Conference Agenda

Single-cell analysis without immune-specific labelling is essential across research fields, but conventional flow cytometers (FCMs) struggle with label-free analysis. We introduce a novel microfluidic scanning flow cytometer (µSFC) designed for label-free analysis within a simple microfluidic chip. Our system outperforms traditional FCMs for label-free analysis but its signal-to-noise ratio (SNR) limits the minimum detectable size. We present three modifications to enhance SNR and improve the smallest detectable particle size: additional neutral optical density filtering, a lower noise-equivalent-power photoreceiver, and laser spot size reduction. These improvements enable reliable characterization of particles as small as 3 µm. Experimental results validate the correlation between angular profile oscillations and particle size. While reliable detection down to 1 µm is achieved, further refinement is needed. The simplicity and low setup of the µSFC make it promising for integration into multi-parametric single-cell analysis systems, facilitating comprehensive cellular characterization for diagnostic and point-of-care applications.

Imaging flow cytometry (IFC) integrates flow cytometry with optical microscopy, enabling high-throughput, multi-parameter analysis of single cells. Current 3D IFC systems face limitations related to instrumental complexity that might lead to optical misalignment or mechanical instabilities in day-by-day operation. We propose a fully integrated optofluidic platform combining reconfigurable photonic circuits and cylindrical hollow lenses for structured light sheet microscopy in a microfluidic channel. The components are fabricated using femtosecond laser irradiation and chemical etching, ensuring a high level of integration that allows durable alignment and mechanical stability.

Statistical analysis of properties of single microparticles, such as cells, bacteria or plastic slivers, has attracted increasing interest in recent years. In this field flow cytometry is considered the gold standard technique, but commercially available instruments are bulky, expensive, and not suitable for use in Point-of-Care (PoC) testing. Microfluidic flow cytometers, on the other hand, are small, cheap and can be used for on-site analysis. However, in order to detect small particles, they require complex geometries and the aid of external optical components. To overcome these limitations here we present an opto-fluidic flow cytometer with an integrated 3D in-plane spherical mirror for enhanced optical signal collection. As result the signal-to-noise ratio is increased by a factor of 6, enabling the detection of particle sizes down to 1.5µm. The proposed optofluidic detection scheme allows the simultaneous collection of particles fluorescence and scattering - using a single optical fiber - which is crucial to easily distinguish particle populations with different optical properties. The devices have been fully characterized using fluorescent polystyrene beads of different sizes. As a proof of concept for potential real-world applications, signals from fluorescent HEK cells and Escherichia coli bacteria were analyzed.

This project aims to produce a microfluidic device capable of separating 6 µm and 20 µm diameters particles by inertial sorting. This Lab-on-Chip (LoC) was designed with a trapezoidal cross-section for better fluid control and effective particle manipulation at the microscopic level, as demonstrated by COMSOL simulations. The device was manufactured on a substrate of Polymethyl Methacrylate (PMMA) by femtosecond laser technology and then assembled using an innovative geometry-preserving Isopropyl alcohol-based procedure. The LoC was test with spherical plastic microparticles of two diameters (6 µm and 20 µm) suspended in distilled water. The separation efficiencies were (98.2 ± 1.6) % for 20 µm diameter particles and (70.0 ± 1.8) % for 6 µm diameter particles in good agreement with the simulation results. Finally, after a microfluidic channels’ acetone vapors treatment, the device demonstrated a good ability to separate biological particles (Red Blood Cells) at different concentrations (20%, 30%, 40%, 50%) in a PBS buffer.

Pancreatic ductal adenocarcinoma (PDAC) is a prevalent form of pancreatic cancer, contributing significantly to cancer-related mortality worldwide. Early lesions manifest within the exocrine pancreatic gland. Thus, in vitro fully human models of the exocrine pancreatic unit are needed to foster the development of more effective diagnosis and treatments. However, it is challenging to make these models anatomically and functionally relevant. Here, tomographic volumetric bioprinting was used to biofabricate human fibroblast-laden gelatin methacrylate-based pancreatic models, mimicking glandular structure. Indeed, this technique is an optically based method that uses reverse optical tomography to construct 3D objects in a layerless fashion.

Pancreatic epithelial cells, healthy or cancerous, were then seeded, and the development of a thin epithelium inside the lumen of the 3D model was demonstrated. Immunofluorescence and ELISA assays revealed higher activation of fibroblasts when they were co-cultured with cancer cells, replicating the realistic situation in vivo. To our knowledge, this is the first demonstration of a 3D bioprinted portion of pancreas that recapitulates its physiological 3-dimensional microanatomy, and which shows tumor triggered inflammation. It opens new avenues to the application of light-based additive manufacturing in tissue engineering, overcoming the difficulties associated with light scattering from cells in hydrogels.

Non-linear excitation microscopy offers superior in-vivo imaging but faces challenges in deep tissue. High numerical aperture beams suffer spherical aberrations, while tissue scattering impacts image quality. To address this, we propose implantable microlenses for precise focusing below the skin in lab animals. By using low numerical aperture lasers, we avoid spherical aberrations induced by high NA objectives. Our study presents various microlens designs differing in size, shape, and fabrication methods, all on glass or organo-hybrid ceramic substrates. This approach shows promise for enhancing deep tissue imaging, facilitating better understanding of biological processes in vivo.