volumes (<1 mu L) and the measurement of viscosity as a function of temperature. Thanks to the precise control of the force exerted on dielectric spheres by optical beams, the viscosity of fluids is assessed by comparing the experimentally observed movement of dielectric beads produced by the optical forces with that expected by numerical calculations. The chip and the developed technique are validated by analyzing several fluids, such as Milli-Q water, ethanol and water-glycerol mixtures. The results show a good agreement between the experimental values and those reported in the literature. The extremely reduced volume of the sample required and the high flexibility of this technique make it a good candidate for measuring a wide range of viscosity values as well as for the analysis of nonlinear viscosity in complex fluids

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 [...