This PDF file contains the front matter associated with SPIE Proceedings Volume 8792, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Tablet coating is a common pharmaceutical technique to apply a thin continuous layer of solid on the top of a tablet or a granule containing active pharmaceutical ingredients (APIs). Coating thickness and homogeneity are critical parameters regarding the drug release rate, and consequently a direct or indirect monitoring strategy of these critical process parameters is essential. With the aid of Optical Coherence Tomography (OCT) it is not only possible to measure the absolute coating thickness, but also to detect inhomogeneities in the coating or substrate material. In this work the possible application of OCT as in-line method for monitoring pharmaceutical tablet film coating is studied. Firstly, the feasibility of OCT for analysis tablet coating is examined. Seven different commercially available film-coated tablets with different shapes, formulations and coating thicknesses were investigated off-line. OCT images were acquired by two different spectral-domain OCT systems operating at center wavelengths of 830 and 1325 nm. Since the images of both systems allow the analysis of the coatings, the OCT system employing the shorter wavelength and thus providing a higher axial resolution was selected for the further experiments. The influence of a moving tablet bed on OCT images was analyzed by considering a static tablet bed and moving the sensor head along the tablet bed. The ability to analyze the coating homogeneity is limited to a speed up to 0.3 m/s. However, determining the coating thickness and inter-coating uniformity is still possible up to a speed of 0.7 m/s.

The method of dynamic data processing in Fourier-domain optical coherence tomography based on the Kalman filtering algorithm is proposed. It is shown the possibility of adaptive selection of the required number of spectral samples at different wavelengths to provide necessary resolution of an investigated object micro structure. Experimental results of dynamic data processing in the swept-source optical coherence tomography are presented.

In this paper we present a method for numerical correction of phase images captured in a digital holographic microscopy (DHM) setup adapted to tomographic measurement of biological objects. The purpose of the correction is a removal of the object wave deformation associated with a fluid filled fiber capillary, which is used in DHM system to enable manipulation of a specimen. The proposed correction procedure is based on a simple concept of the phase subtraction, preceded by an estimation of the aberration profile using areas of a hologram that have not been affected by the object. The phase subtraction methodology, developed on the ground of the thin element approximation, is very effective in the visual enhancement of phase images; however, its application to quantitative measurement of micro-objects is questionable. Therefore, in this paper we verify the possible use of the phase subtraction methodology in DHM by performing a numerical experiment, supported with the finite difference time domain method (FDTD), which allows us to identify the residual error of the correction. The FDTD computation reveals that the phase subtraction methodology is insufficient to properly remove the influence of a capillary, in particular to compensate for two effects associated with the focusing properties of the aberration: a transversal shift of the image and the change of its magnification. Nevertheless, the possibility of the visual improvement of holographic images of a living human leukemia cell using the outlined method is demonstrated.

The optical coherence tomography (OCT) is an important technology for non-invasive, in vivo medical diagnostics. It enables the high-resolution recording of two-dimensional tomograms or three-dimensional volumes of biological tissue. Two mechanisms help separating the signal from the scattering background. First, reflected or backscattered light from outside the focal spot is suppressed by confocal discrimination. Additionally, the signal modulation is enhanced due to identical optical path lengths of both branches of the white light interferometry setup. Since the OCT relies on the interference between reference light and scattered light, this method cannot be readily extended for fluorescence measurements. An alternative approach is the confocal fluorescence microscopy, which uses confocal microscopy to suppress the fluorescent light from outside the focal spot. Hence, only the fluorescent light in the focal plane, which is 3 to 4 magnitudes lower in intensity than the excitation light, is detected. However, the surrounding area is illuminated with full intensity, which might cause photo-bleaching. There are also other promising approaches such as the two-photon excitation microscopy or fluorescence lifetime microscopy, which we will not cover in more detail. For fluorescence measurements of strongly-scattering samples such as biological tissue but also for technical surfaces, we propose a structured white-light illumination. We present two different approaches for the sample illumination utilizing a white light laser or a white light LED, respectively. We show first simulations of the individual illumination setups and their impact on the scattering within the sample. Furthermore, we investigated the distribution of the fluorescent light that reaches the detection part of the device when excited within a scattering medium, for this purpose we implemented a novel fast-converging algorithm for conditional fluence rate in our Monte Carlo algorithm.

A Quadri-Wave Lateral Shearing Interferometer (QWLSI) is an efficient tool for measuring phase gradients of optical beams along two perpendicular directions. Post-processing integration then allows obtaining the complete phase spatial distribution of the beam. By placing a QWLSI on the exit image plane of such a microscope, we are able to measure the complex field spatial distribution in this plane, and then to retrieve the quantitative optical path difference (OPD) of the observed sample. Here, we demonstrate that we can extend the technique to new applications, were different physical phenomena produce a given sample-induced change in the phase of the exit optical beam that modulates the incident wavefront. More precisely, we used direct refractive-induced OPD, thermal-induced OPD, and resonant vibrational-induced OPD to produce phase contrast images of living cells, temperature distribution of complex patterns of nanostructures, and Raman spectra of polystyrene beads, respectively. In the case of refractive-induced OPD of living cells, we also show that the OPD distribution of a living cell can be used to monitor its dry mass during the cell cycle.

In this contribution we propose a new technique for deformation measurement based upon a multipoint speckle imaging using the correlation statistics of speckle patterns. The system is capable of interferometric accuracy, although it relies on self-interference, shown as speckle patterns on the detector plane. Therefore, most of the constraints imposed by interferometric setups no longer apply. A camera is used to capture images at the desired frame rate, a collimated laser and a diffractive optical element, achieving a high number of inspection points opens the possibility for analyzing simultaneously a plurality of inspected points. Proper adjustment of the optical parameters (aperture size and shape) can deal with the measurements at different locations of the object’s surface with no crosstalk between the outputs for each inspected point. The data from the different inspected locations can be analyzed separately or integrated to provide a global surface change in shape. The system has two major advantages. On one hand, it uses few hardware elements, making the system easily portable and compact. On the other hand the system needs a laser source with relatively low degree of coherence, as interference is done on the tested surface itself and no external coherent reference is needed. The system can be packed in a compact enclosure and it can operate at an arbitrary distance from the inspected object, limited only by intensity available on the detector and sensor’s sensitivity. The system can work at frame rate allowed by the camera in the selected region of interest.

In this paper, we propose a simple method for reconstructing the corneal surface profile by using the Talbot effect, projection moiré method, and heterodyne interferometry. A linear grating is obliquely illuminated by an expanding collimated light, and a self-image of this grating can be generated and projected on the corneal surface. The deformed grating fringes are imaged on the second grating to form the moiré fringes. If the first grating is moved with a constant velocity along the grating plane, a series of sampling points of the sinusoidal wave which behave like the heterodyne interferometric signal can be recorded by a CMOS camera. The phase distribution of the corneal surface then can be obtained with the IEEE 1241 least-square sine fitting algorithm and 2D phase unwrapping. Finally, the corneal surface profile can be reconstructed by substituting the phase distribution into special derived equation. This method provides the advantages of a simple optical setup, ease of operation, high stability, and high resolution.

An optical noninvasive inspection tool is presented to, in vivo, better characterize biological tissues such as human skin. The method proposed exploits a multispectral imaging device to acquire a set of images in the visible and NIR range. This kind of information can be very helpful to improve early diagnosis of melanoma, a very aggressive cutaneous neoplasm, incidence and mortality of which continues to rise worldwide. Currently, noninvasive methods (i.e. dermoscopy) have improved melanoma detection, but the definitive diagnosis is still achieved only by invasive method (istopathological observation of the excised lesion). The multispectral system we developed is capable of imaging layers of structures placed at increasing depth, thanks to the fact that light propagates into the skin and reaches different depths depending on its wavelength. This allows to image many features which are less or not visible in the clinical and dermoscopic examination. A new semeiotics is proposed to describe the content of multispectral images. Dermoscopic criteria can be easily applied to describe each image in the set, however inter-images correlations need new suitable descriptors. The first group of new parameters describes how the dermoscopic features, vary across the set of images. More aspects are then introduced. E.g. the longest wavelength where structures can be detected gives an estimate of the maximum depth reached by the pigmented lesion. While the presence of a bright-to-dark transition between the wavebands in the violet to blue range, reveals the presence of blue-whitish veil, which is a further malignancy marker.

Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample giving insight into the vibration eigenmodes of the excited molecules. For these reasons, Raman spectroscopy has been used as a powerful tool to investigate different biological tissues and living cells. In this paper, we present a Raman spectroscopy-based method for sensitive biochemical characterization of bovine sperm cells. Importantly, by analysing separate Raman spectra from the nucleus, acrosomale vesicle and tail of single sperm cells, we are able to identify characteristic Raman features associated with DNA, protein and lipid molecular vibrations for discriminating among different locations inside the cell with sub-micrometric resolution (∼0.3 μm). We demonstrate that our Raman spectroscopy facilitates spectral assignment and increases detection sensitivity, opening the way for novel bio-imaging platforms.

The purpose of this paper is to present the current activities of a research collaborative program between three institutions from Zagreb (School of Dental Medicine, Institute of Physics, and Institute Ruđer Bošković). Within the scope of this program, it is planned to investigate and find guidelines for the refinement of the properties of dental biomaterials (DBs) and of procedures in restorative dental medicine. It is also planned to identify and model the dominant mechanisms which control polymerization of DBs. The materials to be investigated include methacrylate based composite resins, new composite materials with amorphous calcium phosphate, silorane based composite resins, glass-ionomer cements, and giomer.

Vascular prostheses are widely used devices fundamental to avoid the effect of life-threatening diseases and defects. Besides a long experience in the fabrication of biomaterials for vascular applications, many issues still remain unattended. In particular, obtaining a bio-resorbable and bio-active scaffold is a challenge of paramount importance. We present a novel application in which a promising biodegradable polymer, poly-propylene fumarate (PPF), is printed using three dimensional laser-induced cross-linking micromachining device. To enhance the biological role of the scaffold, a bio-inspired approach was taken, by coating the surface of the PPF with elastin, the main constituent of the innermost layer of natural veins and arteries.

In this study we realized a three-dimensional human dermis equivalent (3D-HDE) and, by exploiting multi-photon microscopy (MPM) we validated its use as an in vitro model to study collagen network re-arrangement under simulated solar exposure. The realization of 3D-HDE has been pursed by means of a bottom-up tissue engineering strategy that comprises firstly the fabrication of micron sized tissue building blocks and then their assembly in a 3D tissue construct. The building blocks injected in a maturation chamber, and cultured under optimized culture condition, were able to fuse due to the establishment of cell-cell and cell-extra cellular matrix (ECM) interactions that induced a biological sintering process resulting in 3D-HDE production. The final 3D tissue was made-up by fibroblasts embedded in their own ECM rich in endogenous collagen type I, resembling the composition and the architecture of native human dermis. Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) imaging have been exploited to assess modification in collagen assembly before and after UV irradiation. Textural features and SHG to TPFE ratio of the endogenous ECM within 3D-HDE have been shown to vary after UVA irradiation, proving the hypothesis that the 3DHDE realized can be used as biological platform in vitro to study ECM modifications induced by photo-damage.

Diatoms are monocellular algae responsible of 20-25% of the global oxygen produced by photosynthetic processes. The protoplasm of every single cell is enclosed in an external wall made of porous hydrogenated silica, the frustule. In recent times, many effects related to photonic properties of diatom frustules have been discovered and exploited in applications: light confinement induced by multiple diffraction, frustule photoluminescence applied to chemical and biochemical sensing, photonic-crystal-like behavior of valves and girdles. In present work we show how several techniques (e.g. digital holography) allowed us to retrieve information on light manipulation by diatom single valves in terms of amplitude, phase and polarization, both in air and in a cytoplasmatic environment. Possible applications in optical microsystems of diatom frustules and frustule-inspired devices as active photonic elements are finally envisaged.

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.

A method for 3D tracking has been developed exploiting Digital Holographic Microscopy (DHM) features. In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of specimen with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the issue of amplitude refocusing in traditional tracking processing. Our technique belongs to the video tracking methods that, conversely from Quadrant Photo-Diode method, opens the possibility to track multiples probes. All the common used video tracking algorithms are based on the numerical analysis of amplitude images in the focus plane and the shift of the maxima in the image plane are measured after the application of an appropriate threshold. Our approach for video tracking uses different theoretical basis. A set of interferograms is recorded and the complex wavefields are managed numerically to obtain three dimensional displacements of the probes. The procedure works properly on an higher number of probes and independently from their size. This method overcomes the traditional video tracking issues as the inability to measure the axial movement and the choice of suitable threshold mask. The novel configuration allows 3D tracking of micro-particles and simultaneously can furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. In this paper, we show a new concept for a compact interferometric microscope that can ensure the multifunctionality, accomplishing accurate 3D tracking and quantitative phase-contrast analysis. Experimental results are presented and discussed for in vitro cells. Through a very simple and compact optical arrangement we show how two different functionalities can be accomplished by the same optical setup, i.e. 3D tracking of micro-object and quantitative phase contrast imaging.

Intracochlear imaging is of great interest clinically because cochlea is the central organ of hearing. However, intracochlear imaging is technologically challenging due to the cochlea’s small size and encasement in bone. The state-of- the-art imaging techniques are not adequate for high resolution cellular imaging to establish diagnosis without destroying the cochlea. We report in situ imaging of intact mouse cochlea using endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. TPEF eliminates the need for exogenous labeling and eradicating the staining-induced artifacts. We used a natural, membranous opening into the cochlea, the round window, as the optical access to reach the organ of Corti, requiring no additional slicing or opening. Our approach provides the maximum non-invasiveness in the imaging process. TPEF exhibits strong contrast allowing deep imaging of mouse cochlea with cellular and even subcellular resolution. Inner hair cell, outer hair cell and supporting cell are clearly identifiable in TPEF images. Distinct morphological differences are observed between healthy and noise-exposed cochleae, allowing detection of specific, noise-induced pathologic changes. The TPEF images taken through the round window are correlated with the whole mount sections, verifying their reliability. Compared with one-photon excitation fluorescence (OPEF) confocal microscope and wide-field transmission microscope images taken under the same magnification and resolution, TPEF images demonstrate clear advantages in terms of sharpness, signal to noise ratio and contrast. These capabilities provide a working foundation for microendoscopy-based clinical diagnostics of sensorineural hearing loss.

Very interesting effects can be observed in maneuvering nematic liquid crystal (NLC) droplets onto functionalized polar lithium niobate (LN) crystal surfaces, covered with thin films of Polydimethylsiloxane (PDMS). It has been discovered that pyroelectric effect is able to drive a reversible fragmentation process in liquid crystal drops, starting from nanoliter drops and obtaining pico/femtoliter droplets. These small droplets are patterned according to the geometry of the substrate and aligned along the electric field lines. This novel approach for manipulating different classes of liquids by exploiting the pyroelectric effect, where the strong electric fields generated allow to manipulate liquids in 2D on a substrate or even in 3D, has been recently discovered and exploited for different purposes. In particular, manipulation of liquid crystals by a thermal stimulus could be suitable for applications such as spatial modulation of the wettability (i.e. wettability patterning), or, in principle, a dynamical optical element able to switch from a diffuser (fragmentation) state to a microlens array. Moreover, the biocompatibility of some kinds of nematic or cholesteric liquid crystals makes them suitable as biomaterials for applications in biology and tissue engineering.

A digital holographic characterization technique is developed for studying changes in the refractive index when polymerization occurs. This all optical characterization technique enables real-time detection of the photopolymer refractive index changes during the recording process. In this paper, two different new photopolymers, sensitive to light at wavelength of 532 nm, were characterized by means of digital holography. We found a very high refractive index variation for both the new photopolymers, thus this materials could be suitable for holographic recordings with the great advantage of being inexpensive and easy to make.

We propose a new strategy of three-dimensional (3D) tracking of living cells by digital holographic microscopy based on the morphological changes of cells during the migration. The typical strategy adopted in digital holography for the tracking of living cells consists into compute the 3D position dividing the calculation process into two parts: the estimation of the focal plane on the amplitude reconstruction of the digital holograms and the estimation of the transverse coordinates by the phase reconstruction of digital holograms computed at distance equal to the estimated focal plane. We propose to use an approximation of the Tamura coefficient, as image contrast measure, for the estimation of the focal plane and a new morphological operator, that is called minimum boundary filter (MBF), from which we compute the transverse coordinates. A comparison with other 3D tracking methods is accomplished.

This paper proposes a full-field and simultaneous method to visualize acoustic waves at the surface of granular media, using a three color digital holographic set-up. Experimental results permit the quantification of the response of the medium to an acoustic excitation, and exhibit propagative longitudinal waves, as well as transverse waves. This new approach also permits the measurement of the influence of a small buried obstacle.

A CCD-camera based small angle light scattering (SALS) apparatus has been used to characterize single micrometric particles flowing in a micro-channel. The measured scattering vector spans the range 2x10^-2 - 6:8x10¹μm^-1. The incident laser light is collimated to a spot of about 50 μm in diameter at the sample position with a divergence lower than 0.045 rad. Such small collimated laser beam opens the possibility to perform on-line SALS of micron-sized particles flowing in micro-channels. By properly designing the micro-channel and using a viscoelastic liquid as suspending medium we are able to realize a precise 3D focusing of the target particles. The forward scattering emitted from the particle is collected by a lens with high numerical aperture. At the focal point of that lens a homemade beam stop is blocking the incident light. Finally, a second lens maps the scattered light on the CCD sensor, allowing to obtain far field images on short distances. Measurements with mono-disperse polystyrene particles, both in quiescent and in-flow conditions have been realized. Experiments in-flow allow to measure the single particle scattering. Results are validated by comparison with calculations based on the Lorenz-Mie theory. The quality of the measured intensity profiles confirms the possibility to use our apparatus in real multiplex applications, with particles down to 1 μm in radius.

We present a simple and non-invasive experimental procedure to measure the linear viscoelastic properties of cells by passive particle tracking microrheology. In order to do this, a generalised Langevin equation is adopted to relate the timedependent thermal fluctuations of a probe sensor, immobilised to the cell’s membrane, to the frequency-dependent viscoelastic moduli of the cell. The method has been validated by measuring the linear viscoelastic response of a soft solid and then applied to cell physiology studies. It is shown that the viscoelastic moduli are related to the cell’s cytoskeletal structure, which in this work is modulated either by inhibiting the actin/myosin-II interactions by means of blebbistatin or by varying the solution osmolarity from iso- to hypo-osmotic conditions. The insights gained from this form of rheological analysis promises to be a valuable addition to physiological studies; e.g. cell physiology during pathology and pharmacological response.

We propose an off-axis interferometric imaging system as a simple and unique modality for continuous, non-contact and non-invasive wide-field imaging and characterization of drug release from its polymeric device used in biomedicine. In contrast to the current gold-standard methods in this field, usually based on chromatographic and spectroscopic techniques, our method requires no user intervention during the experiment, and only one test-tube is prepared. We experimentally demonstrate imaging and characterization of drug release from soy-based protein matrix, used as skin equivalent for wound dressing with controlled anesthetic, Bupivacaine drug release. Our preliminary results demonstrate the high potential of our method as a simple and low-cost modality for wide-field imaging and characterization of drug release from drug delivery devices.

Laser printing constitutes an interesting alternative to more conventional printing techniques in the microfabrication of biomedical devices. The principle of operation of most laser printing techniques relies on the highly localized absorption of strongly focused laser pulses in the close proximity of the free surface of the liquid to be printed. This leads to the generation of a cavitation bubble which further expansion results in the ejection of a small fraction of the liquid, giving place to the deposition of a well-defined droplet onto a collector substrate. Laser printing has proved feasible for printing biological materials, from single-stranded DNA to proteins, and even living cells and microorganisms, with high degrees of resolution and reproducibility. In consequence, laser printing appears to be an excellent candidate for the fabrication of biological microdevices, such as DNA and protein microarrays, or miniaturized biosensors. The optimization of the performances of laser printing techniques requires a detailed knowledge of the dynamics of liquid transfer. Time-resolved microscopy techniques play a crucial role in this concern, since they allow tracking the evolution of the ejected material with excellent time and spatial resolution. Investigations carried out up to date have shown that liquid ejection proceeds through the formation of long, thin and stable liquid jets. In this work the different approaches used so far for monitoring liquid ejection during laser printing are considered, and it is shown how these techniques make possible to understand the complex dynamics involved in the process.

Microscopes are usually thought of comprising imaging elements such as objectives and eye-piece lenses. A different type of microscope, used for endoscopy, consists of waveguiding elements such as fiber bundles, where each fiber in the bundle transports the light corresponding to one pixel in the image. Recently a new type of microscope has emerged that exploits the large number of propagating modes in a single multimode fiber. We have successfully produced fluorescence images of neural cells with sub-micrometer resolution via a 200 micrometer core multimode fiber. The method for achieving imaging consists of using digital phase conjugation to reproduce a focal spot at the tip of the multimode fiber. The image is formed by scanning the focal spot digitally and collecting the fluorescence point by point.

We illustrate the abilities of an advanced full-field optical coherence microscope (FF-OCM) setup for characterization of technical materials with internal micro-structures and present this technique also for dynamic process monitoring, as strain-stress tests. Additionally we briefly illustrate the potential of image processing in context of the chosen applications. Furthermore, contrast modification techniques based on Fourier plane filtering are discussed.

Scanning ion conductance microscopy (SICM) is a type of scanning probe microscopy based on the continuous measurement of an ion current flowing through a pipette filled with an electrolyte solution, while the pipette apex approaches a non-conductive sample. This technique can be operated in environmental conditions such as those of cell cultures and does not require a direct contact between probe and sample. It is therefore particularly suitable for the investigation of living specimens. SICM was initially proposed as an instrument that could obtain topographic 3D images with high resolution. Later, simple modifications have been devised to apply a mechanical stimulus to the specimen via a solution flux coming out from the pipette aperture. This modified setup has been employed to measure cell membrane elasticity and to guide the growth cones of neurons for tens of micrometers, by means of repeated non-contact scanning. Both these applications require an accurate measurement of the mechanical forces acting on the cell surface, which can be obtained by combining SICM, Atomic force microscopy (AFM) and inverted optical microscopy in the same apparatus. In this configuration, a SICM pipette is approached to an AFM cantilever while monitoring the cantilever deflection as a function of the pressure applied to the pipette and the relative distance. In addition, the pipette aperture can be imaged in situ by exploiting the AFM operation, so that all the experimental parameters can be effectively controlled in the investigation of pressure effects on living cells.

We review our progress on the development of computational lensfree on-chip microscopy and tomography techniques for biomedical imaging, microanalysis and telemedicine applications.

In this manuscript we propose optical lensless configuration for a remote non-contact measuring of mechanical contractions of vast number of cardiac myocytes. All the myocytes were taken from rats, and the measurements were done in an in vitro mode. The optical method is based on temporal analysis of secondary reflected speckle patterns generated in lensless microscope configuration. The processing involves analyzing the movement and the change in the statistics of the generated secondary speckle patterns that are created on top of the cell culture when it is illuminated by a spot of laser beam. The main advantage of the proposed system is the ability to measure many cells simultaneously (approximately one thousand cells) and to extract the statistical data of their movement at once. The presented experimental results also include investigation the effect of isoproteranol on cells contraction process.

In biology and biomedical research fields one of the main topic is the understanding of morphology and mechanics of cells and microorganisms. Biological samples present low amplitude contrast that limits the information that can be retrieved through optical bright-field microscope measurements. Optical transparency is overcame for fixed specimen by means of staining techniques but such well-established methods present the issue to be invasive and not applicable on live cells. Study of microorganism in their natural environment without perturbing their equilibrium is challenging in biology. The main effect on light propagating in such objects is in phase, indeed it is altered respect to the phase of the beam propagating in the surrounding medium. This is known as phase-retardation or phase-shift. Objects are visible by Phase Contrast Imaging (PCI) due to interferometric processes able to transform tiny phase variation in amplitude modulation so that any small differences in the beam optical path can be visualized. Digital Holography (DH) in microscopy present as a powerful tool to overcome all these issues. The main characteristic is the possibility to discern between intensity and phase information performing quantitative mapping of the Optical Path Length. Up to now, DH has been considered as an innovative and alternative approach in microscopy and it’s a good candidate for complete specimen analysis in the framework of no invasive microscopy. In this paper, the flexibility of DH is employed to analyze in a completely and no-invasive way the cell mechanics of live and unstained cell subjected to appropriate stimuli. The potentialities of DH are employed to measure all the parameters useful to understand the deformations induced by external and controlled stress in living cells.

Interferometric phase microscopy (IPM) enables to obtain quantitative optical thickness profiles of transparent samples, including live cells in-vitro, and track them in time with sub-nanometer accuracy without any external labeling, contact or force application on the sample. The optical thickness measured by IPM is a multiplication between the cell integral refractive index differences and its physical thickness. Based on the time-dependent optical thickness profile, one can generate the optical thickness fluctuation map. For biological cells that are adhered to the surface, the variance of the physical thickness fluctuations in time is inversely proportional to the spring factor indicating on cell stiffness, where softer cells are expected fluctuating more than more rigid cells. For homogenous refractive index cells, such as red blood cells, we can calculate a map indicating on the cell stiffness per each spatial point on the cell. Therefore, it is possible to obtain novel diagnosis and monitoring tools for diseases changing the morphology and the mechanical properties of these cells such as malaria, certain types of anaemia and thalassemia. For cells with a complex refractive-index structure, such as cancer cells, decoupling refractive index and physical thickness is not possible in single-exposure mode. In these cases, we measure a closely related parameter, under the assumption that the refractive index does not change much within less than a second of measurement. Using these techniques, we lately found that cancer cells fluctuate significantly more than healthy cells, and that metastatic cancer cells fluctuate significantly more than primary cancer cells.

We present a Nd:YAG CW laser based second-harmonic interferometer with 60 μm spatial resolution. The interferometer is sensitive to the phase shift between fundamental and second harmonic radiation when passing through a dispersive medium. The device performance is tested by measuring the dispersion induced phase shift of laser etched polymeric films resulting in a sensitivity down to 7×10⁻³ Rad for a detector acquisition time of 300 μs. These results demonstrate the feasibility of high spatial resolution second-harmonic interferometry, and an outlook is given for its use as a novel quantitative phase sensitive imaging technique.

We present a type of diffractive lenses “Zernike apodized photon sieves” (ZAPS), which structure is based on the combination of two concepts: apodized photon sieves and Zernike phase-contrast. In combination with the synchrotron light sources, the ZAPS can be used as an objective for high-resolution X-ray phase-contrast microscopy in physical and life sciences. The ZAPS is a single optic that integrates the appropriate ±π/2 radians phase shift through selective zone placement shifts in an apodized photon sieve. The focusing properties of the ZAPS can be easily controlled by apodizing its pupil function. An apodized photon sieve with Gaussian pupil was fabricated by lithographic technique and showed that the side-lobes have been significantly suppressed at the expense of slightly widening the width of the main lobe.

A critical issue in the production of ophthalmic lenses is to guarantee the correct centering and alignment throughout the manufacturing and mounting processes. Aimed to that, progressive addition lenses (PALs) incorporate permanent marks at standardized locations at the lens. Those marks are engraved upon the surface and provide the model identification and addition power of the PAL, as well as to serve as locator marks to re-ink the removable marks again if necessary. Although the permanent marks should be visible by simple visual inspection, those marks are often faint and weak on new lenses providing low contrast, obscured by scratches on older lenses, and partially occluded and difficult to recognize on tinted or anti-reflection coated lenses. In this contribution, we present an extremely simple visualization system for permanent marks in PALs based on digital in-line holography. Light emitted by a superluminescent diode (SLD) is used to illuminate the PAL which is placed just before a digital (CCD) sensor. Thus, the CCD records an in-line hologram incoming from the diffracted wavefront provided by the PAL. As a result, it is possible to recover an in-focus image of the PAL inspected region by means of classical holographic tools applied in the digital domain. This numerical process involves digital recording of the in-line hologram, numerical back propagation to the PAL plane, and some digital processing to reduce noise and present a high quality final image. Preliminary experimental results are provided showing the applicability of the proposed method.