The rapid development of scientific CMOS (sCMOS) technology has greatly advanced optical microscopy for biomedical research with superior sensitivity, resolution, field-of-view, and frame rates. However, for sCMOS sensors, the parallel charge-voltage conversion and different responsivity at each pixel induces extra readout and pattern noise compared to charge-coupled devices (CCD) and electron-multiplying CCD (EM-CCD) sensors. This can produce artifacts, deteriorate imaging capability, and hinder quantification of fluorescent signals, thereby compromising strategies to reduce photo-damage to live samples. Here, we propose a content-adaptive algorithm for the automatic correction of sCMOS-related noise (ACsN) for fluorescence microscopy. ACsN combines camera physics and layered sparse filtering to significantly reduce the most relevant noise sources in a sCMOS sensor while preserving the fine details of the signal. The method improves the camera performance, enabling fast, low-light and quantitative optical microscopy with video-rate denoising for a broad range of imaging conditions and modalities.

Imaging flow cytometry (IFC) combines flow cytometry and fluorescence microscopy to enable high-throughput, multiparametric single-cell analysis with rich spatial details. However, current IFC techniques remain limited in their ability to reveal subcellular information with a high 3D resolution, throughput, sensitivity, and instrumental simplicity. In this study, we introduce a light-field flow cytometer (LFC), an IFC system capable of high-content, single-shot, and multi-color acquisition of up to 5,750 cells per second with a near-diffraction-limited resolution of 400-600 nm in all three dimensions. The LFC system integrates optical, microfluidic, and computational strategies to facilitate the volumetric visualization of various 3D subcellular characteristics through convenient access to commonly used epi-fluorescence platforms. We demonstrate the effectiveness of LFC in assaying, analyzing, and enumerating intricate subcellular morphology, function, and heterogeneity using various phantoms and biological specimens. The advancement offered by the LFC system presents a promising methodological pathway for broad cell biological and translational discoveries, with the potential for widespread adoption in biomedical research.

A Lab-on-a-Chip device for space-time digital holography is presented. Here, computational methods, holography, and microfluidics are intertwined to provide a reliable system for high-throughput counting of RBCs.

Holographic microscopes are emerging as suitable tools for in situ diagnostics and environmental monitoring, providing high-throughput, label-free, quantitative imaging capabilities through small and compact devices. In-line holographic microscopes can be realized at contained costs, trading off complexity in the phase retrieval process and being limited to sparse samples. Here we present a 3D printed, cost effective and field portable off-axis holographic microscope based on the concept of holographic microfluidic slide. Our scheme removes complexity from the reconstruction process, as phase retrieval is non iterative and obtainable by hologram demodulation. The configuration we introduce ensures flexibility in the definition of the optical scheme, exploitable to realize modular devices with different features. We discuss trade-offs and design rules of thumb to follow for developing DH microscopes based on the proposed solution. Using our prototype, we image flowing marine microalgae, polystyrene beads, E.coli bacteria and microplastics. We detail the effect on the performance and costs of each parameter, design, and hardware choice, guiding readers toward the realization of optimized devices that can be employed out of the lab by non-expert users for point of care testing.

The dynamics and stability of thin liquid films have fascinated scientists over many decades. Thin film flows are central to numerous areas of engineering, geophysics, and biophysics and occur over a wide range of lengths, velocities, and liquid property scales. In spite of many significant developments in this area, we still lack appropriate quantitative experimental tools with the spatial and temporal resolution necessary for a comprehensive study of film evolution. We propose tackling this problem with a holographic technique that combines quantitative phase imaging with a custom setup designed to form and manipulate bubbles. The results, gathered on a model aqueous polymeric solution, provide unparalleled insight into bubble dynamics through the combination of a full-field thickness estimation, three-dimensional imaging, and a fast acquisition time. The unprecedented level of detail offered by the proposed methodology will promote a deeper understanding of the underlying physics of thin film dynamics.

Fourier Ptychography probes the sample from different directions to achieve label-free quantitative phase imaging with a large space-bandwidth product. However, special attention has to be paid in the calibration of the optical setup to assure the accurate knowledge of the geometrical parameters involved in the image reconstruction. Any slight misalignment can provoke incorrect synthesis of the observables and, in turn, severe phase errors in the resulting high-resolution image. Here, we present a new processing pipeline that automatically removes such a priori unknown artifacts, thus making Fourier Ptychography miscalibration-tolerant. This result is achieved through a numerical Multi-Look approach that generates and combines multiple reconstructions of the same set of observables where phase artifacts are largely uncorrelated and, thus, automatically suppress each other. The proposed method is non-iterative, fully parallelizable, and completely blind, unlocking the use of Fourier Ptychography as an easy to handle tool or add-on to existing microscopes to be employed by unskilled users, thus paving the way to biomedical and clinical practices.