Showing papers by "Pietro Ferraro published in 2017"

Tomographic flow cytometry by digital holography.

Abstract: High-throughput single-cell analysis is a challenging task. Label-free tomographic phase microscopy is an excellent candidate to perform this task. However, in-line tomography is very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions. We demonstrate that by exploiting the random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain in-line phase-contrast tomography, if smart strategies for wavefront analysis are adopted. In fact, surprisingly, a priori knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely retrieved through digital holography wavefront numerical analysis. This approach makes continuous-flow cytotomography suitable for practical operation in real-world, single-cell analysis and with a substantial simplification of the optical system; that is, no mechanical scanning or multi-direction probing is required. A demonstration is given for two completely different classes of biosamples: red blood cells and diatom algae. An accurate characterization of both types of cells is reported, despite their very different nature and material content, thus showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells.

Endowing a plain fluidic chip with micro-optics: a holographic microscope slide.

Abstract: Lab-on-a-Chip (LoC) devices are extremely promising in that they enable diagnostic functions at the point-of-care Within this scope, an important goal is to design imaging schemes that can be used out of the laboratory In this paper, we introduce and test a pocket holographic slide that allows digital holography microscopy to be performed without an interferometer setup Instead, a commercial off-the-shelf plastic chip is engineered and functionalized with this aim The microfluidic chip is endowed with micro-optics, that is, a diffraction grating and polymeric lenses, to build an interferometer directly on the chip, avoiding the need for a reference arm and external bulky optical components Thanks to the single-beam scheme, the system is completely integrated and robust against vibrations, sharing the useful features of any common path interferometer Hence, it becomes possible to bring holographic functionalities out of the lab, moving complexity from the external optical apparatus to the chip itself Label-free imaging and quantitative phase contrast mapping of live samples are demonstrated, along with flexible refocusing capabilities Thus, a liquid volume can be analyzed in one single shot with no need for mechanical scanning systems

Holographic microscope slide in a spatio-temporal imaging modality for reliable 3D cell counting.

Abstract: In the current trend of miniaturization and simplification of imaging flow cytometry, Lab-on-a-Chip (LoC) microfluidic devices represent an innovative and cost-effective solution. In this framework, we propose for the first time a novel platform based on the compactness of a holographic microscope slide (HMS) in combination with the new computational features of space-time digital holography (STDH) that uses a 1D linear sensor array (LSA) instead of 2D CCD or CMOS cameras to respond to real diagnostic needs. In this LoC platform, computational methods, holography, and microfluidics are intertwined in order to provide an imaging system with a reduced amount of optical components and capability to achieve reliable cell counting even in the absence of very accurate flow control. STDH exploits the sample motion into the microfluidic channel to obtain an unlimited field-of-view along the flow direction, independent of the magnification factor. Furthermore, numerical refocusing typical of a holographic modality allows imaging and visualization of the entire volume of the channel, thus avoiding loss of information due to the limited depth of focus of standard microscopes. Consequently, we believe that this platform could open new perspectives for enhancing the throughput by 3D volumetric imaging.

Direct Writing of Microfluidic Footpaths by Pyro-EHD Printing.

Abstract: In this study, we report a direct writing method for the fabrication of microfluidic footpaths by pyro-electrohydrodynamic (EHD) jet printing. Here, we propose the use of a nozzle-free three-dimensional printing technique for the fabrication of printed structures that can be embedded in a variety of soft, transparent, flexible, and biocompatible polymers and thus easily integrated into lab-on-chip devices. We prove the advantage of the high resolution and flexibility of pyro-EHD printing for the realization of microfluidic channels well below the standard limit in dimension of conventional ink-jet printing technique and simply adaptable to the end-user desires in terms of geometry and materials. Starting from the description of the innovative approach proposed for the channel fabrication, we demonstrate the design, fabrication, and proof of a microfluidic matrix of interconnected channels. The method described here could be a breakthrough technology for the fabrication of in situ implantable, stretchable,...

Investigating fibroblast cells under "safe" and "injurious" blue-light exposure by holographic microscopy.

Abstract: The exposure to visible light has been shown to exert various biological effects, such as erythema and retinal degeneration. However, the phototoxicity mechanisms in living cells are still not well understood. Here we report a study on the temporal evolution of cell morphology and volume during blue light exposure. Blue laser irradiation is switched during the operation of a digital holography (DH) microscope between what we call here "safe" and "injurious" exposure (SE & IE). The results reveal a behaviour that is typical of necrotic cells, with early swelling and successive leakage of the intracellular liquids when the laser is set in the "injurious" operation. In the phototoxicity investigation reported here the light dose modulation is performed through the very same laser light source adopted for monitoring the cell's behaviour by digital holographic microscope. We believe the approach may open the route to a deep investigation of light-cell interactions, with information about death pathways and threshold conditions between healthy and damaged cells when subjected to light-exposure. 3D Morphology and quantitative phase information from late stage of necrosis cell death.

Numerical Manipulation of Digital Holograms for 3-D Imaging and Display: An Overview

Abstract: In the last two decades, thanks to the considerable technological development of solid-state sensors, digital holography (DH) has gained credits as the elective imaging technique for applications in various research fields, e.g., material science, biotechnology, as well as a diagnostic tool for applications at lab-on-a-chip scale. However, since its beginning, the intrinsic coherent nature of holography made 3-D imaging and display one of its preferred applications. Still today, several research groups around the world are working to develop novel numerical solutions in the framework of DH-based 3-D imaging and display technology. In this paper, we report an overview of the most important contributions given to this field over the last years.

Digital holography as 3D tracking tool for assessing acoustophoretic particle manipulation.

Abstract: The integration of digital holography (DH) imaging and the acoustic manipulation of micro-particles in a microfluidic environment is investigated. The ability of DH to provide efficient 3D tracking of particles inside a microfluidic channel is exploited to measure the position of multiple objects moving under the effect of stationary ultrasound pressure fields. The axial displacement provides a direct verification of the numerically computed positions of the standing wave's node, while the particles' transversal movement highlights the presence of nodes in the planar direction. Moreover, DH is used to follow the aggregation dynamics of trapped spheres in such nodes by using aggregation rate metrics.

Biolens behavior of RBCs under optically‐induced mechanical stress

Abstract: In this work, the optical behavior of Red Blood Cells (RBCs) under an optically-induced mechanical stress was studied. Exploiting the new findings concerning the optical lens-like behavior of RBCs, the variations of the wavefront refracted by optically-deformed RBCs were further investigated. Experimental analysis have been performed through the combination of digital holography and numerical analysis based on Zernike polynomials, while the biological lens is deformed under the action of multiple dynamic optical tweezers. Detailed wavefront analysis provides comprehensive information about the aberrations induced by the applied mechanical stress. By this approach it was shown that the optical properties of RBCs in their discocyte form can be affected in a different way depending on the geometry of the deformation. In analogy to classical optical testing procedures, optical parameters can be correlated to a particular mechanical deformation. This could open new routes for analyzing cell elasticity by examining optical parameters instead of direct but with low resolution strain analysis, thanks to the high sensitivity of the interferometric tool. Future application of this approach could lead to early detection and diagnosis of blood diseases through a single-step wavefront analysis for evaluating different cells elasticity. © 2017 International Society for Advancement of Cytometry.

Rapid Prototyping of Plastic Lab-on-a-Chip by Femtosecond Laser Micromachining and Removable Insert Microinjection Molding

Abstract: We have introduced a new hybrid fabrication method for lab-on-a-chip devices through the combination of femtosecond laser micromachining and removable insert micro-injection molding. This method is particularly suited for the fast prototyping of new devices, while maintaining a competitive low cost. To demonstrate the effectiveness of our approach, we designed, fabricated, and tested a completely integrated flow cytometer coupled to a portable media device. The system operation was tested with fluorescent plastic micro-bead solutions ranging from 100 beads/μL to 500 beads/μL. We demonstrated that this hybrid lab-on-a-chip fabrication technology is suitable for producing low-cost and portable biological microsystems and for effectively bridging the gap between new device concepts and their mass production.

Simple and Rapid Bioink Jet Printing for Multiscale Cell Adhesion Islands.

Abstract: A simple and rapid process for multiscale printing of bioinks with dot widths ranging from hundreds of microns down to 0.5 μm is presented. The process makes use of spontaneous surface charges generated pyroelectrically that are able to draw little daughter droplets directly from the free meniscus of a mother drop through jetting ("p-jet"), thus avoiding time-consuming and expensive fabrication of microstructured nozzles. Multiscale can be easily achieved by modulating the parameters of the p-jet process. Here, it is shown that the p-jet allows us to print well-defined adhesion islands where NIH-3T3 fibroblasts are constrained to live into cluster configurations ranging from 20 down to single cell level. The proposed fabrication approach can be useful for high-throughput studies on cell adhesion, cytoskeleton organization, and stem cell differentiation.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

Abstract: The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion.

Label-free analysis of mononuclear human blood cells in microfluidic flow by coherent imaging tools.

Abstract: The investigation of the physical properties of peripheral blood mononuclear cells (PBMC) is of great relevance, as they play a key role in regulating human body health. Here we report the possibility to characterize human PBMC in their physiological conditions in a microfluidic-based measurement system. A viscoelastic polymer solution is adopted for 3D alignment of individual cells inflow. An optical signature (OS) acquisition of each flowing cell is performed using a wide angle light scattering apparatus. Besides, a quantitative phase imaging (QPI) holographic system is employed with the aim (i) to check the position in flow of individual cells using a holographic 3D cell tracking method; and (ii) to estimate their 3D morphometric features, such as their refractive index (RI). Results obtained by combining OS and QPI have been compared with literature values, showing good agreement. The results confirm the possibility to obtain sub-micrometric details of physical cell properties in microfluidic flow, avoiding chemical staining or fluorescent labelling.

Investigation of pyroelectric fields generated by lithium niobate crystals through integrated microheaters

Abstract: We present a deep investigation of pyroelectric fields generated by lithium niobate crystals through integrated microheater structures. The microheaters are made of highly compact titanium microcircuits able to dissipate heat through a low-power consuming Joule effect. Microheaters with diverse geometries were designed and fabricated on the + Z face of lithium niobate crystals, in order to characterize pyroelectric fields with different distributions. The pyroelectric effect was studied under ambient conditions analysing the current impulses detected using a metallic probe connected to an oscilloscope. The current impulses were related to the air breakdown induced by the electric field arising between the −Z face of the crystal and the metallic tip. We show that the fabrication technique is relatively easy to accomplish and we analyse the thermal behaviour of the microheaters both theoretically and experimentally. The results show how such microheaters are able to control the intensity and the spatial distribution of the pyroelectric fields at microscale.

Direct Evidence of Polar Ordering and Investigation on Cytophilic Properties of Pyroelectrified Polymer Films by Optical Second Harmonic Generation Analysis

Abstract: Pyroelectrification (PE) has been proposed as a low-cost electrode-free tool for orienting and aligning dipole molecules in polymer layers, by means of the electric field produced by a pyroelectric crystal under appropriate thermal stimulations Probing and assessing the coherent polarization arrangement is of fundamental importance for the process control and optimization but is also a challenging task In fact, the probing operation must be noninvasive and avoid any disarrangement the dipoles and thus without interfering with the resulting surface charges Here we show that the PE-induced polar ordering can be probed in situ by an electrode-free analysis based on the measurement of the intensity of the second-harmonic optical wave generated by the polymer film In fact, the results show a substantial enhancement of the second-order susceptibility of the polymer layer, caused by the PE-induced dipoles alignment Moreover, thanks to this approach, it is demonstrated the ability of PE process to polarize p

Food quality inspection by speckle decorrelation properties of bacteria colonies

Abstract: The development of tools for rapid food quality inspection is a highly pursued goal. These could be valuable devices to be used by food producers in factories or the customers themselves in specific installations at the marketplace. Here we show how speckle patterns in coherent imaging systems can be can be employed as indicators of the presence of bacteria colonies contaminating food or water. Speckle decorrelation is induced by the self-propelling movement of these organisms when they interact with coherent light. Hence, their presence can be detected using a simple setup in a condition in which the single element cannot be imaged, but the properties of the ensemble can be exploited. Thanks to the small magnification factor we set, our system can inspect a large Field-of-View (FoV). We show the possibility to discriminate between fresh and contaminated food, thus paving the way to the rapid food quality testing by consumers at the marketplace.

Interferometric measurement of film thickness during bubble blowing

Abstract: In this paper, we propose digital holography in transmission configuration as an effective method to measure the time-dependent thickness of polymeric films during bubble blowing. We designed a complete set of experiments to measure bubble thickness, including the evaluation of the refractive index of the polymer solution. We report the measurement of thickness distribution along the film during the bubble formation process until the bubble‘s rupture. Based on those data, the variation range and variation trend of bubble film thickness are clearly measured during the process of expansion to fracture is indicated.

Direct fabrication of polymer micro-lens array

Abstract: In order to break the rigidity of classic lithographic techniques, a flexible pyro-electric-electrohydrodynamic (EHD) inkjet printing is presented. In particular, here is showed a method able to manipulate highly viscous polymers, usable for optical integrated devices. The system proposed reaches spatial resolution up to the nano-scale and can print, for instance, nano-particles and high viscous polymer solutions. This technique allows writing patterns directly onto a substrate of interest in 2D or in 3D configuration and is studied in order to overcome limitations in terms of type of materials, geometry and thickness of the substrate. In the present work, we show the potential of pyro-EHD printing in fields as optics and micro-fluidics. A micro-channel chip is functionalized with a PDMS-made micro-lenses array, directly printed on the chip. The geometric properties and the quality of the lenses are evaluated by a Digital Holography (DH) analysis.

Imaging cytometry in a plastic ultra-mobile system

Abstract: We present a cost-effective and highly-portable plastic prototype that can be interfaced with a cell phone to implement an optofluidic imaging cytometry platform. It is based on a PMMA microfluidic chip that fits inside an opto-mechanical platform fabricated by a 3D printer. The fluorescence excitation and imaging is performed using the LED and the CMOS from the cell phone increasing the compactness of the system. A custom developed application is used to analyze the images and provide a value of particle concentration.

Peripheral blood mononuclear cells analysis in microfluidic flow by coherent imaging tools

Abstract: Cell of human blood stream are divided into two groups: Red Blood Cells (RBC) and White Blood Cells (WBC). RBC have a peculiar biconcave disk shape and they are responsible for the delivering of O 2 and CO 2 through the body. WBC are a more widespread class of cell ensuring immunity against pathogens. They can be divided in two main classes: granulocyte cells and A-granulocyte cells. Neutrophils, basophils and eosinophils belong to the granulocyte cell class, while lymphocytes and monocytes belong to A-granulocyte. Both in RBC and WBC, the intrinsic physical properties of a cell are indicators of cell condition and, furthermore, of the overall human body state. Thus, the accurate comprehension of the physiological structure of WBCs is fundamental to recognize diseases. Here we show the possibility to simple and straightforwardly characterize the physical properties of individual RBC and mononuclear WBC in a microfluidic context, using a wide angle light scattering apparatus and a corresponding theoretical simulation of Optical Signature (OS). A non-Newtonian polymer alignment solution for cell is used to ensure an individual cell alignment in the microfluidic flow, thus permitting a precise investigation. Additionally, Quantitative Phase Imaging (QPI) holographic measurements are performed to estimate cell morphometric features, such as their refractive index. We analyzed more than 200 WBCs and 100 RBCs of three different probands. Results showed distinct cell populations according to their measured dimensions and shape, which can be associated to the presence of RBC, lymphocytes and monocytes.

Label-free investigation of the effects of lithium niobate polarization on cell adhesion

Abstract: The determination of contact area is pivotal to understand how biomaterials properties influence cell adhesion. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, we use for the first time Holographic Total Internal Reflection Microscopy (HoloTIRM) to study the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells. The selective illumination of a very thin region directly above the substrate, achieved by Total Internal Reflection, provides high-contrast images of the contact regions. Holographic recording, on the other hand, allows for label-free quantitative phase imaging of the contact areas between cells and LN. Phase signal is more sensitive in the first 100nm and, thus more reliable in order to locate focal contacts. This work shows that cells adhering on negatively polarized LN present a significant increase of the contact area in comparison with cells adhering on the positively polarized LN substrate, as well as an intensification of contact vicinity. This confirms the potential of LN as a platform for investigating the role of charges on cellular processes. The similarity of cell adhesion behavior on negatively polarized LN and glass control also confirms the possibility to use LN as an active substrate without impairing cell behavior.

Laser interrogation techniques for high-sensitivity strain sensing by fiber-Bragg-grating structures

Abstract: Novel in terrogation methods for static and dynamic measurements of mechanical def ormations by fiber Bragg-gratings (FBGs) structures are presented. The sensor-reflected radi ation gives information on suffered strain, with a sen sitivity dependent o n the in terrogation setup. Different approaches have been carried out, based on laser-frequency modulation techniques a nd near-IR lasers, to measure strain in single-FBG and in resonant high-reflectivity FBG arra ys. In particular, for t he fiber resonator, the la ser frequency is actively locked to the cavity resonances by the P ound-Drever-Hall tec hnique, thus tracking any frequency change due to deformations. The l oop error an d correct ion signals fed back to the laser are used as strain monitor. Sen sitivity limits vary between 200 nHz in the q uasi-static d omain (0.52 Hz), and between 1 and 4 nHz in the 0.4-1 kHz range for th e single-FBG scheme, while strain down to 50 p can be detected by using the laser-cavity-locked method. 1. EXPERIMENTAL METHODOLOGY A sketch of the experimental apparatus is represented in Fig. 1. A 1560-nm DFB di ode laser i s driven b y a Peltier-based PID module and a low-noise power source for temperature control and current supply. The diode is fiber coupled t o an array made of two i dentical FBGs with peak reflectivity > 99.5 % and a relative distance of 13 cm. An external optical isolation stage is used to avoid undesired optical feedback to the laser.

A method for total noise removal in digital holography based on enhanced grouping and sparsity enhancement filtering

Abstract: In digital holography (DH), the coherent nature of the employed light sources severely degrades the holographic reconstructions due to a mixture of speckle and incoherent additive noise. These can affect both the visual quality in holographic imaging and display, and the accuracy of quantitative phase-contrast reconstructions. Typically, the noise problem is tackled by reducing the illumination coherence, thus the most intuitive way involves the recording of multiple uncorrelated holograms to be incoherently combined. This framework is known as Multi-Look DH (MLDH). However, single shot recordings are highly desirable in DH, and numerical methods are required to go beyond the improvement bound of ML techniques. Among the existing image processing methods, the 3D Block Matching filtering (BM3D) has shown the best performance. Here we present the MLDH-BM3D, a method specifically suitable to filter DH images that combines the two aforementioned strategies to overcome their respective limitations. We demonstrate the effectiveness of this framework in three different experimental situations, i.e. reconstructions of single wavelength holograms and color holograms in the visible region and the challenging case of the Infrared Radiation Digital Holography (IRDH) reconstructions, where a very severe noise degradation occurs.

Investigation on microfluidic particles manipulation by holographic 3D tracking strategies

Abstract: We demonstrate a 3D holographic tracking method to investigate particles motion in a microfluidic channel while unperturbed while inducing their migration through microfluidic manipulation. Digital holography (DH) in microscopy is a full-field, label-free imaging technique able to provide quantitative phase-contrast. The employed 3D tracking method is articulated in steps. First, the displacements along the optical axis are assessed by numerical refocusing criteria. In particular, an automatic refocusing method to recover the particles axial position is implemented employing a contrast-based refocusing criterion. Then, the transverse position of the in-focus object is evaluated through quantitative phase map segmentation methods and centroid-based 2D tracking strategy. The introduction of DH is thus suggested as a powerful approach for control of particles and biological samples manipulation, as well as a possible aid to precise design and implementation of advanced lab-on-chip microfluidic devices.

Computational tomographic phase microscopy

Abstract: We investigate a computational-based approach in tomographic flow cytometry by digital holography to characterize self-rolling cells in microfluidic channels. Different experimental validations, by employing red blood cells (RBCs) and diatoms, are made.

High-throughput label-free optofluidic imaging and 3D tracking using a pocket holographic microscope slide

Abstract: We designed a LoC with embedded optofluidic holographic microscopy capabilities. Object flow allows unlimited FoV microscopy of samples in a liquid volume. High-throughput counting and 3D tracking of RBCs is demonstrated.