This PDF file contains the front matter associated with SPIE Proceedings Volume 11786, including the Title Page, Copyright information and Table of Contents

OCT adoption is somewhat limited by the lack of an effective means for obtaining adequate image penetration in highly scattering tissue. For example, the ability to observe subtle changes in the layers of skin tissue where microcirculation occurs, generally anywhere between 1-4 mm deep, a depth currently beyond the penetration of traditional OCT systems. DA-OCT presents an attractive potential to image deeper into tissues exposing morphology that otherwise may go undetected. The approach is based on an off-axis scanning approach which uses distinct illumination and collection apertures to accept a larger proportion of quasiballistic signal. Here we present transliation ofDA-OCT to image large tissue volumes using a broadband SLD centered about 1.3 μm paired with a dynamic focus-tracking method to create an enhanced depth of focus.

The European Innovation Council (EIC) is Europe's flagship innovation programme aiming to identify, develop and scale up breakthrough technologies and game changing innovations. EIC has been established under the EU Horizon Europe programme and It has a budget of €10.1 billion. The aim is to support game changing innovations from early stage research, to proof of concept, technology transfer, and the financing and scale up of start-ups and SMEs. The EIC takes a pro-active approach to managing funding by developing the visions for innovation and technology breakthroughs and by steering the portfolios of projects to achieve these goals. In this new environment the well-known FET is transformed to EIC Pathfinder (with the Pathfinder Open and Pathfinder Challenges calls) aiming to develop a diverse portfolio of targeted projects that explore wide-ranging technological potential, inspired by cutting-edge science, unconventional collaboration and innovative practices. Grants of up to 3 to 4 million euros support early stage development of future technologies (e.g. various activities at low Technology Readiness Levels 1-3), up to proof of concept. Pathfinder projects can also receive additional funding for testing the innovation potential of their research outputs.

The goal of the SensApp FET-Open project is to develop an innovative super-sensor that will be able to detect Alzheimer’s disease (AD) biomarkers (β-amyloid, Tau and pTAU) in peripheral blood. Considering that nowadays an accurate diagnosis of AD requires the highly invasive withdrawal and analysis of cerebrospinal fluid, SensApp will represent a breakthrough in the field of AD diagnosis thanks to the ability to detect the early stage of the disease by a simple blood collection. We call Droplet-Split-and-Stack (DSS) the new technology that will emerge from SensApp. The achievement of SensApp goal is enabled by the interdisciplinary cooperation between different research institutions and one company involved in the key fields of the project, Vrije Universiteit Brussels, VTT Technical Research Centre of Finland, University of Linz, Ginolis Ltd, IRCCS Centre “Bonino Pulejo”, under the coordination of CNR-Institute of Applied Sciences and Intelligent Systems. This communication will illustrate the progress of the activities.

Neural probes for electrical imaging and microstimulation of brain networks represent an ideal communication gateway between nanoelectronic devices emulating neuron computation and biological neurons. We show that titanium oxide microelectrodes and memristors can establish synaptic-like connections between biological and silicon spiking neurons across an elementary biohybrid network.

Vision starts when light is captured by photoreceptors, specialized cells in the retina that set fundamental limits on what we can see and are unfortunately lost in many blinding diseases. While photoreceptors carry considerable clinical and scientific importance in ophthalmology and vision science, means to assess their function and health at the level of individual cells remain limited. Recent advances in adaptive optics optical coherence tomography (AO-OCT) imaging systems have enabled photoreceptor cells to be observed and tracked with unprecedented 3D resolution and sensitivity in the living human eye. This imaging capability has allowed the dynamics of these cells to be studied in exquisite detail, in particular nanoscale transients the cells generate after being stimulated by light. These changes have been found to carry fundamental information about the photoreceptor’s physiology. Here, I will describe the capability of AO-OCT to image, track, and quantify these miniscule cell dynamics and how these measurements are being used to study vision and to assess cell dysfunction and health in disease.

I will review the fundamental limiting issues in in-vivo optical imaging and discuss the proposal made by the IN2SIGHT consortium to overcome them and its impact. IN2SIGHT will foster a breakthrough in in-vivo optical imaging that will renovate the biocompatibility tests (ISO10993 EU norm) required for the development of biomaterials for clinical use. These tests are economical and ethical unsustainable for small-medium industries and for the society. The IN2SIGHT approach stands on a micro-structured chip that will recast our thinking of deep tissue in-vivo imaging. I discuss how this proposal will allow unique quantification of the immune reaction to biomaterials, thus reducing time and costs for testing with a potential huge impact on public health systems and our society. The project sees the collaboration of seven partners from five countries and will exploit from the beginning inter-sectorial approaches in an interdisciplinary environment.

The goal of NeuroStimSpinal FETOPEN project is to contribute with a solution for spinal cord injury (SCI). Considering the biological complexity of SCI, a small incremental discovery in this area may represent an improvement that will translate into better clinical care and in the quality of life of these patients. Therefore, a neural tissue engineered scaffold capable of not only combining fibrous and porous topographic cues in order to mimic the morphology of the native spinal cord, but also potentiating the properties of graphenebased materials supported in a protein-rich decellularized matrix is being developed to be coupled with a wireless electrical stimulation device to promote the growth and reconnection of the ruptured nerves. This communication will cover the challenge and the progresses obtained so far.

Soft robotic systems benefit from improved compliance and larger degrees of freedom, allowing more complex motions and safer interaction with their environment than rigid robotic structures. Due to the inherent softness of the materials that are often used to create such soft robotic structures, these systems are highly susceptible to various damage modes, such as cuts or punctures by sharp objects, overloading during actuation, flaws originating from manufacturing process or fatigue failure, drastically limiting their service lifetimes. Various types of synthetic materials that possess the ability to repair damage are being used to create soft robotic systems that are able to recover their performance by repairing incurred damage, either autonomously or after the application of a stimulus. The breakthrough targeted in the SHERO project is the development of complete robotic systems that are able to (1) sense and locate damage and to evaluate the system performance, (2) react intelligently to alleviate the damaging event and prevent catastrophic failure, (3) take the necessary measures to heal the damage to restore all functions by facilitating an autonomous or controlled healing action of the damaged element, and (4) perform a rehabilitation by evaluating the quality of the healing process and the recovery of functional performance, and finally, (5) return to action.

In the course of experiments for our FET open project Lumiblast, we set off to measure the excitation of various photoactive drugs (photosensitizers, PS) by the luminescence emission of luminol. Luminol (5-Amino-2,3-dihydrophthalazine-1,4-dione) is a chemical that interacts with reactive oxygen species (ROS) in basic conditions, and in the presence of metal catalysts like Fe or Cu, gives out a characteristic blue luminescence. When dissolved in organic solvents like DMSO, however, luminol only requires the addition of bases like KOH, NaOH or potassium terbutoxide, to fulfil the conditions for luminescence emission. In the present work we employed a detection system based on a spectrograph coupled to a ccd camera to register fluorescence (Fig 1B) or luminescence (Fig 1 A, C). In the case of characteristic fluorescence registration (Fig. 1B), the PSs investigated were excited by a 532 nm laser with a variable power output. We have documented the energy transfer from chemically induced luminol luminescence to a number of PSs including rose bengal, erythrosin B, hypericin amongst others. In all cases both the luminol emission and the luminol luminescence-induced PS fluorescence were registered as shown in the example of luminol and erythrosine b in Fig. 1C. We further attempted to register the generation of singlet oxygen from luminol-excited PSs. To achieve this, we employed the near-infrared (NIR) photomultiplier tube (PMT) shown in Fig.1 E, with a cut-off filter at 900nm and a bandpass filter at 1270±30 nm. This allowed only radiation within this spectral region to reach the PMT, corresponding to the characteristic phosphorescence of singlet oxygen, spin forbidden de-excitation to ground state triplet oxygen. A characteristic steady state singlet oxygen registration can be seen in Fig. 1D, for erythrosine b which has a high singlet oxygen quantum yield. The luminol luminescence was initiated by addition of terbutoxide to the DMSO luminol solution, at which point we can see a rise of the signal at 1270 nm. Upon addition of the singlet oxygen quencher, L-histidine, the signal dropped steeply to background levels. NOTE: Figures are not available.

The detection of CTCs in a blood sample is a challenging task due to their rarity and variety. We develop a new label-free and all-optical approach at the lab-on-chip scale for the detection of CTCs based on morphological biomarkers. In particular, we design a microfluidic device to be combined with a phase-contrast tomography system to carry out quantitative measurements of the three-dimensional structure of each single cell in a blood sample. In such device, two aspects are conjugated: on the one hand, the cells need to perform at least one complete rotation within the field of view of the imaging apparatus; on the other hand, the highest possible throughput has to be achieved, yet without deforming the cells significantly, which would impede their tomographic reconstruction. In this contribution, the finite-element-simulation-based preliminary design of a microfluidic device that would allow the achievement of the aforementioned objectives for cells with different shape and deformability is presented.

Performance improvements in microfluidic systems depend on accurate measurement and control of fluids on the micro- and nanoscale, and new applications are continuously moving the needle to lower volumetric flow rates. This work focuses on improving an optofluidic system for the measurement and calibration of microflows to the sub-nanoliter per minute range. The experimental measurements rely on an optofluidic system that delivers excitation light and records fluorescence in a precise interrogation region of a microfluidic channel. Using a scaling relationship between flow rate and the fluorescence emission after photobleaching, the system enables real-time determination of flow rates. Here we demonstrate improved calibration of a flow controller to 1 % uncertainty and improved resolution of the optofluidic flow meter to less than 1 nL/min using molecules with lower diffusion coefficients.

We propose a label-free metrology technique that can perform analysis of live individual cells while they are growing individually in droplets. The cells first flow in a microfluidics device and imaged using interferometry. At the end of this device the droplets with the cells accumulate and then the cells can be monitored over time. The device presents the possibility for both high-throughput label-free analysis of individual biological cells and tracking their long-term development.

As cells and tissues are highly dependent on their specific microenvironment, in-vitro models need to incorporate this 3D structure to yield reliable results in biomedical research. Cancer cell spheroids and organoids have become valuable tools in oncology and development studies as they are more representative of in-vivo tissues than the traditional 2D cell culture models and have the potential to improve throughput in drug-screening compared to animal-models and also to go towards personalized medicine. Platforms for both assembly and non-invasive long-term monitoring of such models and also for layered bio-engineered tissues are of great interest. We have developed a sono-optical microfluidic device with 3D acoustic trapping and optical tweezers for non-contact manipulation and imaging of biological samples in liquid suspension. 3D acoustic trapping is achieved with two orthogonal side-transducers and an optically transparent top-transducer that enables optical access for imaging and tweezers to the sample volume. We have demonstrated trapping of biological samples and cancer cell spheroids of several 100µm in size. With acoustics alone or combined with optical tweezers, we can trap samples, change their location and orientation or induce sustained rotation of them, without the sample being in contact with confining structures or embedded in gels. With 3D independent control of the transducers we can adjust the relative strength of the acoustic radiation and viscous torques which will determine whether transient reorientation or continuous rotation of a given sample takes place, and along with numerical simulations and experimental insight we can optimize our strategy to achieve a desired manipulation, within limitations depending on sample size and shape asymmetry. Our technique offers access to optical tomographic information, by rotation of samples around one chosen axis or two axes, and mechanical probing or it can be used for 3D patterning of cells and cell structures in gel precursors for tissue-engineering.

Optofluidic microscopes use fluids’ properties as an additional degree of freedom for optical detection and microfluidics to perform simple and low-cost object manipulation. In particular, several devices have been optimized for fluorescencebased imaging. These are usually based on the adaptation of established microscopy tools to the microfluidic system with different levels of integration. However, these systems can rarely resolve sub-micron details, posing a limit to the structures that can be studied. An exception is represented by systems developed for particle detection, which are capable to quantify protein expression and analyze small molecules even at nanoscale resolution. However, in this case high resolution requires a low emitter density and it cannot be used to visualize densely packed structures such as membranes and organelles. Hence, we have developed a system for sub-diffraction-limited optofluidic scanning microscopy (OSM) that uses the optofluidics paradigm to extract the inherent super-resolution information of a confocal system. OSM uses the optofluidic flow scanning scheme and a multifocal illumination pattern to obtain resolution doubling with minimal system complexity. In addition, it does not require any mechanical part for the scanning, so that it can be readily adapted to different levels of integration from commercial microscopes to on-chip configurations. This makes our system the most viable configuration for super-resolution optofluidics, being both suitable for continuous flow scanning and compatible with on-chip configurations through the adoption of integrated optics.

Holographic Tomography (HT) is an emerging label-free technique for microscopic bioimaging applications, that allows reconstructing the three-dimensional (3D) refractive index (RI) distribution of biological specimens. Recently, an inflow HT technique has been proposed in which multiple digital holograms are recorded at different viewing angles around the sample while it flows and rotates within a microfluidic channel. However, unlike conventional HT methods, there is no a priori information about cell 3D orientations, that are instead requested to perform any tomographic algorithm. Here we investigate a tracking-based rolling angles recovery method, showing robustness against the sample’s features. It is based on a phase images similarity metric recently demonstrated, that exploits the local contrast phase measurements to recognize a full cell rotation within the microfluidic channel. Hence, the orientations of the flowing cells are retrieved from their positions, which are in turn computed through the 3D holographic tracking. The performances of the rolling angles recovery method have been assessed both numerically, by simulating a 3D cell phantom, and experimentally, by reconstructing the 3D RI tomograms of two cancer cells. Both the numerical and the experimental analysis have been performed at different spatial resolutions. This rolling angles recovery method, not depending on the cell shapes, the RI contents, and the optical experimental conditions, could pave the way to the study of circulating tumor cells (CTCs) in the challenging tool of liquid biopsy.

In laser based digital holographic microscopy (DHM) quantitative phase imaging (QPI) is affected by scattering and interference fringes due to internal reflections in the experimental setup. We present a concept for the reduction of such coherence induced disturbances. In our approach, the sample illumination light is modulated by an electrically focus tunable lens while series of digital off-axis holograms are recorded from which subsequently averaged QPI images are calculated. The concept is compatible with Mach-Zehnder interferometer-based off-axis DHM and capable for usage with commercial research microscopes. The performance is illustrated by results from living cells.

We address a multi-spectral holographic imaging of optically transparent objects widely used in biomedical studies and industrial inspection. Instead of complicated optomechanical systems based on several light sources, our setup is based on acousto-optic tunable filter (AOTF) coupled with a broadband light source, which enables fast and highly effective spectral selection within a wide wavelength range. The filtered light enters the Mach-Zehnder interferometer and produces the interference pattern captured by camera and processed in the spatial frequency domain to extract amplitude and phase information about the inspected sample. We compare successive acquisition of several single-wavelength holograms and two single-shot modes for obtaining wavelength-multiplexed holograms using either sequential tuning to several wavelengths during the exposure time of the sensor or multi-frequency AOTF driving. The effectiveness of AOTF-based setup for multi-spectral digital holographic microscopy in all modes is demonstrated by the quantitative phase imaging of red blood cells smear specimens. The results of the study may be helpful to choose the optimal mode based on several criteria, such as required spatial and spectral resolution, field of view and image acquisition time, as well as for the optimization of AOTF-based setups for digital holography.

This paper presents the use of oblique illumination in self-referencing digital holographic microscopy (SR-DHM) to separate the interfering beams and enhance the resolution. SR-DHM is a compact and vibration-immune quantitative phase microscopy technique in which the interference of the object beam with part of itself forms the digital hologram. However, one drawback with SR-DHM is that both interfering beams contain object information that may overlap and hinder proper reconstruction. This study shows that using oblique illumination in off-axis DHM configurations separates the two interfering beams' spectra in the Fourier space, which can be used in SR-DHM to separate the overlapping beams' information without any restriction on the sample or the Field of View (FoV). The technique's capability is first shown in lateral shearing SR-DHM using a glass plate in which the object beams reflected from the front and back surfaces of a thick glass plate interfere and form the hologram. Using oblique illumination in this setup removes the redundant information from the FoV and improves the resolution twice. On the other hand, the technique is applied in SR-DHM using Lloyd's mirror, in which part of the object beam reflected from the Lloyd's mirror interferes with the part that arrives straightly at the camera plane. By tuning the fringe density in this setup using the illumination angle, we show that the interfering beams can be separated and the two simultaneous holograms can be recorded and reconstructed without overlapping information. This feature can be used for single-shot 1D resolution enhancement without overlapping information. The samples used in our validation experiments are a USAF test target and a 1D phase grating.

Quantitative measurement for thin-film thickness has been a widely studied issue, and, visualizing and characterizing for full-field thin-film evolution can provide effective data support for membrane science, polymer chemistry, biomaterials, applied Chemistry, etc. Interferometry was considered the most useful tool to measure film thickness in past decades. However, there were deficiencies in imaging speed and field of view for conventical interferometric measurement. In this paper, we proposed a fusion method to achieve full-field and quantitative analysis of the thin liquid film at nanoscale by using Digital Holography (DH) and White Light Interferometry (WLI) simultaneously. Compared with our previous holographic studies on thin film measurement, this new method can overcome the shortcomings of the holographic method related to registration and reconstruction. In addition, these parallel experiments can also provide a reference when we have to make a choice between DH and WLI. For the thickness reconstruction process, the entire life cycle registration is no longer required to obtain the thickness information. Moreover, the region where the thin film thickness is less than half the wavelength is now measurable. Thanks to the fusion processing, the film thickness calibration can be obtained without any background subtraction, in the meantime, the background hologram becomes not essential in the holographic recording process.

Visualizing the intracellular dynamics of plant cells has been an open challenge for modern botany, agronomy and pharmacy. In this paper, we proposed an approach to improve the phase contrast during plant cell holographic imaging by cells’ dehydration, and used this method to realize the observation of cytoplasmic circulation inside the living onion epithelial cell. The dehydration process can be seen as a sort of label-free contrast agent for better imaging biological processes. We have investigated live onion epidermal cells, observing their inner dynamics during long time recordings using a digital holographic microscopy system. For the experiments, an off-axis digital holography setup in transmission configuration with double spherical wave interference was used to record the digital holograms of onion cells. Then, we performed long-term time lapse holographic recordings of onion epidermal cells, and the results show that the intracellular tissue structure and the dynamic behavior of the cytoskeleton features and nuclei can be better exhibited via high-contrast phase imaging under cell dehydration conditions. In this case, the movements of intracellular filaments and the nucleus are observed via dynamical high-contrast phase imaging during the dehydration process. The experimental results clearly show the positive effect of dehydration process on intracellular imaging quality, and create the possibility to track the movement of plant organelles. In sum, thanks to the dehydration process of plant cells, holographic phase contrast enhancement imaging is realized.

Nano graphene-based materials offer interesting physicochemical and biological properties for biotechnological applications due to their small size, large surface area and ability to interact with cells/tissues. Among carbon-based nanomaterials, graphene oxide is one of the most used in biological field. There is an increasing interest in shedding light on the interaction mechanisms of nanographene oxide (nGO) with cells. In fact, the effects on human health of GO, and its toxicological profile, are still largely unknown. Here we show that, by minimizing the oxidation degree of GO, its toxicity is significantly reduced in NIH 3T3 cells. Moreover, we show that mild oxidation of graphene nanoplatelets produces nGO particles, which are massively internalized into the cell cytoplasm. MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed to analyze cell viability. Transmission electron microscopy (TEM) analysis was performed to evaluate nGO internalization mechanism into the cytoplasm under different oxidation degree and concentrations. For the first time, we evaluated quantitatively, the cell volume variation after nGO internalization in live fibroblasts through a label-free digital holography (DH) imaging technique and in quasi-real-time modality, thus avoiding the time-consuming and detrimental procedures usually employed by electron-based microscopy. In conclusion, here we have demonstrated that DH can be a viable tool to visualize and display 3D distributions of nano graphene oxide (nGO) uptake by fibroblast cells. DH opens the route for high-throughput investigation at single cell level for understanding how in different conditions nanoparticles aggregates distribute inside the cells.

In bone tissue, osteocytes are embedded within a microfluid-filled network which expose them to high levels of fluid shear stress (FSS). The osteocytes’ sensitivity to different levels of FSS has demonstrated. However, there are few attempts to image 3D cellular deformation under FSS by label-free and quantitative microscopy. Digital holographic (DH) microscopy is a powerful imaging technique that can provide rich intracellular information based on the refractive index (RI) contrast, without exogenous contrast agents. However, in DH image recording process, the recorded wave-front contains not only the object’s information but also the aberrations caused by the microscope objective (MO) and the imperfections of optical components of the system. The fitting-based numerical method removes total aberrations by detecting object-free background as reference surfaces. In this paper, we proposed a convolutional neural network (CNN) for multivariate regression to cope with the phase aberration compensation problem automatically thus allows performing long-term monitoring of bone cells morphological response under FSS. We transformed the problem of estimating the coefficients for fitting a phase aberration map to a regression problem. The aberrated phase images are put into this model which can automatically learns the internal features of phase aberrations. Then the optimal coefficients are estimated as an output of the network. Based on these coefficients, the phase aberration map is built by the polynomial fitting, and the phase aberrations are removed by subtracting the aberration phase image with the phase map. The trainning and validation set contain thousands of phase image of cells. The mean square error (MSE) is used as the loss function. Then, the trained model was used for aberrations compensation in the FFS experiment of osteocytes. The results show that the proposed approach can predict the optimal coefficients and automatically compensating the phase aberrations without detecting background regions and knowing any physical parameters.

We proposed a rapid autofocusing method exploiting holographic polarization microscope, which could determine the refocusing distance without multiple reconstructions and complex network training. The defocus distance of the object is calculated rapidly by identifying the separation distance of the two defocus images of the object and using the linear relation between the defocus distance of the object and the separation distance of the two defocus images of the object. The linear relationship is obtained by numerical propagation and linear fitting. Last, we have demonstrated the effectiveness of the proposed method by reconstructing the in-focus images of flowing cells. We believe that our proposed method could be a powerful tool for dynamic observation and extend the application of holographic polarization microscope.

Digital holographic imaging has the characteristics of fast and flexible with simple set up, and it can quantitatively obtain the intensity and phase of objects. The resolution of the reconstructed image is greatly limited by the pixelated imaging detectors during the digitization processing of holograms. Due to the limited diffraction bandwidth, the diffracted beam of each object point can be viewed as a diffractive cone. The lack of diffraction components causes the reconstructed quality to decrease when the detector cannot collect a complete diffracted wave field in the inline holography. In this work, we studied the reconstructed quality of holograms under different sampling forms. The losing diffraction components can be reconstructed by using an iterative algorithm based on energy attenuation and support constraints. We propose three iterative methods. The first is the bicubic interpolation and extrapolation iterative method based on a single small detector. The second is multi-plane extrapolation iterative method. The third is a faithful iterative reconstruction based on a sparse sensor array. When the diffraction component recorded by the hologram is missing, the losing diffraction components can be reconstructed by using the principle of the holographic redundancy. The reduction of reconstruction quality caused by the loss of sampling in the small-sized detector and sparse sensor array in the digital holography can be compensated.

Light manipulation through living sample is one of the most challenging insight of the last ten years. Actually, in nature live matter interacts with light to manage biochemical activities and such capability inspired scientist to couple light into biological system in order to achieve well defined optical and photonic operations. In this framework, we will present the lensing effect of living cells that opens new possible routes in diagnostics and, also, in optics and photonics technologies as BioLithography.

With observation of small objects, a precisely manipulation is also highly desirable, especially for a three-dimensional manipulation of nanoparticles or biomolecules with a size of less than 100 nm. Although optical tweezers have become powerful tools to manipulate microparticles and cells, they have limits when extended to the nanoscale because of the fundamental diffraction limit of light. The emergence of near-field methods, such as plasmonic tweezers and photonic crystal resonators, have enabled surpassing of the diffraction limit. However, these methods are usually used for two-dimensional manipulation and may lead to local heating effects that will damage the biological specimens. Therefore, we propose a near-field technique that uses a photonic nanojet to perform the three-dimensional optical manipulation of sub-100-nm objects. With the photonic nanojet generated by a dielectric microlens bound to an optical fiber probe, three-dimensional manipulations were achieved for fluorescent nanoparticles as well as for plasmid DNA molecules. Backscattering and fluorescent signals from the trapped targets were detected in real time with a strong enhancement. The demonstrated approach provides a potentially powerful tool for nanostructure assembly, biosensing and single-biomolecule studies.

Ferroelectric crystals generate electric fields under optical and thermal excitation. Recently, photo-induced electric fields generated in photovoltaic ferroelectric crystals have been applied to act on liquid droplets, either to manipulate droplets or to generate tiny droplets from a liquid reservoir. In this work, we investigate the migration of water and aqueous biodroplets under the light-induced electric fields generated in ferroelectric platforms based on iron doped lithium niobate. Different parameters affecting droplet manipulation have been analysed in detail. Light intensity, distance to the photovoltaic substrate and orientation of the polar axis of the active substrate play a key role. The behaviour and analysis of the droplet motion combined with numerical simulations allow to characterize the droplets and, in particular to distinguish between neutral and charged droplets. The results show the potential of this technique in biotechnology and biomedicine for applications such as bio-droplet analysis or sensing.

The effect of ultrashort laser radiation parameters on the process of biomaterial formation based on dispersed media of bovine serum albumin and carbon nanotubes was studied. It is noted that, in contrast to the use of continuous laser radiation of comparable power, the composite nanomaterial is formed when lower temperatures are reached, which provides additional advantages and a smaller change in the protein structure. This effect can also be associated with a pulse repetition rate of 80 MHz, in the interval, which is about 12.5 ns, partial cooling of the material can occur, while this effect is sufficient for the formation of a biomaterial. The hardness of this material is comparable to that of native tissue. A strong twofold change in the elasticity of such a material in comparison with drying in air indicates the formation of an inner framework of carbon nanotubes. The possibility of this effect is also confirmed by spectral studies, according to which, at the used wavelength of 810 nm, the radiation is absorbed mainly by carbon nanotubes and only as a result of heat transfer is transmitted to bovine serum albumin and is spent on water evaporation. In vitro studies of cell growth in the presence of biomaterial were carried out by quantitative (MTT test) and qualitative (microscopy) methods. It was found that the number of cells grown on the composites exceeds the number of cells in the control after 72 hours of incubation. The cells on the composites formed a monolayer, their morphology does not differ from the morphology of the cells in the control. In vitro studies indicate a positive effect of the biomaterial on cell adhesion and proliferation and, consequently, on the possibility of their use for tissue regeneration.

Materials play a key role in tissue engineering for the construction of 3-dimensional scaffolds that support the formation of a new extracellular matrix. They should be biocompatible, and for the fabrication of functional scaffolds, they should be mechanically robust, provide high resolution and printability factors to use in one-photon polymerization microstereolithography (OPP μ-SLA) technologies. Furthermore, applications where those materials are used, such as tissue regeneration or tissue substitutes, require fabrication approaches that allow the scalability of 3D scaffold for their clinical use. Therefore, both materials and technology need to be optimized and improved. We tackle two research tracks, one to provide high resolution and biocompatible materials that can be used in OPP- μ-SLA, and a second one to obtain a new μ- SLA configuration that can boost large-size scaffold fabrication for tissue engineering applications. In this work, we report our progress towards the formulation of a hydrogel based on the prototype resin X HYDRORES INX X100 from XPECT INX and we describe the configuration based on the beam shaping of a laser source to print centimeter scale scaffolds while conserving micro-scale features. We printed scaffolds with a commercial ABS resin and the bio-inert hydrogel from XPECT INX that allow us to compare resolution, printability, and mechanical stability. Our results evidence structures with voxel widths up to 20 μm and lengths up to 23 mm by using uniform light sheets illumination patterns. This work set new alternatives for the material and the fabrication aspects of additive manufacturing for 3D biofabrication.

Intensity Diffraction Tomography (IDT) is a new computational microscopy technique providing quantitative, volumetric phase imaging of biological samples over a large field-of-view. This approach uses computationally efficient inverse scattering models to recover the 3D phase objects from a set of intensity measurements taken under diverse illumination at a single focal plane. IDT is easily implemented in a standard microscope equipped with an LED array source and requires no exogeneous contrast agents, making the technology easily accessible to the biological research community. Here, I will discuss physics-embedded deep learning-based strategies for improving the imaging capabilities of IDT for handling highly scattering 3D objects.

While traditional optical coherence tomography (OCT) uses near-infrared light, visible-light OCT or vis-OCT takes advantage of the much higher tissue contrast within visible light spectral range to extract physiological and pathological information in addition to ultra-high (1.3 µm) resolution anatomical imaging. Using vis-OCT, for example, we detected retinal ischemia well before the onset of angiogenesis in diabetic mouse models; produced the first three-dimensional imaging of the entire Schlemm’s canal and limbal vascular network noninvasively; and detected ganglion cell damage when clinical biomarkers were normal in glaucoma mouse models. Working with collaborators, we are also aggressively developing human vis-OCT systems and clinical protocols to improve clinical management of diabetic retinopathy and glaucoma.

Phase imaging is a widely used tool in biology with clinical applications. Various phase sensitive imaging techniques exist, ranging from phase contrast microscopy to quantitative schemes such as spatial light interference microscopy and off-axis holography. Here, we discuss these techniques in terms of the Fisher information content they provide, and the resulting Cramer Rao bounds of phase measurement accuracy [1]. We introduce the theoretical framework assuming that shot-noise is the dominant source of noise, and deduce the necessary conditions required to perform optimal phase estimations. This approach brings insights to design maximally sensitive microscopes for photon-limited applications, such as high-speed measurements, or the imaging of ultra-cold atoms or fragile biostructures. We further discuss how local wavefront shaping, adapted to the sample under study, can maximize Fisher information and enable optimal phase estimations [1,2]. We observe the largest improvement when imaging thick samples and demonstrate it experimentally. [1] Fundamental bounds on the precision of classical phase microscopes, D. Bouchet, D. Maestre, J. Dong, and T. Juffmann, https://arxiv.org/abs/2011.04799 [2] Local Optimization of Wave-fronts for optimal sensitivity PHase Imaging (LowPhi), T. Juffmann, A. de los Ríos Sommer & S. Gigan, Opt. Commun., 454, 124484 (2020), DOI: 10.1016/j.optcom.2019.124484

Fourier Ptychographic Microscopy (FPM) probes biological samples from multiple directions and provides amplitude and quantitative phase-contrast imaging in label-free modality with large space-bandwidth product. FPM is suitable to analyze tissues in hospitals and analysis labs by unskilled users. Whenever the FPM setup is misaligned, phase artifacts can prevent a correct retrieval of the sample complex amplitude. Here we show a blind method, named Multi-Look FPM, which eliminates the unwanted artifacts and allows non-expert users skipping the recalibration process. Multi-Look FPM is proved effective in the case of neural tissue slides, cell layers, and marine microalgae with complex inner structures.

Quantitative phase imaging, employing the refractive index as endogenous contrast agent, opens wide possibilities to perform high-contrast cell measurements enabling reliable diagnostics and assessable examination. This capability can be rather easily obtained in a common-path total-shear regime after introducing a grating or beam-splitter into a regular microscope layout with a (partially-)coherent light source. Although these solutions are attractive due to straightforward implementation, overall good stability (thanks to common-path configuration) and high contrast, they are limited in timespace- bandwidth product (TSBP) as multi-frame phase reconstruction is needed to ensure sufficient accuracy and robustness. Alternative single-shot Fourier transform based approaches do allow for dynamic imaging; however they need off-axis recording and thus limit detector bandwidth and can severely truncate object spectrum (phase lateral resolution), which stays virtually intact in the multi-frame approach. Additionally, low signal-to-noise ratio of recorded fringe pattern significantly adds to the error budget via low contrast, high noise and strong incoherent background. In this contribution we study an interesting way to bypass mentioned shortcomings by recording two out-of-phase interferograms simultaneously to subtract them and thus experimentally increase the signal-to-noise ratio of otherwise low-quality dimmed fringe patterns. Ideal subtraction should yield background-rejected and modulation-doubled π- hologram, however in reality additional processing is needed. We will investigate three algorithms for such processing. Filtered interferogram is then analyzed employing Hilbert spiral transform, which is not sensitive to the carrier frequency like the Fourier transform and preserves object spectrum also in quasi on-axis configurations. Finally, sacrificing the field of view to record two interferograms at once, we gain unique feature of significantly increased TSBP enabling realtime investigation of broad-spectrum (highly detailed) transparent objects with enhanced phase resolution and signal-tonoise ratio. We corroborate the claims successfully analyzing prostate cancer cells and flowing microbeads otherwise measurable only in static regime using time-consuming phase-shifting. The technique has been validated utilizing 20x/0.46NA objective in a regular Olympus BX-60 upright microscope.

One in six couples suffer from infertility problems. I will present our recent advances in the development of acquisition methods that enable high-resolution, fine-detail full reconstruction of the 3D movement and structure of individual human sperm cells swimming freely, in thousands of frames per second. We achieved the retrieval of the 3D refractive-index profile of the sperm head, revealing its fine internal organelles and orientation, and the detailed 4D localization of the thin and highly-dynamic sperm tail. We have also achieved virtual staining of individual sperm cells using deep learning. These stain-free imaging methods have great potential for both biological assays and clinical use of intact sperm cells during in-vitro fertilization procedures.

Light is incredible. For imaging, the field of biophotonics has made astonishing advances in the last few decades due to the fact we can extract intricate knowledge of the sample, namely its morphology, molecular composition and metabolism. A prominent outcome is the 2014 Nobel Prize in Chemistry that acknowledged one of the most momentous physics developments for imaging – surpassing the spatial resolution barrier. Whilst today an impressive array of optical modalities exist and are used extensively, it is also recognised that a key barrier to optical imaging is the limited penetration depth maintaining high resolution Current all optical imaging may fail to capture high spatial resolution at significant depth. The depth of penetration for light in biological tissues and materials is limited by attenuation due to scattering and absorption. Depth may be a challenging parameter to quantify as it is highly dependent on the characteristic of the cell/tissue type and level of scattering that is present. As such quoting a distance may not always be helpful and one should also consider scattering lengths. In this talk I will describe approaches that push the use of light further in depth and allow us to gain wide fields of view with high resolution. In particular I will describe the use of shaped light in the form of propagation invariant light beams as well as the use of temporal focusing with single pixel detection. Finally progress in using more advance computational approaches such as deep learning to recover faithful images will be discussed

We propose a multimodal quantitative, label-free and nondestructive diagnostic metrology technique by integrating off-axis interferometric phase microscopy (IPM) and Raman spectroscopy (RS), for analyzing normal and malignant bladder tissue samples. We built a Mach–Zehnder interferometer connected to a commercial confocal microscope for imaging a large area of tissue slices, up to a few millimeters, by semi-automatic scanning of the tissue. Bright-field image of hematoxylin and eosin stained tissue slice of the same area was also acquired. Measurements of Raman spectra were acquired using our RS system with excitation wavelength of 561 nm. Using the quantitative phase information, we obtained various spatial and morphological parameters of the tissues such as the anisotropy factor, which demonstrated their direct correlation with tumor presence. This method is expected to be useful for stain-free cancer diagnosis, while obtaining both quantitative information about tissue morphological modifications and changes in tissue Raman scattering properties induced by cancer.

Recently, we proposed a simple, fast subtraction method to determine 1-D image displacement with subpixel accuracy. In this paper, we provide a theoretical basis for our method and simulate 2-D motion estimation. Results are also analysed using the correlation technique. The results show that for any general motion in 2-D, involving any combination of motion Δx, and Δy, the Subtraction Method can be used to determine the values of Δx, and Δy using 1-D calibration.

Measuring the mechanical properties of living tissue is a challenging task due to the small sizes and the fragility of the living organisms. A promising method, which works best on small scales, is the passive microrheology, which observes the motion of tracing beads within the sample. The video imaging method observes this motion by imaging the tracer particles with suitable optics (e.g. a microscope). As living tissue is a complex material, the viscoelastic properties are highly frequency dependent; therefore, a fast high-speed camera is needed to resolve the important frequencies in the 100 to 1000 Hz regime. As the data rate of high-speed cameras exceed the storage speed, only short burst of measurements can be carried out. This leads to a limited dynamic range of frequencies and missed measurement opportunities. It normally is not possible to track all the particles in real time to avoid the storage requirement of the video, as the tracking needs to be very precise and thus has a high computing demand. In this presentation, a combination of a CMOS imaging sensor with an FPGA is presented, which, in combination, allows for virtually unlimited long high-speed tracking of up to eight particles at up to 10 kHz. First, the sensor and the FPGA combinations are laid out. Secondly, the used particle tracking algorithm and its implementation is explained and benchmarked with a known state-of-the-art algorithm. Finally, this integrated sensor solution is mounted on a standard microscope and hour long tracking experiments on living 3T3 fibroblasts are carried out, studying the impact of blebbistatin on the mobility of polystyrene beads within the cell.

The effective detection of very low abundant biomarkers can enable fast diagnosis of many severe and disabling diseases (e.g. Alzheimer’s Disease) at an early stage. To develop a cost-efficient, super-sensitive optical fluorescence detection microscope, we have proposed an optical modelling approach to predict the signal-noise-ratio that considers various noise sources introduced by the components of the detection system. After the optimal design is identified, a tolerance analysis regarding typical perturbations is performed for further mechanical design and assembly. Finally, experiments have demonstrated a limit of detection at a low abundant concentration reaching 0.05 pmol/ml.

Here, we present a multimodal imaging approach based on Raman spectroscopy and PSDH imaging to detect circulating tumor cells. Particularly, the ability of tumor cells to internalize glucose faster than normal cells was investigated. Deuterated-glucose was used as Raman vibrational tag since its signal is present in the “silent” zone of Raman cell spectra. Raman information were correlated with the visualization of the cell birefringence by PSDH imaging. Each technique provides different information which combination gives a molecular and morphological signature of each cell, allowing to discriminate between normal and cancer cells with a sensitivity and specificity up to 80%.

In the last few years, gold nanoparticles (AuNPs) have been widely used in biomedical applications; unfortunately, in some cases, they induce toxic effects on biological systems. Among these, the activation of the innate immune system (inflammatory response) is considered a central issue for assessing health risks. Although the origin of nanotoxicity is not well known, the cause could be associated with the presence of contaminants on nanoparticles surface, such as bacterial endotoxin. The bacterial endotoxin, also known as Lipopolysaccharide (LPS), is the main component of gram-negative bacteria cell walls and is considered one of the major contaminants in the environment. Therefore, AuNPs can be easily contaminated during the synthesis process or their manipulation, and nanotoxicological assessment has to be performed before using AuNPs for biomedical purposes. Traditional assays for LPS detection are not always effective because AuNPs could interfere with other components and alter the final readout. Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for the analysis of molecules on metallic NPs, allowing to detect low concentration LPS with excellent sensitivity and reproducibility. In this work, we demonstrate that SERS technique can detect low amount of LPS (fg) on AuNPs (50 nm); moreover, we studied the internalization of bare and LPS-coated AuNPs in human monocyte-derived macrophages, by Raman Imaging, and their inflammatory effect by in vitro evaluation of pro and anti-inflammatory cytokine production.

Deflectometry has been widely used in topography measurement of specular surface due to the merits of full-field data acquisition, automatic data processing, high accuracy, and large dynamic range. Existing deflectometry-based methods, such as model phase measuring deflectometry, direct phase measuring deflectometry, stereo deflectometry, employ a plane screen. Due to the size and shape of the used screen, reconstruction of specular surface with large curvature can’t be carried out in single measurement. The characteristics of curved screens effectively expand the ranges of the gradient and height field of mirror. Ideally, curved screens are a part of normal cylinders. However, initial experiment shows that the commercialized curved screen changes in radius, instead of a constant value. In order to obtain the model of the curved screen, two CCD cameras capture fringes displayed on the curved screen. Stereo technique is applied to compute screen’s point cloud containing curved screen’s location and phase. The stereo technique is rectifying pairs of fringe map based on epipolar constraint to the same rotating matrix. The epipolar line of fringe map pair lies in the same row. Then disparity is computed through block match in column coordinate and refined through curve fitting. Some experiments have been carried out and results show that rectifying fringe map helps get more accurate and stable point cloud of curved screen to be suitably employed in deflectometry by a curved screen.

Multimodal nonlinear imaging techniques have proven to be a versatile tool to understand morpho-chemical properties of tissues and biomaterials. Illuminating a sample with strong laser fields gives rise to a variety of nonlinear processes. The corresponding imaging modalities are inherently label-free, non-invasive, and have intrinsic z-sectioning capabilities. By choosing appropriate wavelengths, the resulting signals can be assigned to specific molecules and structures and a combination of techniques thus provides deep insight into a samples structure and composition. Our research focuses on implementations of two-photon excited fluorescence (TPEF) arising from autofluorophores such as NADH, second harmonic generation (SHG) which can be used to specifically address non-centrosymmetric compounds like collagen/myosin, and coherent anti-Stokes Raman scattering (CARS) as a tool for imaging lipids and certain biomolecules like vitamin A. Particularly interesting is the establishment of multimodal nonlinear microscopy for medical purposes. Investigating tissues of different morpho-chemistry like human spinal disk, dura mater, liver, and colon underlines the versatility and high potential of this imaging approach, e.g. to distinguish between healthy tissue and tissue altered by mechanical stress, drugs, diseases, or tumors. Translating the knowledge gained from pathological sections and in-vitro measurements to bulk and in-vivo samples eventually helps to overcome the boundary between research and diagnostics/intraoperative application. To bring multimodal nonlinear imaging from university to clinics, setups adapted to clinical needs will be introduced within this presenation. In the past years, miniaturization and innovative optical components promoted rapid progress in custom-designed small, flexible, and sterilizable setups for fast, easy, and high-resolution imaging applicable to a broad scope of questions. Examples from our group are the compact microscope MediCARS as well as our recent developments in the field of endoscopic fiber probe designs for multimodal nonlinear microscopy such as all-optical multi-core, or piezo-tube controlled double-core-double-cladding scanning fiber probes.

We present our latest work into the modelling and fabrication of 3D auxetic metamaterials, and their evaluation as scaffolds for cell growth. Auxetic metamaterials are materials that display a negative Poisson’s ratio; when stretched, they become thicker perpendicular to the applied force. Their properties are mainly due to their architecture, rather than their chemical composition. Natural biological tissues display auxetic characteristics in organs such as skin, artery, tendon, and cancellous bone. Auxetic porous materials facilitate mass transport and, therefore, there is potential for optimal flow or delivery of nutrients, metabolic wastes and therapeutic agents. Some of their potential application are as scaffolds for blood vessels, esophageal stents, skin grafts, scaffolds for artificial lungs and cardiac patches. To make these metamaterials scaffolds, we employ 3D multiphoton polymerisation (MPP) of photopolymers. This is a technique which allows the fabrication of 3D microstructures with sub-100 nm resolution. It is based on the phenomenon of multiphoton absorption (MPA). When the beam of an ultrafast laser tightly focused inside the volume of a transparent photopolymer, it can initiate polymerisation via MPA. Moving the beam in a three-dimensional manner will result in the direct writing of 3D structures inside the volume of the photopolymer. The material employed for the fabrication of the 3D metamaterial is a homemade zirconium silicate, known to be biocompatible. Finally, we investigate the suitability of these metamaterials as scaffolds for cell growth.

Additive manufacturing technologies support the realization of surgical training devices using, typically, photopolymersbased materials. Unfortunately, the material jetting family, able to print a large range of soft and hard polymers, requires expensive machines and materials, which are not always available. On the other hand, vat polymerization fails in the resolution/volume ratio and in the mechanical properties reconstruction. Stereolithographic 3D printers, mostly used in dental surgery, make possible to realize cheap and sustainable models for training activity using only one material, reducing the possibility to obtain different mechanical characteristics. Moreover, the printed objects have to be treated (i.e. curing post-processing) in order to obtain the required performances, that could be preserved for long term storing. The aim of the proposed approach is to assure the surgeons’ skills improvement through bionic-based surgical 3D printed models and smart devices, able to reproduce the same perception of a real surgical activity. We demonstrated how it is possible develop smart devices capable to take into account the same characteristics of different materials (i.e. bone and spongy bone) even if stored for a long time.

In this work, we present an ultra-high sensitivity (S) refractive index (RI) sensor for biological applications. Silicon on insulator (SOI) ring resonator (RR) based RI sensors have been extensively investigated. Light propagation is studied in the small SOI RRs based on sub-wavelength grating (SWG) waveguides with aqueous claddings. For the device analysis, the two-dimensional conformal transformation method is jointly used with the effective index method. Furthermore, FEM simulations of this structure have been also performed in both the air and water claddings. These good-matched results are further compared with experimental and FDTD results in other valid references. Having found a high data matching between our results and experimental results, we repeat our calculation for different side-widths (d=150-225 nm) and ring radii (7-10 µm). For each ring radius, the best amount of side-width is found. Then bulk refractive index sensing experiments are simulated using changing cladding RI in the range of nc=1.332-1.350 RIU. These RI changes correspond to concertation changes of glucose-water solutions in the cladding. Finally, we obtain the highest sensitivity of 24946 nm/RIU in the case of a radius 7 µm, 500 nm width, 220 nm height, 150 nm side-width, and T of 50 nm between the grating arrays on the RR. It is worth noting that this proposed structure shows 150 times sensitivity enhancement compared to a standard SOI RR-based biosensor with the same radius, w, and h. In addition, the RI sensor suggested in this work shows ultra-high sensitivity in a dynamic range of 1.332-1.352 RIU. This range of RI variation covers almost all the RI variations of the blood components. These results show excellent potential for employing the proposed SOI RR based on SWG waveguides in a liquid-biopsy analysis of patients with cancer. These might pave the way toward the early detection of cancers.

We disclose a new composite featuring noble-metal nanorods in porous chitosan/polyvinyl alcohol mats or sponges for applications in wound healing and monitoring. The plasmonic component provides synergistic opportunities for the optical activation of functions as near-infrared laser welding, and the remote assessment of parameters of prognostic relevance in wound monitoring, like the environmental level of oxidative stress. At the same time, the polymer blend is ideal to bind connective tissue upon photothermal activation, and to support fabrication processes that ensure high porosity, such as electrospinning, thus paving the way to cellular repopulation and antimicrobial protection. In particular, we address the stabilization of the electrospun mats by cross-linking in a vapor of glutaraldehyde, and their cytotoxicity to a model of relevance in wound dressing like human fibroblasts.

Electrospinning technologies (e.g traditional electrospinning and near-field electrospinning) have been widely considered for the design of biomimetic scaffolds for tissue engineering and wound healing. The great advantage of electrospinning technologies lies in the possibility of controlling the surface area, porosity and pore size of the final materials as well as the loading and the release kinetics of the drugs. In addition, the processing of proteins by electrospinning technologies is a promising approach to design functional scaffolds offering better bioctivity (ability to support cell adhesion and growth), better drug stability and safer applicability on skin. In this work, keratin proteins of mammal origin, obtained from wool wastes were processed into electrospun mats. In particular, electrospun mats made of keratin nanofibers, keratin/hydrotalcites hybrid nanofibers and keratin/poly- (butylene succinate) blend nanofibers, loaded with diclofenac were compared as drug delivery systems and scaffolds for cell growth. All the electrospun mats are characterized by layers of interconnected web of nanofibers having a mean diameter of about 200 nm. They are able to adhere to wet skin to support fibroblast cells adhesion and growth. The diclofenac is almost totally released from the keratin nanofibers through a matrix swelling controlled mechanism and from keratin/hydrotalcites hybrid nanofibers through a totally Fickian diffusion mechanism. Instead, only the 50% of drug is released from the keratin/PBS blend nanofibers through a totally Fickian diffusion mechanism. These preliminary obtained results can be exploited to design functional patches with desired bioactivity and drug release profiles through an additive electrospinning process, to be applied in tissue engineering and wound healing. Moreover, the use of a chemical sensors in addition to drugs could be a promising strategy to develop functional patches acting as drug delivery and sensors systems.

An ultrafast laser assisted mesoscale lithography will be introduced by presenting its technological principles, current state-of-the-art and potential in 3D printing of diverse materials ranging from biocompatible, biodegradable and renewable organics to amorphous, ceramic and crystalline inorganics. Its applications towards prototyping and producing bio-medical implants, micro-optics and nano-photonics as well as creating micro-fluidic sensors will be shown. A special emphasis on the development and applications of microfabricated structures for life-sciences will be given, namely customization of laser direct write lithography-made 3D scaffolds for optimized in vivo outcome. Furthermore, the possibility to employ the technique for precision additive manufacturing out of plant-based resins and pure inorganics will be demonstrated. Finally, some unique functional properties of selected prototypes will be provided in detail validating their high efficiency performance.

In this work we present a microscope 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 device is 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.

Oral drug delivery is a preferred method for drug administration because it is economical and convenient to the patient [1]. Uptake from the gut to the blood is mediated by the intestinal barrier, which exhibits some selectivity, i.e., mechanisms in the intestinal barrier define which compounds are absorbed and which are not. The limited understanding of these mechanisms impedes the design and development of new oral drugs. On the horizon: Emerging organ-on-a-chip models of the intestine may provide new insights into structure-function relationships in biological barriers like the gut-blood interface of the intestine. These small ‘artificial organs’ can host biological cells, which mimic the natural behavior of intestinal cells, thus providing a highly controlled platform for fundamental bioresearch and, ultimately, drug-screening [2]. Our group develops and implements optical imaging technologies specifically geared for imaging organ-on-a-chip systems. At the SPIE conference “Optical Methods for Inspection, Characterization, and Imaging of Biomaterials V” we will present the application of two-photon microscopy for imaging of relatively thick 3D printed organ-on-a-chip systems and discuss imaging-challenges related to sample-induced aberrations and scattering. We will also present our ongoing work to implement an adaptive optics-enabled lattice light sheet microscope [3], which (we anticipate) will enable high-resolution imaging inside complex organ-on-a-chip systems by compensating the sample-induced aberrations. [1] P. Viswanathan, Y. Muralidaran, and G. Ragavan, "Challenges in oral drug delivery: a nano-based strategy to overcome", Nanostructures for Oral Medicine, Elsevier (2017) 173-201. [2] R. Zhang and N.B. Larsen, "Stereolithographic hydrogel printing of 3D culture chips with biofunctionalized complex 3D perfusion networks", Lab on a Chip 17.24 (2017) 4273-4282. [3] T. Liu, S. Upadhyayula, et al., "Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms", Science 360.6386 (2018) eaaq1392.

We describe several bioengineered cell models developed by our group. We develop tools for cell culture allowing fluorescence diagnostics on the cellularised constructs cultured within, both in 3D and prolonged culture times extending to several weeks. These cell models proved able to recapitulate in vitro several slowly developing biological processes such as the regeneration of a cartilaginous tissue by cartilage cells, the formation of a bone metastasis by breast cancer cells, the instruction of adaptive immune cells as occurs in a lymphnode, and the neuroprotective effect on pathological neurons of mesenchymal stem cell secretome. We also scaled down these tools in the aim to better control stem cell function in our models, by applying two-photon laser polymerization to fabricate micro scaffolds for cell expansion. We were able to condition mesenchymal stem cells, neural precursor cells and embryonic stem cells towards maintenance of a greater stemness and multipotency or pluripotency, compared to conventional flat culture. This result opens up an avenue towards a safer use of these cells for stem cells therapies. Finally, we describe a revolutionary concept to implant the cell model in a living organism, to be revascularized by the host allowing for studies involving interactions with the host immune system.

We exploit two-photon laser writing to fabricate 3D biocompatible proteinaceous microstructures (∼1 to 50 𝜇m in lateral size) with tunable elasticity and photo-thermal activity in the near-infrared. Structure printing relies on the photo-crosslinking of the protein bovine serum albumin (BSA, 50 mg/mL) initiated by the Rose Bengal dye (2 mM concentration), whereas photo-thermal functionality is achieved by the dispersion of non-spherically symmetric metallic nanoparticles into the ink. Aiming at a subsequent application of the fabricated microstructures as platforms for cell growth and stimulation, we carry out a thorough characterization of their mechanical and photo-thermal properties. Preliminary data obtained by AFM indentation have quantified the structures Young modulus in the broad 100-1000 kPa range depending on the BSA concentration. Stiffness is further characterized here by subjecting the fabricated microstructures to steady flow in a microfluidic device, and by quantifying their real-time bending by a conventional transmitted light microscope. In parallel, we focus on the optimization of the photo-thermal activity of the structures. Anisotropic gold nanoparticles, dispersed in the ink, get trapped into the structure during photo-crosslinking and lead to localized heat release upon excitation in the near-infrared. The temperature increment is rapidly (∼1 s) reached and maintained under continuous wave laser irradiation at 800 nm; the amplitude of the temperature variation is quantified as a function of the incident laser power by means of infrared thermography and is correlated to both the structure thickness and the nanoparticles concentration. The resulting spatially confined heat loads could be exploited to induce highly localized responses in cells. In this direction, proteinaceous photo-thermal microstructures can be used to physically induce the differentiation of cells (e.g. neurons or fibroblasts) in a spatially controlled manner.

Ferroelectric platforms based on photovoltaic LiNbO3:Fe are a powerful tool for trapping and patterning of micro- and nano-particles. By using this tool, metallic structures have been assembled from Ag and Au nanoparticles on the surface of the ferroelectric crystal. Their plasmonic response has been checked through the measurement of the reflectance extinction spectra. Then, the fluorescence enhancement of relevant biomaterials such as DNA and spermatozoa tagged with a variety of fluorophores such as DAPI, fluorescein, or rhodamine B has been investigated. Relevant plasmonic enhancement extended along the visible spectral range have been obtained, particularly with Ag nanoparticle structures. The results suggest the potential of the obtained metallic structures to improve bio-imaging procedures. Finally, we have found that the metal nanoparticle patterns are able to induce an organization of the sperm cells along the nanoparticle fringe structures.

Oral drug delivery is a preferred method for drug administration because it is economical and convenient to the patient. Uptake from the gut to the blood is mediated by the intestinal barrier, which exhibits some selectivity, i.e., mechanisms in the intestinal barrier define which compounds are absorbed and which are not. The limited understanding of these mechanisms impedes the design and development of new oral drugs. On the horizon: Emerging organ-on-chip models of the intestine may provide new insights into structure-function relationships in biological barriers like the gutblood interface of the intestine. These small ‘artificial organs’ can host live cells, which mimic the natural behavior of intestinal cells, thus providing a highly controlled platform for fundamental bioresearch and, ultimately, drug-screening. Our groups develop and implement optical imaging technologies specifically geared for imaging organ-on-chip systems. At the SPIE conference “Optical Methods for Inspection, Characterization, and Imaging of Biomaterials V” we will present the application of two-photon microscopy for imaging of relatively thick 3D printed organ-on-chip systems and discuss imaging-challenges related to sample-induced aberrations. We will also present our ongoing work to implement an adaptive optics-enabled lattice light sheet microscope, which (we anticipate) will enable long-term, high-resolution imaging inside complex organ-on-chip systems by reducing phototoxicity and compensating sample induced aberrations.