Lock-in Raman difference spectroscopy.

Shifted Excitation Raman Difference Spectroscopy (SERDS) is a non-destructive chemical analysis method capable of removing the fluorescence background and other disturbances from the Raman spectrum, thanks to the independence of the fluorescence with respect to the small difference in excitation wavelength. The spectrum difference is computed in a post-processing step. Here, we demonstrate the use of a lock-in camera to obtain an on-line analog SERDS spectra allowing longer exposure times and no saturation, leading to an improved Signal-to-Noise Ratio (SNR) and reduced data storage. Two configurations are presented: the first one uses a single laser and can remove excitation-independent disturbances, such as ambient light; the second employs two-wavelength shifted sources and removes fluorescence background similarly to SERDS. In both cases, we experimentally extrapolate the expected SNR improvement.

Filamented Light (FLight) Biofabrication of Highly Aligned Tissue-Engineered Constructs.

Cell-laden hydrogels used in tissue engineering generally lack sufficient 3D topographical guidance for cells to mature into aligned tissues. A new strategy called filamented light (FLight) biofabrication rapidly creates hydrogels composed of unidirectional microfilament networks, with diameters on the length scale of single cells. Due to optical modulation instability, a light beam is divided optically into FLight beams. Local polymerization of a photoactive resin is triggered, leading to local increase in refractive index, which itself creates self-focusing waveguides and further polymerization of photoresin into long hydrogel microfilaments. Diameter and spacing of the microfilaments can be tuned from 2 to 30 µm by changing the coherence length of the light beam. Microfilaments show outstanding cell instructive properties with fibroblasts, tenocytes, endothelial cells, and myoblasts, influencing cell alignment, nuclear deformation, and extracellular matrix deposition. FLight is compatible with multiple types of photoresins and allows for biofabrication of centimeter-scale hydrogel constructs with excellent cell viability within seconds (<10 s per construct). Multidirectional microfilaments are achievable within a single hydrogel construct by changing the direction of FLight projection, and complex multimaterial/multicellular tissue-engineered constructs are possible by sequentially exchanging the cell-laden photoresin. FLight offers a transformational approach to developing anisotropic tissues using photo-crosslinkable biomaterials.

Controlling Light in Scattering Materials for Volumetric Additive Manufacturing.

3D printing has revolutionized the manufacturing of volumetric components and structures in many areas. Several fully volumetric light-based techniques have been recently developed thanks to the advent of photocurable resins, promising to reach unprecedented short print time (down to a few tens of seconds) while keeping a good resolution (around 100 µm). However, these new approaches only work with homogeneous and relatively transparent resins so that the light patterns used for photo-polymerization are not scrambled along their propagation. Herein, a method that takes into account light scattering in the resin prior to computing projection patterns is proposed. Using a tomographic volumetric printer, it is experimentally demonstrated that implementation of this correction is critical when printing objects whose size exceeds the scattering mean free path. To show the broad applicability of the technique, functional objects of high print fidelity are fabricated in hard organic scattering acrylates and soft cell-laden hydrogels (at 4 million cells mL-1 ). This opens up promising perspectives in printing inside turbid materials with particular interesting applications for bioprinting cell-laden constructs.

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver-Like Metabolic Biofactories.

Organ- and tissue-level biological functions are intimately linked to microscale cell-cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi-scale living constructs, with cellular microenvironments formed by stem cell self-assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid-laden constructs, which capture key functions of the human liver. Volumetric bioprinting via optical tomography shapes organoid-laden gelatin hydrogels into complex centimeter-scale 3D structures in under 20 s. Optically tuned bioresins enable refractive index matching of specific intracellular structures, countering the disruptive impact of cell-mediated light scattering on printing resolution. This layerless, nozzle-free technique poses no harmful mechanical stresses on organoids, resulting in superior viability and morphology preservation post-printing. Bioprinted organoids undergo hepatocytic differentiation showing albumin synthesis, liver-specific enzyme activity, and remarkably acquired native-like polarization. Organoids embedded within low stiffness gelatins (<2 kPa) are bioprinted into mathematically defined lattices with varying degrees of pore network tortuosity, and cultured under perfusion. These structures act as metabolic biofactories in which liver-specific ammonia detoxification can be enhanced by the architectural profile of the constructs. This technology opens up new possibilities for regenerative medicine and personalized drug testing.

High-resolution tomographic volumetric additive manufacturing.

In tomographic volumetric additive manufacturing, an entire three-dimensional object is simultaneously solidified by irradiating a liquid photopolymer volume from multiple angles with dynamic light patterns. Though tomographic additive manufacturing has the potential to produce complex parts with a higher throughput and a wider range of printable materials than layer-by-layer additive manufacturing, its resolution currently remains limited to 300 µm. Here, we show that a low-étendue illumination system enables the production of high-resolution features. We further demonstrate an integrated feedback system to accurately control the photopolymerization kinetics over the entire build volume and improve the geometric fidelity of the object solidification. Hard and soft centimeter-scale parts are produced in less than 30 seconds with 80 µm positive and 500 µm negative features, thus demonstrating that tomographic additive manufacturing is potentially suitable for the ultrafast fabrication of advanced and functional constructs.

Volumetric Bioprinting of Complex Living-Tissue Constructs within Seconds.

Biofabrication technologies, including stereolithography and extrusion-based printing, are revolutionizing the creation of complex engineered tissues. The current paradigm in bioprinting relies on the additive layer-by-layer deposition and assembly of repetitive building blocks, typically cell-laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Overcoming such limitations, the volumetric bioprinting of clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds is described. An optical-tomography-inspired printing approach, based on visible light projection, is developed to generate cell-laden tissue constructs with high viability (>85%) from gelatin-based photoresponsive hydrogels. Free-form architectures, difficult to reproduce with conventional printing, are obtained, including anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. The latter undergoes maturation in vitro as the bioprinted chondroprogenitor cells synthesize neo-fibrocartilage matrix. Moreover, free-floating structures are generated, as demonstrated by printing functional hydrogel-based ball-and-cage fluidic valves. Volumetric bioprinting permits the creation of geometrically complex, centimeter-scale constructs at an unprecedented printing velocity, opening new avenues for upscaling the production of hydrogel-based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.

Selective femtosecond laser ablation via two-photon fluorescence imaging through a multimode fiber.

We demonstrate the ability of a multimode fiber probe to provide two-photon fluorescence (TPF) imaging feedback that guides the femtosecond laser ablation (FLA) in biological samples for highly selective modifications. We implement the system through the propagation of high power femtosecond pulses through a graded-index (GRIN) multimode fiber and we investigate the limitations posed by the high laser peak intensities required for laser ablation. We demonstrate that the GRIN fiber probe can deliver laser intensities up to 1.5x1013 W/cm2, sufficient for the ablation of a wide range of materials, including biological samples. Wavefront shaping through an ultrathin probe of around 400 µm in diameter is used for diffraction limited focusing and digital scanning of the focus spot. Selective FLA of cochlear hair cells is performed based on the TPF images obtained through the same multimode fiber probe.

Raman imaging through multimode sapphire fiber.

We report on a sapphire fiber Raman imaging probe's use for challenging applications where access is severely restricted. Small-dimension Raman probes have been developed previously for various clinical applications because they show great capability for diagnosing disease states in bodily fluids, cells, and tissues. However, applications of these sub-millimeter diameter Raman probes were constrained by two factors: first, it is difficult to incorporate filters and focusing optics at such small scale; second, the weak Raman signal is often obscured by strong background noise from the fiber probe material, especially the most commonly used silica, which has a strong broad background noise in low wavenumbers (<500-1700 cm-1). Here, we demonstrate the thinnest-known imaging Raman probe with a 60 µm diameter Sapphire multimode fiber in which both excitation and signal collection pass through. This probe takes advantage of the low fluorescence and narrow Raman peaks of Sapphire, its inherent high temperature and corrosion resistance, and large numerical aperture (NA). Raman images of Polystyrene beads, carbon nanotubes, and CaSO4 agglomerations are obtained with a spatial resolution of 1 µm and a field of view of 30 µm. Our imaging results show that single polystyrene bead (~15 µm diameter) can be differentiated from a mixture with CaSO4 agglomerations, which has a close Raman shift.

Multimode optical fiber transmission with a deep learning network.

Multimode fibers (MMFs) are an example of a highly scattering medium, which scramble the coherent light propagating within them to produce seemingly random patterns. Thus, for applications such as imaging and image projection through an MMF, careful measurements of the relationship between the inputs and outputs of the fiber are required. We show, as a proof of concept, that a deep neural network can learn the input-output relationship in a 0.75 m long MMF. Specifically, we demonstrate that a deep convolutional neural network (CNN) can learn the nonlinear relationships between the amplitude of the speckle pattern (phase information lost) obtained at the output of the fiber and the phase or the amplitude at the input of the fiber. Effectively, the network performs a nonlinear inversion task. We obtained image fidelities (correlations) as high as ~98% for reconstruction and ~94% for image projection in the MMF compared with the image recovered using the full knowledge of the system transmission characterized with the complex measured matrix. We further show that the network can be trained for transfer learning, i.e., it can transmit images through the MMF, which belongs to another class not used for training/testing.

Single-photon three-dimensional microfabrication through a multimode optical fiber.

Two-photon polymerization (TPP) processes have enabled the fabrication of advanced and functional microstructures. However, most TPP platforms are bulky and require the use of expensive femtosecond lasers. Here, we propose an inexpensive and compact alternative to TPP by adapting an endoscopic imaging system for single-photon three-dimensional microfabrication. The wavefront of a visible continuous-wave laser beam is shaped so that it focuses into a photoresist through a 5 cm long ultra-thin multimode optical fiber (∅70 µm, NA 0.64). Using this device, we show that single-photon polymerization can be confined to the phase-controlled focal spot thanks to the non-linearity of the photoresist, likely due to oxygen radical scavenging. Thus, by exploiting this non-linearity with a specific overcuring method we demonstrate single-photon three-dimensional fabrication of solid and hollow microstructures through a multimode fiber with a 1.0-µm lateral and 21.5-µm axial printing resolution. This opens up new possibilities for advanced and functional microfabrication through endoscopic probes with inexpensive laser sources.

High power, ultrashort pulse control through a multi-core fiber for ablation.

Ultrashort pulse ablation has become a useful tool for micromachining and biomedical surgical applications. Implementation of ultrashort pulse ablation in confined spaces has been limited by endoscopic delivery and focusing of a high peak power pulse. Here we demonstrate ultrashort pulse ablation through a thin multi-core fiber (MCF) using wavefront shaping, which allows for focusing and scanning the pulse without requiring distal end optics and enables a smaller ablation tool. The intensity necessary for ablation is significantly higher than for multiphoton imaging. We show that the ultimate limitations of the MCF based ablation are the nonlinear effects induced by the pulse in the MCFs cores. We characterize and compare the performance of two devices utilizing a different number of cores and demonstrate ultrashort pulse ablation on a thin film of gold.

Bend translation in multimode fiber imaging.

Light propagation in multimode fibers is typically assumed to be extremely sensitive to changes in geometry. We study here a particular configuration where an S-shaped bend is translated between two sections of fiber. In this sliding bend configuration, we show that nearly constant propagation characteristics can be obtained in certain fibers. Several fibers were tested using a bend with a peak radius of curvature of 25 mm. We found large differences in bending behavior between fibers of varying core diameters and numerical apertures. Fibers with a large numerical aperture are found to be more stable. In several fibers, the bend can be translated over a distance of 25 mm with a limited impact on imaging performance. The experimental results are confirmed using simulations. Our findings shed a new light on bending sensitivity in multimode fibers, and open up more possibilities for their use as imaging devices.

Confocal microscopy through a multimode fiber using optical correlation.

We report on a method to obtain confocal imaging through multimode fibers using optical correlation. First, we measure the fiber's transmission matrix in a calibration step. This allows us to create focused spots at one end of the fiber by shaping the wavefront sent into it from the opposite end. These spots are scanned over a sample, and the light returning from the sample via the fiber is optically correlated with the input pattern. We show that this achieves spatial selectivity in the detection. The technique is demonstrated on microbeads, a dried epithelial cell, and a cover glass.

Digital confocal microscopy through a multimode fiber.

Acquiring high-contrast optical images deep inside biological tissues is still a challenging problem. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. However, it suffers from low sensitivity in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images are acquired through a multimode fiber. We achieve this by digitally engineering the excitation wavefront and then applying a virtual digital pinhole on the collected signal. In this way, we are able to acquire images through the fiber with significantly increased contrast. With a fiber of numerical aperture 0.22, we achieve a lateral resolution of 1.5µm, and an axial resolution of 12.7µm. The point-scanning rate is currently limited by our spatial light modulator (20Hz).