Pietro Ferraro

Bio: Pietro Ferraro is an academic researcher from National Research Council. The author has contributed to research in topics: Digital holography & Holography. The author has an hindex of 61, co-authored 653 publications receiving 12666 citations. Previous affiliations of Pietro Ferraro include Aeritalia & Centre national de la recherche scientifique.

Monitoring cell morphology during necrosis and apoptosis by quantitative phase imaging

Abstract: Cellular morphology changes and volume alterations play significant roles in many biological processes and they are mirrors of cell functions. In this paper, we propose the Digital Holographic microscope (DH) as a non-invasive imaging technique for a rapid and accurate extraction of morphological information related to cell death. In particular, we investigate the morphological variations that occur during necrosis and apoptosis. The study of necrosis is extremely important because it is often associated with unwarranted loss of cells in human pathologies such as ischemia, trauma, and some forms of neurodegeneration; therefore, a better elucidation in terms of cell morphological changes could pave the way for new treatments. Also, apoptosis is extremely important because it’s involved in cancer, both in its formation and in medical treatments. Because the inability to initiate apoptosis enhances tumour formation, current cancer treatments target this pathway. Within this framework, we have developed a transmission off-axis DH apparatus integrated with a micro incubator for investigation of living cells in a temperature and CO2 controlled environment. We employ DH to analyse the necrosis cell death induced by laser light (wavelength 473 nm, light power 4 mW). We have chosen as cellular model NIH 3T3 mouse embryonic fibroblasts because their adhesive features such as morphological changes, and the time needed to adhere and spread have been well characterized in the literature. We have monitored cell volume changes and morphological alterations in real time in order to study the necrosis process accurately and quantitatively. Cell volume changes were evaluated from the measured phase changes of light transmitted through cells. Our digital holographic experiments showed that after exposure of cells to laser light for 90-120 min., they swell and then take on a balloon-like shape until the plasma membrane ruptures and finally the cell volume decreases. Furthermore, we present a preliminary study on the variation of morphological parameters in case of cell apoptosis induced by exposure to 10 μM cadmium chloride. We employ the same cell line, monitoring the process for 18 hours. In the vast group of environmental pollutants, the toxic heavy metal cadmium is considered a likely candidate as a causative agent of several types of cancers. Widely distributed and used in industry, and with a broad range of target organs and a long half-life (10-30 years) in the human body, this element has been long known for its multiple adverse effects on human health, through occupational or environmental exposure. In apoptosis, we measure cell volume decrease and cell shrinking. Both data of apoptosis and necrosis were analysed by means of a Sigmoidal Statistical Distribution function, which allows several quantitative data to be established, such as swelling and cell death time, flux of intracellular material from inside to outside the cell, initial and final volume versus time. In addition, we can quantitatively study the cytoplasmatic granularity that occurs during necrosis. As a future application, DH could be employed as a non-invasive and label-free method to distinguish between apoptosis and necrosis in terms of morphological parameters.

Investigation on 3D morphological changes of in vitro cells through digital holographic microscopy

Abstract: We report the investigation of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps (QPMs) of biological cells by digital holographic microscopy (DHM), with the aim to analyze the 3D positions and 3D morphology together. We consider as test case for our tool the in vitro bull sperm head morphometry analysis. Extraction and measurement of various morphological parameters are performed by using two methods: the anisotropic diffusion filter, that is based on the Gaussian diffusivity function which allows more accuracy of the edge position, and the simple thresholding filter. In particular we consider the calculation of area, ellipticity, perimeter, major axis, minor axis and shape factor as a morphological parameter, instead, for the estimation of 3D position, we compute the centroid, the weighted centroid and the maximum phase values. A statistical analysis on a data set composed by N = 14 holograms relative to bovine spermatozoa and its reference holograms is reported.

Exploiting a holographic polarization microscope for rapid autofocusing and 3D tracking.

Abstract: We report a fast autofocusing and accurate 3D tracking scheme for a digital hologram (DH) that intrinsically exploits a polarization microscope setup with two off-axis illumination beams having different polarization. This configuration forms twin-object images that are recorded in a digital hologram by angular and polarization multiplexing technique. We show that the separation of the two images on the recording plane follows a linear relationship with the defocus distance and indicates the defocus direction. Thus, in the entire field of view (FOV), the best focus distance of each object can be directly retrieved by identifying the respective separation distance with a cross-correlation algorithm, at the same time, 3D tracking can be performed by calculating the transverse coordinates of the two images. Moreover, we estimate this linear relationship by utilizing the numerical propagation calculation based on a single hologram, in which the focus distance of one of the objects in the FOV is known. We proved the proposed approach in accurate 3D tracking through multiple completely different experimental cases, i.e., recovering the swimming path of a marine alga (tetraselmis) in water and fast refocusing of ovarian cancer cells under micro-vibration stimulation. The reported experimental results validate the proposed strategy’s effectiveness in dynamic measurement and 3D tracking without multiple diffraction calculations and any precise knowledge about the setup. We claim that it is the first time that a holographic polarization multiplexing setup is exploited intrinsically for 3D tracking and/or fast and accurate refocusing. This means that almost any polarization DH setup, thanks to our results, can guarantee accurate focusing along the optical axis in addition to polarization analysis of the sample, thus overcoming the limitation of the poor axial resolution.

Optical-force-controlled red-blood-cell microlenses for subwavelength trapping and imaging.

Abstract: We demonstrate that red blood cells (RBCs), with an adjustable focusing effect controlled by optical forces, can act as bio-microlenses for trapping and imaging subwavelength objects. By varying the laser power injected into a tapered fiber probe, the shape of a swelled RBC can be changed from spherical to ellipsoidal by the optical forces, thus adjusting the focal length of such bio-microlens in a range from 3.3 to 6.5 µm. An efficient optical trapping and a simultaneous fluorescence detecting of a 500-nm polystyrene particle have been realized using the RBC microlens. Assisted by the RBC microlens, a subwavelength imaging has also been achieved, with a magnification adjustable from 1.6× to 2×. The RBC bio-microlenses may offer new opportunities for the development of fully biocompatible light-driven devices in diagnosis of blood disease.

Self-organized and high-density filamentous nanodomain patterns fabricated in lithium niobate by discharge poling

Abstract: We report on spontaneous formation of nanoscale surface domains obtained in lithium niobate substrates by electric field discharge poling. A constant high voltage was applied at room temperature to virgin and unpatterned crystals in air, by using metal tip-shaped electrodes. An enhancement of the electric field in correspondence of the electrode tips was expected due to the fringing field effect, and electric discharge was observed during the process. The standard wet etching in hydrofluoric acid solution revealed the formation of nanoscale domain structures that exhibit filamentous self-organized geometries. Topographic measurements of the fabricated structures were performed by an atomic force microscope, and the corresponding results are presented and discussed. The investigation provides remarkable information about the shortest domain size possibly obtainable by electric field poling.

Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry

Abstract: A fast-Fourier-transform method of topography and interferometry is proposed. By computer processing of a noncontour type of fringe pattern, automatic discrimination is achieved between elevation and depression of the object or wave-front form, which has not been possible by the fringe-contour-generation techniques. The method has advantages over moire topography and conventional fringe-contour interferometry in both accuracy and sensitivity. Unlike fringe-scanning techniques, the method is easy to apply because it uses no moving components.

Fiber grating sensors

Abstract: We review the recent developments in the area of optical fiber grating sensors, including quasi-distributed strain sensing using Bragg gratings, systems based on chirped gratings, intragrating sensing concepts, long period-based grating sensors, fiber grating laser-based systems, and interferometric sensor systems based on grating reflectors.