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Despite an increasing gain of knowledge regarding small extracellular vesicle (sEV) composition and functions in cell-cell communication, the mechanism behind their biogenesis remains unclear. Here, we revealed for the first time that the sEV biogenesis and release into the microenvironment are tightly connected with another important organelle: Lipid Droplets (LD). We have observed this correlation using different human cancer cell lines as well as patient-derived colorectal cancer stem cells (CR-CSCs). Our results showed that the use of external stimuli such as radiation, pH, hypoxia, or lipid interfering drugs, known to affect the LD content, had a similar effect in terms of sEV secretion. Additional validations were brought using multiple omics data, at the mRNA and protein levels. Altogether, the possibility to fine-tune sEV biogenesis by targeting LDs, could have a massive impact on the amount, the cargos and the properties of those sEVs, paving the way for new clinical perspectives.Significance Statement

Stimulated Raman scattering (SRS) microscopy is increasingly employed for highly specific, label-free, and high-speed bioimaging. Despite its benefits, SRS is susceptible to spurious background signals caused by competing effects, which lower the possible imaging contrast and sensitivity. An efficient approach to suppress these undesired background signals is frequency-modulation (FM) SRS, which exploits the competing effects’ weak spectral dependence compared to the SRS signal’s high spectral specificity. We propose an FM-SRS scheme realized with an acousto-optic tunable filter, which presents a few advantages compared to other solutions presented in the literature. In particular, it can perform automated measurements from the fingerprint to the CH-stretching region of the vibrational spectrum without any manual adjustment of the optical setup. Moreover, it allows simple all-electronic control of the spectral separation and relative intensities of the pair of probed wavenumbers.

We report high efficiency, miniaturized, ultra-fast coherent beam combining with 3D printed micro-optics directly on the tip of a multicore fiber bundle. The highly compact device foot-print (180 micron diameter) facilitates its incorporation into a minimally invasive ultra-thin nonlinear endoscope to perform two-photon imaging, Comment: Published in Optics Letters

Lipid Droplets (LDs) are emerging as crucial players in colon cancer development and maintenance. Their expression has been associated with high tumorigenicity in Cancer Stem Cells (CSCs), so that they have been proposed as a new functional marker in Colorectal Cancer Stem Cells (CR-CSCs). They are also indirectly involved in the modulation of the tumor microenvironment through the production of pro-inflammatory molecules. There is growing evidence that a possible connection between metabolic alterations and malignant transformation exists, although the effects of nutrients, primarily glucose, on the CSC behavior are still mostly unexplored. Glucose is an essential fuel for cancer cells, and the connections with LDs in the healthy and CSC populations merit to be more deeply investigated. Here, we showed that a high glucose concentration activated the PI3K/AKT pathway and increased the expression of CD133 and CD44v6 CSC markers. Additionally, glucose was responsible for the increased amount of Reactive Oxygen Species (ROS) and LDs in both healthy and CR-CSC samples. We also investigated the gene modulations following the HG treatment and found out that the healthy cell gene profile was the most affected. Lastly, Atorvastatin, a lipid-lowering drug, induced the highest mortality on CR-CSCs without affecting the healthy counterpart.

We report the use of 3D-printed microscale spiral phase plates to generate orbital angular momentum (OAM) carrying beams. We confirm that the generated beams have high purity, and we have successfully tested them to convey data signals with low bit error rates at the wavelength of 980 nm. This method will open new opportunities for generating OAM beams for many applications in optical communications, including free-space optics, as well as underwater, chip-tochip, and quantum communications.

The field of 3D micro-optics is rapidly expanding, and essential advances in femtosecond laser direct-write 3D multi-photon lithography (MPL, also known as two-photon or multi-photon polymerization) are being made. Micro-optics realized via MPL emerged a decade ago and the field has exploded during the last five years. Impressive findings have revealed its potential for beam shaping, advanced imaging, optical sensing, integrated photonic circuits, and much more. This is supported by a game-changing and increasing industrial interest from key established companies in this field. In this review, the origin and the advancement of micro-optics fabrication with MPL are detailed by describing the chronology, distinguishing discrete application groups, providing generalized technical data on the processes and available materials, and discussing the foreseen near-future advances.

Microstructured optical fibers (MOFs) have interesting properties such as high birefringence, high nonlinearity, and endlessly single-mode operation [1] . MOFs are optical fibers made with a single material, in which an array of micron-sized longitudinal holes enables light guidance. The design of the hole pattern allows controlling the optical properties of the MOFs.

Optical tweezers (OT) are non-destructive, contactless tools that use light to trap and manipulate microscopic objects. They have applications in diverse fields, particularly in biomedicine and physics [1] . Due to their unique applications and the ability to analyze sub-micron biological systems, the Nobel prize for Physics in 2018 was awarded to Arthur Ashkin, the inventor of OT. Conventional single-beam OT setups require the focusing of a laser beam with a high-Numerical Aperture (NA) objective. However, these setups are bulky and restrict the trapping capabilities to table-top arrangements. The miniaturization of OT to be implemented with a single optical fiber is vital as it opens up new possibilities such as trapping in turbid media or in-vivo. Several groups demonstrated single-fiber OT but the proposed approaches use non-standard fibers and grant only limited design flexibility in controlling the optical trap geometry [2] .

Conventional stimulated Raman scattering (SRS) microscopes perform spectral acquisitions probing one single Raman shift (wavenumber) at a time. A multiplexed approach, however, would allow to perform faster spectral acquisitions, either measuring several wavenumbers in parallel or using reconstruction techniques that increase the signal to noise ratio (SNR). While the first approach often requires the use of complex technical solutions, the latter can be implemented with minor changes to an already existing broadband setup.

Bessel beams have gained prominence due to their diffraction-free propagation and self-healing properties [1] . The zeroth-order Bessel beam has maximum intensity at its center and has found many applications, including optical trapping and bio-imaging. On the other hand, higher-order Bessel beams have a node due to a phase singularity and carry orbital angular momentum (OAM). Over the past years, Bessel beams have been generated by using different approaches, namely by Fourier transforming a narrow circular beam with a lens, by using axicons and with holographic beam shaping techniques. The need to miniaturize optics has led to efforts towards the on-fiber generation of Bessel beams. The successful attempts include the fabrication of a customized annular-core fiber along with a polymer lens [2] , or modifying the end of the fiber to an axicon profile with chemical or lithographic methods [3] . These techniques have limited capability on generating Bessel beams with on-demand optical parameters and are only focused on creating zeroth-order Bessel beams from optical fibers.

We investigated terahertz resonant gold nanocones for out-of-plane field-driven photoemission and compare their performance with a standard non-resonant nanotip.

We present a novel configuration for high spectral resolution multiplexing acquisition based on the Hadamard transform in stimulated Raman scattering (SRS) microscopy. The broadband tunable output of a dual-beam femtosecond laser is filtered by a fast, narrowband, and multi-channel acousto-optic tunable filter (AOTF). By turning on and off different subsets of its 8 independent channels, the AOTF generates the spectral masks given by the Hadamard matrix. We demonstrate a seamless and automated operation in the Raman fingerprint and CH-stretch regions. In the presence of additive noise, the spectral measurements using the multiplexed method show the same signal-to-noise ratio of conventional single-wavenumber acquisitions performed with 4 times longer integration time.

We present a high spectral resolution multiplexing acquisition mode for SRS microscopy based on a dual-beam femtosecond laser. A multi-channel Acousto Optical Tunable Filter (AOTF) generates spectral masks, given by the Hadamard matrix, by turning on and off different subsets of its 8 independent channels, corresponding to different wavelengths available within the broad bandwidth of the “pump” femtosecond laser. The SRS spectrum is retrieved by using the inverse Hadamard matrix. When additive noise is dominating, spectral measurements using the multiplexed method show the same signal to noise ratio of conventional single-wavenumber acquisitions performed with 4 times longer integration time.

The miniaturization of optical fiber polarization beam splitters (PBS) is highly desired, especially in optical communications. Here, we demonstrate the first-ever PBS based on Photonic Crystal Fibers (PCFs). The device is enabled by high-resolution 3D printing based on two-photon lithography, a method that grants unprecedented design flexibility to realize PCF segments with arbitrary geometries. The PCF-PBS is composed of three segments, for a total length of 210 µm, and is directly printed in a single step on a single-mode fiber. The PCF-PBS has an extinction ratio greater than 10 dB on a 100 nm bandwidth centered at around 1550 nm.

Optical tweezers based on single optical fibers are typically fabricated with methods that grant only limited design flexibility in the trapping geometry. Here, we present a novel hybrid micro-optical structure 3D printed in a single step on a standard single-mode optical fiber, to create an ultra-compact fiber tweezer. The structure is made of stacked refractive, reflective, and waveguiding optical elements. Our approach allows an easy and independent tuning of trapping parameters as the numerical aperture and the working distance of the optical trap. We experimentally illustrate the 3D trapping capabilities of the proposed fiber optical tweezers.

We exploit the strong field enhancement offered by vertical gold nanocones resonating at 1 THz to induce THz field-driven electron emission. The nanocones are fabricated via an advanced 3D printing technique on a photopolymer and are successively gold coated. We demonstrate the clear advantage offered by nanocones featuring a monopolar resonance at THz frequencies with respect to traditional non-resonant tips via numerical modelling, THz far-field characterization, and the analysis of electron-induced argon gas fluorescence. Finally, we show that a further degree of optimization is enabled by tailoring the collective response of the nanocones when arranged in an array geometry.

Photonic crystal fibers (PCF) have revolutionized the field of optical fibers. Their unique properties, as endlessly single-mode operation, high birefringence, and hollow-core optical guidance, are enabled and controlled by their hole-array geometry. Thanks to these properties, optical waveguides based on geometrically unbound PCF designs could be exploited to realize miniaturized complex devices which implement advanced photonic operations. However, arbitrary PCF geometries are difficult or even impossible to produce using current PCF fabrication methods. Here, we show how to fabricate optical waveguide segments with PCF designs by 3D microprinting and how the combination of these segments can realize complex photonic devices.

Bessel beams (BBs) have gained prominence thanks to their diffraction-free propagation and self-healing properties. These beams are conventionally generated using different approaches, namely by transforming a narrow circular beam with a lens, using axicons or holographic beam-shaping techniques. These methods involve space-consuming optics. To overcome this limitation, in the past, efforts have been made to create BBs directly from optical fibers. However, these solutions have limited capability to generate BBs with on-demand optical parameters and only focused on creating zeroth-order BBs. Here, we propose a photonic structure that uses stacked miniaturized optical elements 3D printed in a single step on the fiber facet. Our design allows for the generation of both zeroth- and high-order BBs and fully controllable tailoring of the beams’ parameters, such as their diffraction-free propagation distance or the width of their central peak or node. Remarkably, we report for the first time, to the best of our knowledge, the generation of high-order BBs from optical fibers. We expect our approach to be useful in applications such as optical and quantum communications, fiber-based sensors, microscopy, spectroscopy, and optical trapping.

We numerically and experimentally investigate out-of-plane gold nanostructures resonating at terahertz frequencies for field-driven photoemission and compare their performance with a traditional non-resonant nanotip geometry.

We present a fully 3D printed, on-fiber hybrid photonic structure to create customizable single-fiber optical tweezers. The structure contains waveguiding, reflecting, and refracting micro-optical elements stacked to generate a high-NA focal spot.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤ 500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

In 2018 Arthur Ashkin shared half of the Nobel prize for physics "for the optical tweezers and their application to biological systems." Indeed, Optical Tweezers (OT) have found relevant applications in bioscience, thanks to the capability of trapping and manipulating small entities like viruses, bacteria, and DNA strands, in a non-destructive way. The balance of optical forces creating a stable all-optical trap is usually achieved by focusing a Gaussian beam with a high-NA microscope objective. Nevertheless, more efficient optical traps can be obtained by focusing a "donut" annular beam instead of a Gaussian beam [1], for particles larger than the focused beam size. Additionally, the need for bulky high-NA optics such as microscope objective may limit the use of OT in potential applications, like in-vivo trapping, and prompts the development of optical fiber-based OT. Here, we 3D printed a hybrid micro-optical structure on a single-mode optical fiber to create an ultra-compact annular beam fiber tweezer [2], with long working distance and a small probe diameter. A cascade of refractive, reflective, and waveguiding optical elements are microfabricated in a single step to transform the beam output from the fiber. The sequence is constituted by a gaussian to annular waveguide converter, a reflective annular beam expander, and a lensed prism deflector. The gaussian to annular waveguide converter transforms the single-mode fiber output into an annular beam with an inner radius of 4.5 µm and an outer radius of 9 µm. Then, a reflective inverted truncated cone expands the annular beam to an inner radius of 47 µm and an outer radius of 57 µm, by two total internal reflections. Afterward, a total internal reflection prism focuses the annular beam along its radius with a numerical aperture of 1 in water immersion. The output surface has a rounded profile with a radius of curvature of 7 µm to create a lens with a focal length of 50 µm that focuses the annular beam at the trapping point. All the total internal reflection surfaces are designed to work in water immersion, where the critical angle is around 60deg. The final result of this cascaded micro-optical structure is a sharply focused annular beam which forms an optical trap with a working distance of 30 µm. The microstructure is 275 µm long with a diameter of 116µm, smaller than the single-mode optical fiber which has a diameter of 125 µm. The structure has been 3D printed with a commercial Two-photon lithography system (Photonic Professional GT, Nanoscribe GmbH), by using the proprietary material Ip-Dip (Nanoscribe). We demonstrate the all-optical 3D trapping capabilities of this device by trapping polystyrene beads with different diameters. This device demonstrates the potential of 3D printing through two-photon lithography as a flexible tool to realize complexly integrated micro-optical systems. [1] O'Neil, A.T., Padgett, M.J., 2001. Axial and lateral trapping efficiency of Laguerre–Gaussian modes in inverted optical tweezers. Optics Communications, 193(1-6), pp.45-50. [2] Liberale, C. et al. Miniaturized all-fibre probe for three-dimensional optical trapping and manipulation. Nature Photonics, 1(12), p. 723.