Francesco Merola

Super-resolution in digital holography by a two-dimensional dynamic phase grating

An approach that uses an electro-optically tunable two dimensional phase grating to enhance the resolution in digital holographic microscopy is proposed. We show that, by means of a flexible hexagonal phase grating, it is possible to increase the numerical aperture of the imaging system, thus improving the spatial resolution of the images in two dimensions. The augment of the numerical aperture of the optical system is obtained by recording spatially multiplexed digital holograms. The grating tuneability allows one to adjust the intensity among the spatially multiplexed holograms maximizing the grating diffraction efficiency. Furthermore we demonstrate that the flexibility of the numerical reconstruction allows one to use selectively the diffraction orders carrying useful information for increasing the spatial resolution. The proposed approach can improve the capabilities of digital holography in three-dimensional imaging and microscopy.

3D lithography by rapid curing of the liquid instabilities at nanoscale.

In liquids realm, surface tension and capillarity are the key forces driving the formation of the shapes pervading the nature. The steady dew drops appearing on plant leaves and spider webs result from the minimization of the overall surface energy [Zheng Y, et al. (2010) Nature 463:640–643]. Thanks to the surface tension, the interfaces of such spontaneous structures exhibit extremely good spherical shape and consequently worthy optical quality. Also nanofluidic instabilities generate a variety of fascinating liquid silhouettes, but they are however intrinsically short-lived. Here we show that such unsteady liquid structures, shaped in polymeric liquids by an electrohydrodynamic pressure, can be rapidly cured by appropriate thermal treatments. The fabrication of many solid microstructures exploitable in photonics is demonstrated, thus leading to a new concept in 3D lithography. The applicability of specific structures as optical tweezers and as novel remotely excitable quantum dots–embedded microresonators is presented.

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.

Tomographic flow cytometry by digital holography

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.

Breakthroughs in Photonics 2013: Holographic Imaging

Although holography is topic that goes back to the 1950s, the research in this field continues to be very active worldwide. A continuous growth is confirmed by the publication of more than 2000 papers each year in archival journal on different holographic issues. Here we describe shortly what appeared to us to be the most significant achievements reached in 2013 on holographic imaging.

Diagnostic Tools for Lab-on-Chip Applications Based on Coherent Imaging Microscopy

Today, fast and accurate diagnosis through portable and cheap devices is in high demand for the general healthcare. Lab-on-chips (LoCs) have undergone a great growth in this direction, supported by optical imaging techniques more and more refined. Here we present recent progresses in developing imaging tools based on coherent imaging microscopy that can be very useful when applied into biomicrofluidics. In some cases, the optical tweezers (OT) technique is combined with digital holography (DH), thus offering the possibility to manipulate, analyze, and measure fundamental parameters of different kinds of cells. This approach can open the route for rapid and high-throughput analysis in label-free microfluidic devices and for prognostic based on cell examination, thus allowing advancements in biomedical science.

Driving and analysis of micro-objects by digital holographic microscope in microfluidics

We propose an optical configuration in which floating particles in a microfluidic chamber can be characterized by an interference microscopy configuration to obtain quantitative phase-contrast maps. The configuration is simply made by two laser beams from the same laser source. One beam provides the optical forces for driving the particle along appropriate paths, but at same time works as the object illumination beam in the holographic microscope. The second beam plays the role of the reference beam, allowing recording of an interference fringe pattern (i.e., the digital hologram) in an out-of-focus image plane. The system and method are illustrated and experimental results are offered for polymeric particles as well as for in vitro cells with the aim to demonstrate the approach.

Simultaneous Optical Manipulation, 3-D Tracking, and Imaging of Micro-Objects by Digital Holography in Microfluidics

Recent advancements in the fields of interferometric microscopy and optical trapping are presented. In particular, the possibility to integrate these techniques in microfluidic and lab-on-chip devices is taken into account. We review the latest results concerning the realization of compact platforms to perform accurate measurements and particle trapping. In this framework, we present the performance of a novel and compact holographic microscope that can ensure multifunctionality accomplishing, at the same time and by the same configuration, 3-D tracking, optical manipulation, and quantitative phase-contrast analysis. Experimental results obtained on in vitro cells in microfluidic devices are presented. The system is based on twin laser beams coming from a single laser source. Through this simple and compact optical setup design, we show how multitasking can be accomplished by a single apparatus.

Holographic imaging of unlabelled sperm cells for semen analysis: a review

Male reproductive health in both humans and animals is an important research field in biological study. In order to characterize the morphology, the motility and the concentration of the sperm cells, which are the most important parameters to feature them, digital holography demonstrated to be an attractive technique. Indeed, it is a label-free, non-invasive and high-resolution method that enables the characterization of live specimen. The review is intended both for summarizing the state-of-art on the semen analysis and recent achievement obtained by means of digital holography and for exploring new possible applications of digital holography in this field. Quantitative phase maps of living swimming spermatozoa.

Characterization of Bessel beams generated by polymeric microaxicons

We present a quick, simple and accurate digital holographic characterization of the Bessel beams produced by polymeric microaxicons. This technique allows the numerical reconstruction of both intensity and phase of the beam at whichever point starting from a single acquired hologram. From these data, it is possible to go back to the axicon structure, and to gather information about their characteristics. In particular, the focal length and the depth of focus of the axicon lens are experimentally measured, and the full width at half maximum of the beam is obtained too. The depth of focus, very large for a Bessel beam with respect to a Gaussian one, is successfully exploited for optical trapping of micrometric objects.