Holographic tracking of living cells by three-dimensional reconstructed complex wavefronts alignment.

We propose here a new three-dimensional (3D) holographic tracking method capable to track, simultaneously and in a single step, all the spatial coordinates of micro-objects. The approach is based on the enhanced correlation coefficient (ECC) maximization method but applied, for the first time to the best of our knowledge, directly on the holographic reconstructed complex wave fields. The key novelty of the proposed strategy is its ability to calculate simultaneously the 3D coordinates of cells, without decoupling the contribution of amplitude and phase. The proposed strategy is tested on living cells (i.e., NIH 3T3 mouse fibroblast) flowing into a microfluidic channel and compared with classical holographic tracking approach. Theoretical description and experimental validation of the proposed strategy are reported.

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.

Enhancing depth of focus in tilted microfluidics channels by digital holography.

In this Letter we propose a method to enhance the limited depth of field (DOF) in optical imaging systems, through digital holography. The proposed approach is based on the introduction of a cubic phase plate into the diffraction integral, analogous to what occurs in white-light imaging systems. By this approach we show that it is possible to improve the DOF and to recover the extended focus image of a tilted object in a single reconstruction step. Moreover, we demonstrate the possibility of obtaining well-focused biological cells flowing into a tilted microfluidic channel.

Quantitative phase maps denoising of long holographic sequences by using SPADEDH algorithm.

We propose a denoising method for digital holography mod 2π wrapped phase map by using an adaptation of the SPArsity DEnoising of Digital Holograms (SPADEDH) algorithm. SPADEDH is a l(1) minimization algorithm able to suppress the noise components on digital holograms without any prior knowledge or estimation about the statistics of noise. We test our algorithm with either general numerical simulated wrapped phase, quantifying the performance with different efficiency parameters and comparing it with two popular denoising strategies, i.e., median and Gaussian filters, and specific experimental tests, by focusing our attention on long-sequence wrapped quantitative phase maps (QPMs) of in vitro cells, which aim to have uncorrupted QPMs. In addition, we prove that the proposed algorithm can be used as a helper for the typical local phase unwrapping algorithms.

On the holographic 3D tracking of in vitro cells characterized by a highly-morphological change.

Digital Holography (DH) in microscopic configuration is a powerful tool for the imaging of micro-objects contained into a three dimensional (3D) volume, by a single-shot image acquisition. Many studies report on the ability of DH to track particle, microorganism and cells in 3D. However, very few investigations are performed with objects that change severely their morphology during the observation period. Here we study DH as a tool for 3D tracking an osteosarcoma cell line for which extensive changes in cell morphology are associated to cell motion. Due to the great unpredictable morphological change, retrieving cell's position in 3D can become a complicated issue. We investigate and discuss in this paper how the tridimensional position can be affected by the continuous change of the cells. Moreover we propose and test some strategies to afford the problems and compare it with others approaches. Finally, results on the 3D tracking and comments are reported and illustrated.

Clear coherent imaging in turbid microfluidics by multiple holographic acquisitions.

Recently it has been demonstrated that digital holography is a powerful means allowing imaging of both amplitude and phase objects in turbid flowing media. However, in quasi-static turbid microfluidics, multiple scattering contributions through the colloids superimpose coherently to the recording device, resulting in speckle noise and hindering a clear vision of the objects. In this Letter we exploit the Brownian motion of the colloidal particles to get multiple uncorrelated holograms, and we combine them to reduce the speckle contrast. In this way we get a multi-look gain without losing image resolution.

Terahertz tuning of whispering gallery modes in a PDMS stand-alone, stretchable microsphere.

We report on tuning the optical whispering gallery modes (WGMs) in a poly dimethyl siloxane-based (PDMS) microsphere resonator by more than 1 THz. The PDMS microsphere system consists of a solid spherical resonator directly formed with double stems on either side. The stems act like tie-rods for simple mechanical stretching of the microresonator, resulting in tuning of the WGMs by one free spectral range. Further investigations demonstrate that the WGM shift has a higher sensitivity (0.13 nm/µN) to an applied force when the resonator is in its maximally stretched state compared to its relaxed state.

Twin-beams digital holography for 3D tracking and quantitative phase-contrast microscopy in microfluidics.

We report on a compact twin-beam interferometer that can be adopted as a flexible diagnostic tool in microfluidic platforms with twofold functionality. The novel configuration allows 3D tracking of micro-particles and, at same time, can simultaneously furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. Experimental demonstration is given on for in vitro cells in a microfluidic environment.

Light induced patterning of poly(dimethylsiloxane) microstructures.

A new method for direct patterning of Poly(dimethylsiloxane) (PDMS) microstructures is developed by taking advantage of photorefractive effect in a functionalized substrate. Here we show that when a x-cut Iron doped Lithium Niobate (LN) crystal is exposed to appropriate structured laser light, a charge density pattern builds-up in the crystal and a space charge field arise that is able to induce self-patterning of the PDMS liquid film deposited on its surface via the dielectrophoretic effects. Proper heating treatment allows to achieve polymeric linking process creating a solid and stable PDMS microstructures. The self-patterned structures replicate the illuminating light pattern. We show that 1D and 2D patterning of PDMS gratings can be achieved. This new soft-lithographic approach can pave the way for realizing PDMS micro-structures with high degree of flexibility that avoids the need of moulds fabrication.

Synthesis and display of dynamic holographic 3D scenes with real-world objects.

A 3D scene is synthesized combining multiple optically recorded digital holograms of different objects. The novel idea consists of compositing moving 3D objects in a dynamic 3D scene using a process that is analogous to stop-motion video. However in this case the movie has the exciting attribute that it can be displayed and observed in 3D. We show that 3D dynamic scenes can be projected as an alternative to complicated and heavy computations needed to generate realistic-looking computer generated holograms. The key tool for creating the dynamic action is based on a new concept that consists of a spatial, adaptive transformation of digital holograms of real-world objects allowing full control in the manipulation of the object's position and size in a 3D volume with very high depth-of-focus. A pilot experiment to evaluate how viewers perceive depth in a conventional single-view display of the dynamic 3D scene has been performed.

Compression of digital holograms via adaptive-sparse representation.

Efficient storage and transmission of digital holograms (DHs) requires the development of appropriate compression techniques for such a special class of images. In this Letter, we investigate a method to compress DHs using a sparse matrix representation. Using digital holography to numerically manage complex wave fields, we are able to apply an adaptive mask, based on a threshold filter, to the object wave field. From there, we store the result of this filtering by sparse representation. In this Letter, we demonstrate that using sparse representation allows for a high compression factor with minimal loss in the quality of the reconstructed image. This technique is efficient for storage and transmission of DHs.

Optical reconstruction of digital holograms recorded at 10.6 microm: route for 3D imaging at long infrared wavelengths.

We demonstrate the optical reconstruction in the visible range (0.532 microm) of digital holograms recorded at long IR wavelengths (10.6 microm) by means of a spatial light modulator. By using an integrated recording-reconstruction system, it is, in fact, feasible to achieve direct imaging of holograms acquired outside the visible range, i.e., in the IR spectrum. By choosing a Fourier recording configuration, the reconstructed image, obtained at about a 20 times shorter wavelength than the acquisition image, exhibits minor aberrations, which do not significantly affect the optical reconstruction. The high NA achievable at a long IR wavelength allows us to image large objects at reasonable distances.

Spatially resolved refractive index profiles of electrically switchable computer-generated holographic gratings.

We describe a spatially resolved interferometric technique combined with a phase reconstruction method that provides a quantitative two-dimensional profile of the refractive index and spatial distribution of the optical contrast between the on-off states of electrically switchable diffraction gratings as a function of the external electric field. The studied structures are holographic gratings optically written into polymer/liquid crystal composites through single-beam spatial light modulation by means of computer-generated holograms. The electro-optical response of the gratings is also discussed. The diffraction efficiency results to be dependent on the incident light polarization suggesting the possibility to develop polarization dependent switching devices.

Surgical Excision of Keloids Followed by In-office Superficial Radiation Therapy: Prospective Study Examining Clinical Outcomes.

BACKGROUND: Keloids are benign proliferative scars that often occur among individuals of color, and are thought to be the result of excessive collagen deposition that occurs after injury to the skin. The treatment of these scars is difficult with often poor outcomes. This study aimed to evaluate the effectiveness of surgical excision followed by in-office superficial radiation therapy (SRT) as a method to improve keloid remission. METHODS: Participants for this study were recruited from June 2016 through February 2017 with 48 subjects enrolled and completed this study. All keloids were surgically resected and participants received 3 consecutive days of a customized dose of SRT, with a maximum cumulative dosage of 18 Gy. Patients were followed over the course of 12 months to monitor outcomes. RESULTS: In this cohort, we found 39 (81%) to have achieved successful remission with 9 (19%) being classified as refractory. There were no adverse effects or medical complications reported as a part of this study. CONCLUSION: Study outcomes support the clinical benefits of surgical excision followed by SRT as a practical and efficient treatment for keloids.

Biophysical investigation of living monocytes in flow by collaborative coherent imaging techniques.

We implemented a completely label-free biophysical (morphometric and optical) property characterization of living monocytes in flow, using measurements obtained from two coherent imaging techniques: a pure light scattering approach to obtain an optical signature (OS) of cells, and a digital holography (DH) approach to achieve optical cell reconstructions in flow. A precise 3D cell alignment platform, taking advantage of viscoelastic fluid properties and microfluidic channel geometry, was used to investigate the OS of cells to achieve their refractive index, ratio of the nucleus over cytoplasm, and overall cell dimension. Further quantitative phase-contrast reconstructions by DH were employed to calculate surface area, dry mass, and biovolume of monocytes by using the OS outcomes as input parameters. The results show significantly different biophysical cell properties, confirming the possibility to differentiate monocytes from other cell classes in flow, thus avoiding chemical cell staining or labeling, which are nowadays used.

Three-dimensional color object visualization and recognition using multi-wavelength computational holography.

In this paper, we address 3D object visualization and recognition with multi-wavelength digital holography. Color features of 3D objects are obtained by the multiple-wavelengths. Perfect superimposition technique generates reconstructed images of the same size. Statistical pattern recognition techniques: principal component analysis and mixture discriminant analysis analyze multi-spectral information in the reconstructed images. Class-conditional probability density functions are estimated during the training process. Maximum likelihood decision rule categorizes unlabeled images into one of trained-classes. It is shown that a small number of training images is sufficient for the color object classification.

Label-free analysis of mononuclear human blood cells in microfluidic flow by coherent imaging tools.

The investigation of the physical properties of peripheral blood mononuclear cells (PBMC) is of great relevance, as they play a key role in regulating human body health. Here we report the possibility to characterize human PBMC in their physiological conditions in a microfluidic-based measurement system. A viscoelastic polymer solution is adopted for 3D alignment of individual cells inflow. An optical signature (OS) acquisition of each flowing cell is performed using a wide angle light scattering apparatus. Besides, a quantitative phase imaging (QPI) holographic system is employed with the aim (i) to check the position in flow of individual cells using a holographic 3D cell tracking method; and (ii) to estimate their 3D morphometric features, such as their refractive index (RI). Results obtained by combining OS and QPI have been compared with literature values, showing good agreement. The results confirm the possibility to obtain sub-micrometric details of physical cell properties in microfluidic flow, avoiding chemical staining or fluorescent labelling.

Quasi noise-free digital holography.

One of the main drawbacks of Digital Holography (DH) is the coherent nature of the light source, which severely corrupts the quality of holographic reconstructions. Although numerous techniques to reduce noise in DH have provided good results, holographic noise suppression remains a challenging task. We propose a novel framework that combines the concepts of encoding multiple uncorrelated digital holograms, block grouping and collaborative filtering to achieve quasi noise-free DH reconstructions. The optimized joint action of these different image-denoising methods permits the removal of up to 98% of the noise while preserving the image contrast. The resulting quality of the hologram reconstructions is comparable to the quality achievable with non-coherent techniques and far beyond the current state of art in DH. Experimental validation is provided for both single-wavelength and multi-wavelength DH, and a comparison with the most used holographic denoising methods is performed.

Extended focused image in microscopy by digital Holography.

In microscopy, high magnifications are achievable for investigating micro-objects but the paradigm is that higher is the required magnification, lower is the depth of focus. For an object having a three-dimensional (3D) complex shape only a portion of it appears in good focus to the observer who is essentially looking at a single image plane. Actually, two approaches exist to obtain an extended focused image, both having severe limitations since the first requires mechanical scanning while the other one requires specially designed optics. We demonstrate that an extended focused image of an object can be obtained through digital holography without any mechanical scanning or special optical components. The conceptual novelty of the proposed approach lies in the fact that it is possible to completely exploit the unique feature of DH in extracting all the information content stored in hologram, amplitude and phase, to extend the depth of focus.

On the origin of internal field in Lithium Niobate crystals directly observed by digital holography.

We show the defect dependence of the internal field in Lithium Niobate using a full-field interferometric method and demonstrate that it can be directly measured on some clusters of defects embedded in a stoichiometric matrix. Results show that the value of the internal field grows in proximity of defects and vanishes far from them, which addresses the long-standing issue about its origin in Lithium Niobate crystal.