Simonetta Grilli

Digital self-referencing quantitative phase microscopy by wavefront folding in holographic image reconstruction.

A completely numerical method, named digital self-referencing holography, is described to easily accomplish a quantitative phase microscopy for microfluidic devices by a digital holographic microscope. The approach works through an appropriate numerical manipulation of the retrieved complex wavefront. The self-referencing is obtained by folding the retrieved wavefront in the image plane. The folding operation allows us to obtain the correct phase map by subtracting from the complex region of interest a flat area outside the microfluidic channel. To demonstrate the effectiveness of the method, quantitative phase maps of bovine spermatozoa and in vitro cells are retrieved.

Quantitative Label-Free Animal Sperm Imaging by Means of Digital Holographic Microscopy

A digital holographic microscope (DHM) has been employed in the retrieval and analysis of morphological images of bovines sperm cells. Digital holography is a noncontact technique capable of investigating the shape of the sample without altering its characteristics and has been used for the first time in retrieving quantitative morphological information of sperm cells. Different spermatozoa have been analyzed by means of this technique allowing us to obtain 3-D images with precise topographical details and valuable information about morphological defects, provided with biological considerations. Moreover, by making use of a microfluidic system, the digital holographic technique has been employed to analyze unstained spermatozoa in their natural physiological surroundings. Detailed information on morphological images of spermatozoa acquired by DHM is expected to provide a better understanding of various reproductive pathways, which, in turn, can help in improving infertility management. This could constitute the basis of an alternative method for the zoothecnic industry aimed at the investigation of morphological features and the sorting of the motile sperm cells.

Wavefront aberration analysis in two-beam reversal shearing interferometry by elliptical Zernike polynomials

We present a modal phase-reconstruction method for analyzing wave front aberrations of rotationally symmetric optical components in two beam shearing interferometry. The optical configuration requires only two mutually coherent off-axis plane wave fronts transmitted through or reflected by the optical component under test. Finite moire beating between the interferograms and the CCD array is used to subtract the linear carrier introduced by defocus and the tilt making the presense of high order aberrations more evident. The difference wave front is described by elliptical Zernike polynomials as a function of the amount of lateral displacement between the two aberrated wave fronts. The method allows for accurate wave front reconstruction inside the quasi-elliptical overlap area between the laterally sheared interfering beams. Results of numerical experiments and applications of the technique for measuring aberrations of simple biconvex spherical lenses are presented.

Investigation on overpoled lithium niobate patterned crystal

We investigate the domain structure obtained on overpoled both sides polished z-cut Lithium Niobate crystal sample by applying an electric field on periodic liquid electrodes. Lithium Niobate sample was spatially patterned with photoresist by use of interference photolithographic process. The sample was then etched in a mixture of hydrofluoric and nitric acid to reveal the reversed domains. The different etch rate of the two opposite z faces of the crystal domains allowed to measure the surface profile of the sample by using a profilometer. Overpoling the crystal caused the appearance of high density and submicrometer spacing single dot domains vertically aligned along the direction of the interference fringes in the patterned region. (Summary only available)

Monitoring cell morphology during necrosis and apoptosis by quantitative phase imaging

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.

Quantitative Phase Contrast in Holographic Microscopy Through the Numerical Manipulation of the Retrieved Wavefronts

In this chapter we show how digital holography (DH) can be efficiently used as a free and noninvasive investigation tool capable of performing quantitative and qualitative mapping of biological samples. A detailed description of the recent methods based on the possibility offered by DH to numerically manage the reconstructed wavefronts is reported. Depending on the sample and on the investigation to be performed it is possible, by a single acquired image, to recover information on the optical path length changes and, choosing the more suitable numerical method, to obtain quantitative phase map distributions. The progress achieved in the reconstruction methods will certainly find useful applications in the field of the biological analysis.

Imaging and phase measurements of 3D objects at 10.6 microns by digital holography

Digital holography in the mid infrared range is shown to be a feasible technique for optical metrological applications. The technique allows to reconstruct both amplitude and phase of wavefronts scattered by a 3D object. Experimental results of the method applied to the reconstruction of digitally holograms recorded at CO2 laser wavelength of 10.6 micron are reported. It is show that good reconstructions can be obtained even with the lower spatial resolution of IR recording detectors compared to visible CCD array. The results show that new prospective can be exploited by using high power CO2 laser sources in optical metrological applications.

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.

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.

Quantitative Phase Microscopy for Accurate Characterization of Microlens Arrays

Microlens arrays are of fundamental importance in a wide variety of applications in optics and photonics. This chapter deals with an accurate digital holography-based characterization of both liquid and polymeric microlenses fabricated by an innovative pyro-electrowetting process. The actuation of liquid and polymeric films is obtained through the use of pyroelectric charges generated into polar dielectric lithium niobate crystals.