Mona Mihailescu

Automated imaging, identification, and counting of similar cells from digital hologram reconstructions

This paper presents our method, which simultaneously combines automatic imaging, identification, and counting with the acquisition of morphological information for at least 1000 blood cells from several three-dimensional images of the same sample. We started with seeking parameters to differentiate between red blood cells that are similar but different with respect to their development stage, i.e., mature or immature. We highlight that these cells have different diffractive patterns with complementary central intensity distribution in a given plane along the propagation axis. We use the Fresnel approximation to simulate propagation through cells modeled as spheroid-shaped phase objects and to find the cell property that has the dominant influence on this behavior. Starting with images obtained in the reconstruction step of the digital holographic microscopy technique, we developed a code for automated simultaneous individual cell image separation, identification, and counting, even when the cells are partially overlapped on a slide, and accurate measuring of their morphological features. To find the centroids of each cell, we propose a method based on analytical functions applied at threshold intervals. Our procedure separates the mature from the immature red blood cells and from the white blood cells through a decision based on gradient and radius values.

Laser micro-patterning of biodegradable polymer blends for tissue engineering

We propose a multistep all laser, maskless, and solvent free synthesis of micro-patterned substrates of biodegradable polymer blends, with applicability for guided cell adhesion and localized hyaluronic acid (HA) immobilization. The polymer blends comprised polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), and polylactide-polyethylene glycol-polylactide (PPP) in 1:1:1 blending ratios. Polymer patterning was performed by laser processing in two steps. First, the polymers were patterned with periodic micro-channels by direct femtosecond laser ablation, which provided flexibility in design and spatial accuracy for the patterns. As a second step, the micro-patterned polymers were coated with thin layers of polymer blends using matrix assisted pulsed laser evaporation (MAPLE). The resulted sandwich substrates were composed of a bottom, micro-patterned layer and thin, top layer which conserved the patterns from the underlying layer and preserved the polymers chemical composition. Depending on the bottom/top layers, the substrates were denominated PU/PU:PLGA:PPP and PU:PLGA:PPP/PU:PLGA:PPP, respectively. The laser generated micro-patterns were used for selective attachment of oral keratinocyte stem cells and for HA immobilization. The highest cellular density was found on the PU:PLGA:PPP/PU:PLGA:PPP substrate, where the spongy-like micro-channels provided multiple anchoring points for the cells. For both substrates, the micro-channels enabled localized immobilization of HA. The effectiveness of HA immobilization was tested against cell adhesion and protein adsorption.

Laser-assisted fabrication and non-invasive imaging of 3D cell-seeding constructs for bone tissue engineering

We report on laser-assisted fabrication and non-invasive imaging of porous 3D cell-seeding constructs (3D-CSCs) for bone tissue engineering. The 3D structures were built by two-photon polymerization-direct writing (2PP_DW) of IP-L780 photopolymer and consist in arrays of vertical microtubes arranged in triangular lattices. The microtubes were tightly, medium, and rarely packed, according to the constants of the triangular lattices of 8, 12, and 24 μm, respectively. The efficiency of the laser-generated 3D-CSCs for new bone formation was assessed in MG63 osteoblast-like cells cultures. High spatial resolution 3D images of the cell-seeded 3D-CSCs were obtained by digital holographic microscopy (DHM). The recorded holograms allowed the simultaneous evaluation of the 3D-CSCs and of the seeded cells, in terms of 3D shapes and dimensions, without intruding into the cells natural environment. The seeded cells, in particular the cells nuclei, conformed to the micro-architectures of the 3D-CSCs. Furthermore, the osteogenic potential of the 3D-CSCs was assessed in terms of cell morphology, viability, and level of mineralization. The microtubes packing density that allowed the seeded osteoblasts to reach the highest level of mineralization was established.

Electrically stimulated osteogenesis on Ti-PPy/PLGA constructs prepared by laser-assisted processes.

This work describes a versatile laser-based protocol for fabricating micro-patterned, electrically conductive titanium-polypyrrole/poly(lactic-co-glycolic)acid (Ti-PPy/PLGA) constructs for electrically stimulated (ES) osteogenesis. Ti supports were patterned using fs laser ablation in order to create high spatial resolution microstructures meant to provide mechanical resistance and physical cues for cell growth. Matrix Assisted Pulsed Laser Evaporation (MAPLE) was used to coat the patterned Ti supports with PPy/PLGA layers acting as biocompatible surfaces having chemical and electrical properties suitable for cell differentiation and mineralization. In vitro biological assays on osteoblast-like MG63 cells showed that the constructs maintained cell viability without cytotoxicity. At 24 h after cell seeding, electrical stimulation with currents of 200 μA was applied for 4 h. This treatment was shown to promote earlier onset of osteogenesis. More specifically, the alkaline phosphatase activity of the stimulated cultures reached the maximum before that of the non-stimulated ones, i.e. controls, indicating faster cell differentiation. Moreover, mineralization was found to occur at an earlier stage in the stimulated cultures, as compared to the controls, starting with Day 6 of cell culture. At later stages, calcium levels in the stimulated cultures were higher than those in control samples by about 70%, with Ca/P ratios similar to those of natural bone. In all, the laser-based protocol emerges as an efficient alternative to existing fabrication technologies.

Independent and combined information transfer from axicon and helical phase distributions.

Helical phase distributions used for optical information transfer increase its capacity by offering a characteristic spatial intensity arrangement for the diffracted beam. Here we propose the superposition between helical phase distribution with an axicon type. They form a composed object placed in the object arm to generate holographic masks. The diffracted patterns from these masks exhibit asymmetric shapes and peaks along the optical axis, with two kinds of spots, which contain independent or combined information from both phase distribution constructive parameters. To read these parameters based only on the diffraction patterns analysis, we generate the match reading masks (RMs) to be inserted in the optical path. In this proof-of-concept experiment, we demonstrate that one can sort constructive parameter values of each phase distribution, from both kinds of spots, using specific RMs.

Microchannel-pinhole parameters investigation for cells visualization in holographic microscopy

Here we propose a compact system built around a microchannel aiming to analyze individual blood cells (BCs), using digital in-line holographic microscopy (DIHM). Under the constraints of achieving maximum resolution for BC images, the study is focused on analytical investigations of the main elements constructive dimensions and the distances between them. In the simulations, we used models of oblate spheroids for BC shapes. The iso-intensity curves in the diffraction pattern of the BCs which flow in the microchannel at different distances are studied and the technically possible dimensions were selected to record the signal from one BC.

Evaluation of the metastatic potential of malignant cells by image processing of digital holographic microscopy data

The cell refractive index has been proposed as a putative cancer biomarker of great potential, being correlated with cell content and morphology, cell division rate and membrane permeability. We used digital holographic microscopy to compare the refractive index and dry mass density of two B16 murine melanoma sublines of different metastatic potential. Using statistical methods, the distribution of phase shifts within the reconstructed quantitative phase images was analyzed by the method of bimodality coefficients. The observed correlation of refractive index, dry mass density and bimodality profile with the metastatic potential of the cells was validated by real time impedance‐based assay and clonogenic tests. We suggest that the refractive index and bimodality analysis of quantitative phase image histograms could be developed as optical biomarkers useful in label‐free detection and quantitative evaluation of cell metastatic potential.

Free space optical communications system with helical beams

We propose an experimental model of free space optical communicator in which the information is carried by different orbital angular momentum values. In this sense independent helical beams are superposed in the same channel of communication in order to increase the capacity of the transmitted information. The information is encoded with optical vortices generated by reflective spiral phase plates placed in a cascade configuration. The decoding consists in the reconstruction of initial Gaussian beams and it is performed with fork-like computer generated holograms.

A Generalized Devil Staircase-Based Generator for the JPY-USD Exchange Rate?

Abstract By exploiting the linear similarity between two series, namely the exchange rate Japanese Yen - United States Dollar (JPY-USD), and the generalized devil staircase (GDS) recursive plot, the possibility of synthesizing the real series by using the GDS generator is investigated. The properties of the real and simulated series are compared from both fractal and chaotic perspective using a new multifractal deviation factor, and a crossing analysis based on correlation dimension and non linear prediction error, respectively. Given the encouraging results supported by good fitting values, there are arguments to consider the method an alternative for time series modeling on deterministic assumptions.

Empirical quantitative characterization of holographic phase images of normal and abnormal cervical cells by fractal descriptors

AbstractThe paper proposes a way to examine whether cervical cells qualify as normal or abnormal on the basis of phase profiles, and to subsequently perform local fractal descriptions of their thre...