Oriella Gennari

3D morphometry of red blood cells by digital holography

Three dimensional (3D) morphometric analysis of flowing and not‐adherent cells is an important aspect for diagnostic purposes. However, diagnostics tools need to be quantitative, label‐free and, as much as possible, accurate. Recently, a simple holographic approach, based on shape from silhouette algorithm, has been demonstrated for accurate calculation of cells biovolume and displaying their 3D shapes. Such approach has been adopted in combination with holographic optical tweezers and successfully applied to cells with convex shape. Nevertheless, unfortunately, the method fails in case of specimen with concave surfaces. Here, we propose an effective approach to achieve correct 3D shape measurement that can be extended in case of cells having concave surfaces, thus overcoming the limit of the previous technique. We prove the new procedure for healthy red blood cells (RBCs) (i.e., discocytes) having a concave surface in their central region. Comparative analysis of experimental results with a theoretical 3D geometrical model of RBC is discussed in order to evaluate accuracy of the proposed approach. Finally, we show that the method can be also useful to classify, in terms of morphology, different varieties of RBCs.

Effects of Lithium Niobate Polarization on Cell Adhesion and Morphology

Understanding how the interfacial effects influence cell adhesion and morphology is of fundamental interest for controlling function, growth, and movement of cells in vitro and in vivo. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells is investigated. The spontaneous polarization of LN has one of the largest known magnitudes at room temperature (∼78 μC/cm(2)), and its orientation can be patterned easily by an external voltage, this motivating highly the investigation of its interaction with cells. Immunofluorescence and migration assays show strong evidence that the surface polarity regulates the adhesion functions, with enhanced spreading of the cytoskeleton on the negative face. The results suggest the potential of LN as a platform for investigating the role of charges on cellular processes, thus favoring new strategies in fabricating those biocompatible constructs used for tissue engineering. In fact, the orientation of the high-magnitude polarization can be patterned easily and, in combination with piezoelectric, pyroelectric, and photorefractive properties, may open the route to more sophisticated charge templates for modulating the cell response.

Electrohydrodynamic Assembly of Multiscale PDMS Microlens Arrays

In this paper, we introduce an easy multiscale approach for the fabrication of polymer microlens arrays through a self-assembling process driven by the electrohydrodynamic (EHD) pressure. This method represents a simple alternative to the conventional soft lithography techniques. A thin layer of liquid polymer is deposited on a microengineered ferroelectric crystal and can be self-assembled and cross-linked in a single-step process as a consequence of the pyroelectric effect activated by simply heating the substrate. Although the EHD instability induced by the pyroelectric effect was discovered in principle few years ago, here we demonstrate a systematic investigation for fabrication of microlens arrays in a multiscale range (i.e., between 25 to 200 μm diameter) with high degree of uniformity. By controlling the polymer instability driven by EHD, we report on two different microoptical shapes can be obtained spontaneously, i.e., spherical or toroidal. Here, we show how the geometrical properties and the focal length of the lens array are modulated by controlling two appropriate parameters. Such microlenses can be useful also as polymer patterned arrayed microstructures for optical data interconnections, OLEDs efficient light extraction, concentrating light in energy solar cells, imaging and 3-D display solutions, and other photonics applications.

Investigation on cone jetting regimes of liquid droplets subjected to pyroelectric fields induced by laser blasts

Electrical conductivity and viscosity play a major role in the tip jetting behaviour of liquids subjected to electrohydrodynamic (EHD) forces, thus influencing significantly the printing performance. Recently, we developed a nozzle- and electrode-free pyro-EHD system as a versatile alternative to conventional EHD configurations and we demonstrated different applications, including inkjet printing and three-dimensional lithography. However, only dielectric fluids have been used in all of those applications. Here, we present an experimental characterization of the pyro-EHD jetting regimes, induced by laser blasts, of sessile drops in case of dielectric and conductive liquids in order to extend the applicability of the system to a wider variety of fields including biochemistry and biotechnology where conductive aqueous solutions are typically used.

Label-free quantification of the effects of lithium niobate polarization on cell adhesion via holographic microscopy

The surface of a c- cut ferroelectric crystal at room temperature is characterized by the so-called screening surface charges, able to compensate the charge due to the spontaneous polarization. Recently, these charges inspired the investigation of the interaction affinity of live cells with lithium niobate and lithium tantalate crystals. However, different knowledge gaps still remain that prevent a reasonable application of these materials for biological applications. Here, a label-free holographic total internal reflection microscopy is shown; the technique is able to evaluate quantitatively the contact area of live fibroblast cells adhering onto the surface of a ferroelectric lithium niobate crystal. The results show values of contact area significantly different between cells adhering onto the positive or negative face of the crystal. This reinforces the reasons for using the polarization charge of these materials to study and/or control cellular processes and, thus, to develop an innovative platform based on polar dielectric functional substrates.

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

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.

Pyroelectric Effect Enables Simple and Rapid Evaluation of Biofilm Formation

Biofilms are detrimental to human life and industrial processes due to potential infections, contaminations, and deterioration. Therefore, the evaluation of microbial capability to form biofilms is of fundamental importance for assessing how different environmental factors may affect their vitality. Nowadays, the approaches used for biofilm evaluation are still poor in reliability and rapidity and often provide contradictory results. Here, we present what we call biofilm electrostatic test (BET) as a simple, rapid, and highly reproducible tool for evaluating in vitro the ability of bacteria to form biofilms through electrostatic interaction with a pyroelectrified carrier. The results show how the BET is able to produce viable biofilms with a density 6-fold higher than that on the control, after just 2 h incubation. The BET could pave the way to a rapid standardization of the evaluation of bacterial resistance among biofilm-producing microorganisms. In fact, due to its simplicity and cost-effectiveness, it is well suited for a rapid and easy implementation in a microbiology laboratory.

Lab on chip optical imaging of biological sample by quantitative phase microscopy

Quantitative imaging and three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes at Lab on Chip scale. Diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. In recent years digital holography (DH) has been improved to be considered as suitable diagnostic method in several research field. In this paper we demonstrate that DH can be used for retrieving 3D morphometric data for sorting and diagnosis aims. Several techniques exist for 3D morphological study as optical coherent tomography and confocal microscopy, but they are not the best choice in case of dynamic events as flowing samples. Recently, a DH approach, based on shape from silhouette algorithm (SFS), has been developed for 3D shape display and calculation of cells biovolume. Such approach, adopted in combination with holographic optical tweezers (HOT) was successfully applied to cells with convex shape. Unfortunately, it’s limited to cells with convex surface as sperm cells or diatoms. Here, we demonstrate an improvement of such procedure. By decoupling thickness information from refractive index ones and combining this with SFS analysis, 3D shape of concave cells is obtained. Specifically, the topography contour map is computed and used to adjust the 3D shape retrieved by the SFS algorithm. We prove the new procedure for healthy red blood cells having a concave surface in their central region. Experimental results are compared with theoretical model.

Electrohydrodynamic Dispenser for Delivering Multiphase Samples at Nanoscale

Manipulation of liquids on micro- and nanoscale is a key issue in many fields of technology and biotechnology. Electric field induced formation of microliter and nanoliter droplets is very useful in lab-on-chip applications and would represent a new and contact-less way for functionalizing smart materials [1, 2, 3]. Ink-jet printing, manipulation of biomolecules, deposition of inorganic, organic and biological inks [4, 5], dispensing of small amounts of material into well-defined areas would be a further possibility for functionalizing sensing area for lab-on-a-fiber devices and related applications.

Easy Printing of High Viscous Microdots by Spontaneous Breakup of Thin Fibers

Electrohydrodynamic jetting is emerging as a successful technique for printing inks with resolutions well beyond those offered by conventional inkjet printers. However, the variety of printable inks is still limited to those with relatively low viscosities (typically <20 mPa s) due to nozzle clogging problems. Here, we show the possibility of printing ordered microdots of high viscous inks such as poly(lactic-co-glycolic acid) (PLGA) by exploiting the spontaneous breakup of a thin fiber generated through nozzle-free pyro-electrospinning. The PLGA fiber is deposited onto a partially wetting surface, and the breakup is achieved simply by applying an appropriate thermal stimulation, which is able to induce polymer melting and hence a mechanism of surface area minimization due to the Plateau-Rayleigh instability. The results show that this technique is a good candidate for extending the printability at the microscale to high viscous inks, thus extending their applicability to additional applications, such as cell behavior under controlled morphological constraints.