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An online field verification method of road vehicle speed meters used for traffic speeding enforcement is proposed in this paper, and an off-board reference facility for the proposed online field verification method based on 3D tracking radar is developed. The principle of the online field verification method and the design requirement of the off-board reference facility are firstly introduced. Then the working principles of speed, distance and angle measurement used in the off-board reference facility based on 3D tracking radar are analyzed, and the traceability of speed measurement of the off-board reference facility is described. Finally, the full-range speed calibration and uncertainty evaluation in the laboratory are carried out. The speed calibration results show that the speed measurement errors of the off-board reference facility are within ±0.5 km/h at the speed value in the range of (10~100) km/h and ±0.5 % at the speed value in the range of (100~300) km/h, which can satisfy the requirement for the online field verification of road vehicle speed meters.

We proposed a rapid autofocusing method exploiting holographic polarization microscope, which could determine the refocusing distance without multiple reconstructions and complex network training. The defocus distance of the object is calculated rapidly by identifying the separation distance of the two defocus images of the object and using the linear relation between the defocus distance of the object and the separation distance of the two defocus images of the object. The linear relationship is obtained by numerical propagation and linear fitting. Last, we have demonstrated the effectiveness of the proposed method by reconstructing the in-focus images of flowing cells. We believe that our proposed method could be a powerful tool for dynamic observation and extend the application of holographic polarization microscope.

Digital holographic reconstruction of diffraction patterns recorded using large aperture parabolic mirrors in a Z-configuration provide accurate, highly detailed, video-rate 3D tracking of mosquito flight in a 250×250×1200mm chamber.

Behaviors of platonic bacteria individuals are profoundly influenced by their interplay. However, probing such interplay still remains a challenge since identification and tracking of bacterial individuals becomes difficult as they come close and interact with each other. Herein, we report 3D tracking of the motions of multiple bacteria by using digital holographic microscopy (DHM), where the subtle 3D behaviors can be characterized as bacteria approach and run away from each other. An algorithm was developed to identify and recover the gap between 3D trajectory segments raising by the interruption from other bacteria through lateral image recognition and axial loalization utilizing cost function. We value the performance of the algorithm in terms of the statistics in trajectory length and correct rate. The study clearly shows how the interplaying Escherichia coli alter their motions.

Background Local computed tomography (CT) reconstruction is achievable with portal images acquired during volumetric-modulated arc therapy (VMAT) delivery and was named as VMAT-CT. However, the application of VMAT-CT is limited because it has limited field of view and no density information. In addition, the new generation of multi-leaf collimator with faster speed and various collimator angles used in patients’ plans could cause more artifacts in VMAT-CT. The goal of this study was to extend VMAT-CT concept, generate complete three-dimensional (3D) CT images, calculate new 3D dose, track and adapt VMAT plan based on updated images and dose. Materials and methods VMAT-CT and planning CT of phantoms were fused by rigid or deformable registration to create VMAT-CT+ images. Trackings based on planning CT, VMAT-CT+, and cone beam CT (CBCT) were compared. When prescription dose was not met for planning target volume (PTV), re-planning was demonstrated on an in-house deformable phantom. Possible uncertainties were also evaluated. Results Tracking based on VMAT-CT+ was accurate and superior to those based on planning CT and CBCT since VMAT-CT+ can detect changes during treatment. PTV coverage in the deformable phantom decreased after deformations but went up and met the prescription goal after re-planning. The impact of uncertainties on dose was minimal. Conclusion 3D tracking and adaptation of VMAT based on VMAT-CT are feasible. Our study has the potential to increase the confidence of beam delivery, catch and remedy errors during VMAT.

Based on the working principle of 3D tracking radar Doppler velocimeter for traffic speed enforcement, a new calibration method based on the Doppler shift simulation method is proposed in this paper. A new moving-target speed simulator is specifically designed and realized to verify the proposed new calibration method. The simulated speed range of the simulator is up to (10~250) km/h and the maximum permissible error is ±0.1 km/h, which can satisfy the calibration requirements of 3D tracking radar Doppler velocimeter. The calibration results of a selected sample validate the effectiveness and feasibility of the new calibration method.

This chapter reviews recent progress in single molecule 3D tracking in living cells. It begins with a historical overview of single particle tracking methods, moving to discussing advances necessary to move from tracking particles to molecules. We also discuss the technical means of encoding or measuring the motion of a single molecule in three spatial dimensions over time. We describe 3D single molecule tracking with both wide-field, camera-based approaches and 3D tracking based upon modified confocal microscope designs. This chapter also discusses various ways of analyzing or classifying molecular trajectories. Biophysical applications, from following protein transport in living cells to studying individual virus infection pathways, are discussed. We end with a view toward potential future improvements of 3D tracking in living cells.

Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information determines the 3D location of single objects at a speed only limited by the array detector’s frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads; the 3D positions and trajectories of single fluorescence beads can be determined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were determined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is superdiffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

Thin-film drainage process has been studied by a digital holographic recording system. In this study, trajectories of three particles inside a bubble film are revealed by holographic 3D tracking during the bubble growth.

We demonstrate that ultrasound field calibration and imaging are achievable in a vertical resonator using digital holography, showing that it is a flexible tool to assist the diffusion of acoustophoresis microfluidic devices.

A holographic 3D particle tracking method based on numerical diffraction propagation and correlation recognition is applied on film drainage analysis and red blood cells counting. Location of particles in different depth layers are revealed accurately.

Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but such recordings are compromised by brain motion that often accompanies behavior. Two-photon point scanning microscopy is especially sensitive to motion artifacts, and to date, two-photon recording of activity has required rigid mechanical coupling between the brain and microscope. To overcome these difficulties, we developed a two-photon tracking microscope with extremely low latency (360 μs) feedback implemented in hardware. We maintained continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s2 both in-plane and axially, allowing high-bandwidth measurements with modest excitation power. We recorded from motor- and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentation and behavioral outputs. Our technique can be extended to stabilize recordings in a variety of moving substrates.

This paper presents a smart cage platform using magnetic sensors for 3D tracking of the location and orientation of a magnetically marked capsule (MMC) for targeted drug delivery. For this study, a small permanent magnet is placed inside a capsule to be tracked. A smart cage is developed using eight 3-axis magnetometers that tracks the location and orientation of the capsule using a magnetic source imaging (MSI) algorithm. The capsule location was estimated within 2–3 mm in XY and 6 mm in the Z direction. The results presented here demonstrate that this noninvasive tracking system holds great promise for monitoring the transit of any device (such as a smart pill) that is magnetically marked.

We demonstrate a 3D holographic tracking method to investigate particles motion in a microfluidic channel while unperturbed while inducing their migration through microfluidic manipulation. Digital holography (DH) in microscopy is a full-field, label-free imaging technique able to provide quantitative phase-contrast. The employed 3D tracking method is articulated in steps. First, the displacements along the optical axis are assessed by numerical refocusing criteria. In particular, an automatic refocusing method to recover the particles axial position is implemented employing a contrast-based refocusing criterion. Then, the transverse position of the in-focus object is evaluated through quantitative phase map segmentation methods and centroid-based 2D tracking strategy. The introduction of DH is thus suggested as a powerful approach for control of particles and biological samples manipulation, as well as a possible aid to precise design and implementation of advanced lab-on-chip microfluidic devices.

We designed a LoC with embedded optofluidic holographic microscopy capabilities. Object flow allows unlimited FoV microscopy of samples in a liquid volume. High-throughput counting and 3D tracking of RBCs is demonstrated.

Most location-based services are based on a global positioning system (GPS), which only works well in outdoor environments. Compared to outdoor environments, indoor localization has created more buzz in recent years as people spent most of their time indoors working at offices and shopping at malls, etc. Existing solutions mainly rely on inertial sensors (i.e., accelerometer and gyroscope) embedded in mobile devices, which are usually not accurate enough to be useful due to the mobile devices’ random movements while people are walking. In this paper, we propose the use of shoe sensing (i.e., sensors attached to shoes) to achieve 3D indoor positioning. Specifically, a short-time energy-based approach is used to extract the gait pattern. Moreover, in order to improve the accuracy of vertical distance estimation while the person is climbing upstairs, a state classification is designed to distinguish the walking status including plane motion (i.e., normal walking and jogging horizontally), walking upstairs, and walking downstairs. Furthermore, we also provide a mechanism to reduce the vertical distance accumulation error. Experimental results show that we can achieve nearly 100% accuracy when extracting gait patterns from walking/jogging with a low-cost shoe sensor, and can also achieve 3D indoor real-time positioning with high accuracy.

We recently pioneered the concept of light-driven micro-robotics including the new and disruptive 3D-printed micro-tools coined Wave-guided Optical Waveguides that can be real-time optically trapped and "remote-controlled" in a volume with six-degrees-of-freedom. To be exploring the full potential of this new drone-like 3D light robotics approach in challenging microscopic geometries requires a versatile and real-time reconfigurable light coupling that can dynamically track a plurality of "light robots" in 3D to ensure continuous optimal light coupling on the fly. Our latest developments in this new and exciting area will be reviewed in this invited paper.

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fi elds, are combined with selfinterference digital holographic microscopy. This biophotonic holographic workstation enables non-contact, minimally invasive, fl exible, high-precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a fi rst model experiment, internalized polystyrene microspheres with 1 μ m diameter are three-dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.

Border cells are a cluster of cells that migrate from the anterior tip of the Drosophila egg chamber to the border of the oocyte in stage 9. They serve as a useful model to study collective cell migration in a native tissue environment. Here we describe a protocol for preparing ex vivo egg chamber cultures from transgenic flies expressing fluorescent proteins in the border cells, and using confocal microscopy to take a multi-positional time-lapse movie. We include an image analysis method for tracking border cell cluster dynamics as well as tracking individual cell movements.

Bacteria employ diverse motility patterns in traversing complex three-dimensional (3D) natural habitats. 2D microscopy misses crucial features of 3D behaviour, but the applicability of existing 3D tracking techniques is constrained by their performance or ease of use. Here we present a simple, broadly applicable, high-throughput 3D bacterial tracking method for use in standard phase contrast microscopy. Bacteria are localized at micron-scale resolution over a range of 350 × 300 × 200 μm by maximizing image cross-correlations between their observed diffraction patterns and a reference library. We demonstrate the applicability of our technique to a range of bacterial species and exploit its high throughput to expose hidden contributions of bacterial individuality to population-level variability in motile behaviour. The simplicity of this powerful new tool for bacterial motility research renders 3D tracking accessible to a wider community and paves the way for investigations of bacterial motility in complex 3D environments., Microscopy techniques used to study the movement of swimming microbes are limited to two dimensions or require sophisticated devices. Here, Taute et al. present a simple method for high-throughput 3D tracking of bacteria using standard phase contrast microscopy.

The Light Scattering Profile (LSP) of an individual cell provides a fast and accurate characterization of its morphological properties. By combining a camera-based small angle light scattering apparatus with a microfluidic-induced particle migration technique, it is possible to characterize cells in microfluidic flows. The scattering profile of an individual cell can be fully characterized by our optimized optical light collection system. Viscoelastic-induced particle migration by polyethylene oxide implemented in a low-cost microfluidic device composed of an alignment section and a measuring section opens the possibility of precise, label-free, individual cell analysis. We have studied living cells in microfluidic flows by our light scattering apparatus and by a Digital Holographic Microscope (DHM) system. Our DHM measurements provided an accurate 3D position tracking even in multiple cell conditions.

Service providers perform regular borescope inspection of field products to ensure system performance and prevent accidents. A full borescope inspection for large equipment usually takes several days to cover every region of interest in the turbine. One of the challenges that causes this long inspection time is the difficulty in navigating the borescope tip to some position of interest and aiming the view of the borescope in given direction. The image from the borescope tip is the only information available to the operator to judge the position of tip. In some cases, the operator can get lost due to the limited field-of-view and illumination provided by the borescope. It is very hard to tell the borescope tip position from one borescope image. This increases the difficulty of correlating the inspection results obtained at different times that might be used to predict potential machinery failure. This paper discusses various methods that have been investigated for 3D borescope tracking and presents a new approach using a shape sensor integrated in the borescope tube used with image based location determination to determine the position of the borescope tip during inspections. The shape sensor provides a real time estimate of the borescope tip position and orientation then the image based analysis uses the part CAD model to fine tune this position information. The tracking result can provide better information of the tip position for the operator. This enhanced position information can be used to better monitor defect changes over time by comparing the inspection result at different times in the parts lifetime.

Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.

Digital Holography (DH) in microscopy allows to retrieve in an accurate way the spatial coordinates of multiple moving particles, performing 3D tracking of the sample in the entire field of view. In particular, a posteriori quantitative multi-focus phase-contrast imaging, suitable for 3D tracking of micro-objects, is one of the main features of the holographic approach. However, classical methods need to decouple amplitude and phase contributions of the reconstructed complex wavefronts to calculate target positions in 3D, due to the fact that the lateral displacements can be calculated only after refocusing step. In order to overcome this limitation, recently, a novel method of the simultaneous calculation of both axial and lateral coordinates of moving particles has been proposed. This is based on the novel concept of wavefronts matching, i.e. the 3D positions of micro-object, moving in 3D volume, are obtained by aligning wo subsequent holographic complex reconstructions, calculated at the same distance. We test this approach in different experimental conditions in order to highlight its effectiveness in bio-microfluidic applications.

In the present paper, Holographic Optical Tweezers (HOT) is employed to trap and manage functionalized micrometric latex beads with the aim at probing cellular forces in no-adherent state. For the first time at best of our knowledge, a suspended cell, subjected to mechanical stress, structures its cytoskeleton when anchored to point-like bonds. We exploit the HOT arrangement to induce mechanical deformation in suspended NIH 3T3 fibroblast. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence. In our experiment, cell adhesion is prevented and the stimulus is applied through latex beads trapped by HOT and positioned externally to the cell membrane. Our aims are devoted to analyze cell response during the transition from an homogeneous and isotropic structure (as it’s in suspension) to a mechanically stressed state. To analyze the cell material interaction we combine the HOT arrangement with two imaging systems: a Digital Holography (DH) setup in microscope configuration that is an investigation method useful for quantitative, label-free and full-field analysis of low contrast object and a fluorescence modulus. HOT are exploited to induce cellular response to specific stimuli while DH allows to measure such responses in no-invasive way. Finally, fluorescence imaging is added to discriminate the inner cell structures.

Digital holographic microscopy (DHM) provides quantitative multi-focus phase contrast for high resolving investigations on reflective and transparent specimens that can be applied for label-free analysis of living cells. Results from application on mouse fibroblasts in collagen demonstrate that DHM enables minimally-invasive automated 3D tracking of multiple cells without mechanical focus control.

Digital holographic microscopy [1-8] is a powerful tool for the imaging of micro-objects contained into a three dimensional (3D) volume. Many techniques accomplish the tracking with good detection accuracy of particle, microorganism and cells [9-13]. The most popular approaches adopted in digital holography for the 3D tracking of micro-objects consist into estimate the 3D position dividing the computation process into two parts: the estimation of the coordinate along the optical axis of the imaging systems through automatic focusing on the amplitude reconstruction of the digital holograms, and the estimation of the transverse coordinates on the phase reconstruction of digital holograms computed at distance d equal to the estimated focal plane. About the estimation of the focal plane, several automatic focusing methods were developed in digital holography for pure phase objects. These techniques are based on a suitable image contrast parameters and perform a numerical scanning of the focal plane [14]. The estimated focus distance is typically achieved in their global maximum or minimum. Instead the localization in the plane can be computed through different image segmentation methods. The accuracy of each of these techniques is closely related to the kind of object to be tracked [11].

The 3D tracking of micro-objects, based on digital holography, is proposed through the analysis of the complex wavefront of the light scattered by the micro-samples. Exploiting the advantages of the off-axis full-field holographic interferometry, the tracking of multiple objects is achieved by a direct wavefront analysis at the focal plane overcoming the limitation of the conventional back focal plane interferometry in which only one object at a time can be tracked. Furthermore, the method proposed and demonstrated here is a step forward with respect to other holographic tracking tools. The approach is tested in two experiments, the first investigates the Brownian motion of particles trapped by holographic optical tweezers, while the second relates to the cell motility in a 3D collagen matrix, thus showing its usefulness for lab-on-chip systems in typical bioassay testing.

A method for 3D tracking has been developed exploiting Digital Holography features in Microscopy (DHM). 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 samples 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 amplitude refocusing in traditional tracking processes. Moreover, 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. The main effect on light propagating in such objects is in phase. This is known as phase-retardation or phase-shift. DHM is an innovative and alternative approach in microscopy, it’s a good candidate for no-invasive and complete specimen analysis because its main characteristic is the possibility to discern between intensity and phase information performing quantitative mapping of the Optical Path Length. In this paper, the flexibility of DH is employed to analyze cell mechanics of unstained cells subjected to appropriate stimuli. DHM is used to measure all the parameters useful to understand the deformations induced by external and controlled stresses on in-vitro cells. Our configuration allows 3D tracking of micro-particles and, simultaneously, furnish quantitative phase-contrast maps. Experimental results are presented and discussed for in vitro cells.

The response of the receptor, IgE-FceRI, to binding of a multivalent antigen begins a cellular signaling cascade resulting in the release of histamine, part of the human allergic response. The receptor's modes of motion and internalization process are investigated using a confocal based 3D tracking microscope. A Nipkow spinning disk has been added to the microscope to allow simultaneous confocal 3D tracking and wide field spinning disk imaging. The receptor is tracked in live cells while simultaneously imaging cellular structures labeled with fluorescent proteins.

Guiding, controlling and studying cellular functions are challenging themes in the biomedical field, as they are fundamental prerequisites for new therapeutic strategies from tissue regeneration to controlled drug delivery. In recent years, multidisciplinary studies in nanotechnology offer new tools to investigate important biophysical phenomena in response to the local physical characteristics of the extracellular environment, some examples are the mechanisms of cell adhesion, migration, communication and differentiation. Indeed for reproducing the features of the extracellular matrix in vitro, it is essential to develop active devices that evoke as much as possible the natural cellular environment. Our investigation is in the framework of studying and clarifying the biophysical mechanisms of the interaction between cells and the microenvironment in which they exist. We implement an optical tweezers setup to investigate cell material interaction and we use Digital Holography as non-invasive imaging technique in microscopy. We exploit Holographic Optical Tweezers arrangement in order to trap and manage functionalized micrometric latex beads to induce mechanical deformation in suspended cells. A lot of papers in literature examine the dynamics of the cytoskeleton when cells adhere on substrates and nowadays well established cell models are based on such research activities. Actually, the natural cell environment is made of a complex extracellular matrix and the single cell behavior is due to intricate interactions with the environment and are strongly correlated to the cell-cell interactions. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence.

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.

We review our recent progress on lensfree holographic on-chip microscopy techniques that are aimed at high-throughput imaging applications including 3D tracking of sperms.

We propose a new strategy of three-dimensional (3D) tracking of living cells by digital holography microscopy based on the morphological changes of cells during the migration. A comparison with other 3D tracking methods is accomplished.

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, fo r 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. Keywords: Digital holographic microscopy, 3D tracking. 1. INTRODUCTION Many studies report on the ability of digital holography [1-8] to track particle, microorganism and cells [9-14] and there are several methodology to obtain the 3D positions. The typical strategy adopted in digital holography for the tracking of living cells consists into estimate the 3D position dividing the calculation process into two parts: the estimation of the focal plane (i.e. the Z coordinate along the optical axis of the imaging systems) on the amplitude reconstruction of the digital holograms [15,16], and the estimation of the transverse coordinates by the phase reconstruction of digital holograms computed at distance d equal to the estimated focal plane. About the estimation of the focal plane, several autofocus approaches were developed in digital holography for pure phase objects for live-cell imaging. These techniques are based on a suitable image contrast coefficients and perform a numer ical scanning of the focus. Instead the localization in the plane can be computed through different image segmentation methods. We propose a new strategy for both focal plane estimation and image segmentation. In particular, we propose to use an approximation of the Tamura coefficient, as image contrast measure, for the estimation of the in-focus distance. This metric has been applied successfully for the amplitude reconstructi on of digital holograms of macroscopic s cattering objects [17]. Instead, for the estimation of the transverse coordinates, we introduce a new morphological operator that we have called minimum boundary filter (MBF) [11]. This operator is robust with resp ect both noise and morphological variations of cells. In fact, the most popular image segmentation techniques used in th e holographic tracking methods introduce position estimation errors caused by these two aforementioned issues. Instead the MBF is built specifically to take into account the morphological changes during the cells migration. We test the proposed method in a typical situation for which extensive changes in cells morphology are associated to cells motion and we perform a comparison between other popular strategies.

Interacting in a small (mobile) auto-stereoscopic display can be difficult because of the lack of accurate tracking of an interaction proxy, and having to maintain a fixed viewpoint and adapt to a different level of depth perception sensitivity. In this paper, we first propose to modify a standard stylus into a mechanical chain with joint sensors for 3D tracking. We also investigate a way to assist the user in selecting an object in the small phone space through supplementary multimodal feedback, such as sound and tactility. We have carried out an experiment comparing the effects of various combinations of multimodal feedback to object selection performance.

A compact twin-beam interferometer that can be adopted as a flexible diagnostic tool in microfluidic platforms is presented. The devise has two functionalities, as explained in the follow, and can be easily integrated in microfluidic chip. The configuration allows 3D tracking of micro-particles and, at same time, furnishes Quantitative Phase-Contrast maps of tracked micro-objects by interference microscopy. Experimental demonstration of its effectiveness and compatibility with biological field is given on for in vitro cells in microfluidic environment. Nowadays, several microfluidic configuration exist and many of them are commercially available, their development is due to the possibility for manipulating droplets, handling micro and nano-objects, visualize and quantify processes occurring in small volumes and, clearly, for direct applications on lab-on-a chip devices. In microfluidic research field, optical/photonics approaches are the more suitable ones because they have various advantages as to be non-contact, full-field, non-invasive and can be packaged thanks to the development of integrable optics. Moreover, phase contrast approaches, adapted to a lab-on-a-chip configurations, give the possibility to get quantitative information with remarkable lateral and vertical resolution directly in situ without the need to dye and/or kill cells. Furthermore, numerical techniques for tracking of micro-objects needs to be developed for measuring velocity fields, trajectories patterns, motility of cancer cell and so on. Here, we present a compact holographic microscope that can ensure, by the same configuration and simultaneously, accurate 3D tracking and quantitative phase-contrast analysis. The system, simple and solid, is based on twin laser beams coming from a single laser source. Through a easy conceptual design, we show how these two different functionalities can be accomplished by the same optical setup. The working principle, the optical setup and the mathematical modeling for 3D tracking is described. Finally, the experimental proof is presented and discussed for in vitro cells in microfluidic chamber.

3D tracking is of fundamental importance to the development of practical visual applications to micromanipulation and microassembly. In this paper, a new 3D tracking method based on CAMSHIFT and Depth-from-Defocus is developed for micromanipulation and microassembly task. CAMSHIFT algorithm is used to find the size and location of moving object even the micro-objects is highly blurred. And Depth-from-defocus method is used to recover the depth information of the object from a measure of the level of defocus. The experimental results of blurring tracking and depth recovery of micro gear validate the feasibility of the proposed 3D tracking method.

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. (C) 2014 Optical Society of America

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. (C) 2012 Optical Society of America

We demonstrate through a unique and novel approach based on holographic imaging the ability to achieve full morphological 3D analysis and 3D visualization of motile cells and their accurate 3D tracking for Lab-on Chip devices.