This PDF file contains the front matter associated with SPIE Proceedings Volume 10887, including the Title Page, Copyright Information, Table of Contents, Author and Conference Committee lists.

Using a coded-illumination LED array microscope and a computational imaging approach to data capture and optical system design, we demonstrate efficient and accurate quantitative phase imaging from minimal measurements with learning-optimized illumination design and reconstruction. In addition, we describe how space-time models for non-rigid sample motion can be used to mitigate sample motion blur in multi-frame phase microscopy.

Optofluidic time-stretch microscopy is a powerful tool in imaging flow cytometry as it enables continuous image acquisition at a frame rate higher than 10,000 frames per second. In addition to bright-field imaging that provides morphological information, attempts have been made to integrate quantitative phase imaging (QPI) with optofluidic time-stretch microscopy to acquire information related to subcellular structure, such as the refractive index and thickness. However, the applicability of such methods is hindered by errors introduced during phase unwrapping and the need for a high-bandwidth photodetector. To overcome these limitations, here we demonstrate optofluidic time-stretch QPI based on an acousto-optic modulator (AOM) that acquires intensity and phase image with a low-bandwidth photodetector without phase-unwrapping errors. In our system, the signal beam that carries cellular information interferes with the reference beam, the frequency of which is shifted by 1/4 of the repetition frequency of the laser by an AOM. The beat note is then detected by a normal photodetector, and its waveform that consists of groups of four successive pulses is converted into phase and intensity images with simple calculations. Therefore, we lower the requirement of the photodetector bandwidth and eliminate the errors in phase unwrapping while maintaining a throughput of 10,000 cells per second. These advantages of our system offer new possibilities for high-throughput label-free cancer cell detection in blood by looking at cellular phase information including structural features, enabling early cancer detection and improving the effectiveness of treatment.

The aim of this work is to develop a holographic method that provides the shape reconstruction with an extended measurement range and preserved high accuracy. The method requires recording of series of fully-coherent holograms generated with varying tilt of plane wave illumination. The captured holograms are numerically processed to obtain the corresponding complex fields. The complex fields are used to produce a new set of holograms, which are used for calculating the longitudinal coherence function. This function allows observing fringes of high contrast at specific heights similar to white light interferometry, and thus, shape reconstruction of the three-dimensional object is carried out. The conclusions of this work are supported with results of numerical simulations.

To digitally decode phase and amplitude images of a sample from its hologram, auto-focusing and phase recovery steps are required, which are in general challenging to compute. Here, we demonstrate fast and robust autofocusing and phase recovery that are simultaneously performed using a deep convolutional neural network (CNN). This CNN is trained with pairs of randomly de-focused back-propagated holograms and their corresponding in-focus phase recovered images (used as ground truth). After its training, the CNN takes a single back-propagated hologram, and outputs an extended depth-of-field (DOF) complex-valued image, where all the objects or points-of-interest within the sample volume are autofocused and phase-recovered all in parallel. Compared to iterative image reconstruction or a CNN trained using only in-focus images, this new approach achieves >25-fold increase in image DOF and eliminates the need to autofocus individual points within the sample volume, thus improving the complexity of holographic image reconstruction from O(nm) to O(1), where n refers to the number of individual object points within the sample volume, and m represents the autofocusing search space. We demonstrated the success of this approach by imaging various samples, including aerosols and human breast tissue sections. Our results highlight some unique capabilities of deep-learning based image reconstruction methods that are powered by data.

As light, or any other wave, converges into focus, the apparent wavelength of the wave increases as the phase velocity surpasses the speed of propagation in that medium. For a Gaussian beam, this causes a phase change Δϕ equal to ±π. This phenomenon, named the Gouy phase shift, can be observed when a plane wave is refracted by a lens and caused to come into focus. The location where this phase shift occurs in the axial direction is the focal length of the lens. Many biological structures are curved and thus can be modeled as lenslets. Bacterial cells have a radius of curvature that can be readily determined from high resolution images. A plane wave passing through them would be refracted and converge at some point after interacting with the bacterium. Altering the refractive index of the cells will change the effective focal length of the lenslet and thus the location of the Gouy phase shift. We have previously shown that purified gas vesicles (GVs) can be transfected into bacterial cells, altering the refractive index in large areas of the cell. In this work, we use off-axis digital holographic microscopy to measure the effect of GVs on the index of refraction of Salmonella cells and relate this to changes in the Gouy phase shift. By observing the location of this phase shift relative to the location of the bacterium, the GV concentration within the cell can be estimated, highlighting the potential of GVs as a quantitative contrast agent for QPI.

Limited angle holographic tomography (LAHT) is currently the most common tool in biomedical applications of 3D quantitative phase imaging. It uses the refractive index (RI) as contrast agent for a single cell or tissue analysis and provides highly accurate RI values in the full measurement volume. Recently several new systems have been built in laboratories and new devices have been released into the market. All of them apply algorithms and processing paths which significantly influence correctness of the results. In our work we perform study of the selected LAHT systems and compare their 3D metrological features and other functional parameters.

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. In this talk, we report two optimum illumination schemes for FPM to achieve high-speed or even single-shot QPI. We present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm2, corresponding to a space–bandwidth–time product of 411 megapixels per second. We also discuss how FPM can be extended to optical diffraction tomography (ODT) under Born or Rytov approximation, achieving super and depth resolved 3D imaging over a wide FOV.

In coherent imaging systems, parasitic fringes and concentric patterns could commonly be found due to the unwanted multiple reflections [1]. One fundamental solution for the coherent noise is to use a temporally incoherent light source. However, maintaining the full-field interference fringe contrast using temporally incoherent light is not straightforward for interferometric imaging techniques such as quantitative phase imaging (QPI). Fortunately, several brilliant incoherent QPI techniques have been realized for wide-field imaging mainly through the common-path interferometer geometries [2-4]. However, it has been more difficult to implement incoherent-light-based optical diffraction tomography (ODT) due to the additional angle-scanning illumination unit that induces severe decoherence over the camera field-of-view [5]. Here, we suggest a temporally low-coherence optical diffraction tomography by angle-scanning broadband illumination based on general Mach-Zehnder interferometric geometry. We have designed an angle-scanning unit composed of two digital micromirror devices (DMDs) to maintain interference fringe contrast across the whole field of view during the angle-scanning sequence. Further, we have developed the theoretical framework for ODT reconstruction using incoherent light. In the light of our recent developments, we will discuss the theoretical and practical constraints, and suggest the best degree of incoherency for incoherent ODT. We will also demonstrate the incoherent optical diffraction tomography of plastic microspheres, human blood cells and rat pheochromocytoma cells. References 1. I. Choi, K. Lee, and Y. Park, "Compensation of aberration in quantitative phase imaging using lateral shifting and spiral phase integration," Opt. Express 25, 30771-30779 (2017). 2. Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. U. Gillette, and G. Popescu, "Spatial light interference microscopy (SLIM)," Opt. Express 19, 1016-1026 (2011). 3. B. Bhaduri, H. Pham, M. Mir, and G. Popescu, "Diffraction phase microscopy with white light," Opt. Lett. 37, 1094-1096 (2012). 4. Y. Baek, K. Lee, J. Yoon, K. Kim, and Y. Park, "White-light quantitative phase imaging unit," Opt. Express 24, 9308-9315 (2016). 5. M. Rinehart, Y. Zhu, and A. Wax, "Quantitative phase spectroscopy," Biomed. Opt. Express 3, 958-965 (2012).

We propose a simple and compact microscope combining phase imaging with fluorescence. This compact setup can be easily inserted in a standard biological incubator and allows observation of cellular cultures over several days. Phase image of the sample is reconstructed from a single, slightly (~50 μm) defocused image taken under semi-coherent illumination. Fluorescence in-focus image is recorded in epi-fluorescence geometry. The phase and fluorescence images are taken sequentially using a single CMOS camera. No mechanical movement of neither sample nor objective is required to change the imaging modality. The only change is the wavelength of illumination and excitation light for phase and fluorescence imaging, respectively. The slight defocus needed for phase imaging is achieved due to specifically introduced chromatic aberration in the imaging system. We present dual modality time-lapse movies of cellular cultures observed over several days in physiological conditions inside an incubator. A field-of-view of 3 mm2 allows observation up to thousands of cells with micro-meter spatial resolution in quasi-simultaneous phase and fluorescence mode. We believe that the simplicity, small dimensions, ease-of-use and low cost of the system make it a useful tool for biological research

Quantitative phase imaging (QPI) allows the monitoring of adherent cell cultures continuously over long time periods and it delivers an image of the cell with pixel intensities corresponding to the optical path difference (OPD). These images can be processed to quantify several cellular features. In particular, cell OPD measurements allows the estimation of the cell dry mass, an important metric to study cell mass and growth kinetics. If the ability of QPI to provide phase-contrast images of cells is taken for granted, the accuracy and the precision of QPI cell OPD measurements can still be questioned. Indeed, the reported QPI cell measurements have not yet been assessed with any reference method (e.g. microfluidic resonators). And there is a lack of independent experimental comparison and validation which can hinder the acceptance of QPI in the realms of live-cell mass profiling. With the aim of filling this gap, here we compare three different methods: digital holographic microscopy, quadriwave lateral sheering interferometry and lens-free microscopy (not yet established as a QPI technique). The experimental design is based on the inter-modality comparisons of OPD measurements performed over several tens of cells. To ensure consistency, we performed OPD measurements on a fixed cell culture the same day on the same location. Importantly, the statistical analysis of these measurements allowed us to estimate the precision of QPI cell OPD measurements without any reference material. In addition, we have evaluated the influence of the post-processing steps (baseline subtraction, cell segmentation) on the precision of QPI cell measurements.

In response to heat stress, triggered by a temperature increase of just a few degrees, cells activate a mechanism called the heat-shock response. While conventional global heating processes lead to an overall and slow increase of the temperature, heating processes based on laser illumination enable to achieve fast dynamics on the sub-second time-scale and spatially localized. Thermal imaging using quadriwave lateral shearing interferometry (TIQSI) has been developped by Institut Fresnel in collaboration with PHASICS SA [1]. By quantitatively measuring the transmitted phase this approach is able to measure temperature fields at the microscopic level from thermal-induced refractive index changes of the medium surrounding laser-illuminated plasmonic nanoabsorber. Phase, intensity and temperature images are measured in parallel thanks to a quadriwave lateral shearing interferometry SID4BIO camera, developed by PHASICS. We propose in this article to study the dynamics of the heat-shock response of living cells by using a TIQSI system. A dynamic control heat stress is induced to retinal pigmented epithelial (RPE) cells by illuminating gold nanoparticles used as nanosources of heat [2]. Intensity images of the heat-shock transcription factors (HSF) of cells fluorescently labelled observed in parallel to the heating process reveal the formation of fluorescent granules within the nucleus, a sign of cellular heat-shock response. By heating successively the RPE cells for different laser powers, we measure rise-time and dynamic of the heat-shock for different magnitudes of the stress response. [1] Baffou, G., Bon, P., Savatier, J., Polleux, J., Zhu, M., Merlin, M., ... & Monneret, S. (2012). Thermal imaging of nanostructures by quantitative optical phase analysis. ACS nano, 6(3), 2452-2458. [2] Robert, H.M.L, Savatier, J., Vial, S., Verghese, J., Wattellier, B., … & Baffou, G. (2018) Small, 1801910

Phase-contrast microscopy detects the optical phase delayed by a specimen, especially by transparent ones. Modern phase imaging techniques are capable of quantifying the phase, enabling a large variety of applications. A missing feature of phase imaging, however, is the lack of chemical information. To fulfill this gap, we developed a bond-selective phase-contrast microscope based on chemical bond vibrational absorption of mid-infrared light. We report high spectral fidelity, nanosecond temporal resolution, submicron spatial resolution, and a speed of 50 frame/sec in a wide field scheme, limited by the camera. Our microscope utilizes a mid-infrared pulsed laser to induce a local temperature rise via absorption, resulting in a transient phase change of probe light that is directly related to the molecular spectroscopy information of the specimen. By tuning the delay between the pump and probe laser, the dynamics of the absorption and heat decay is acquired, adding another dimension of information. Our approach links the missing chemical information to phase contrast, which paves a new avenue for microscopic studies in biology and materials science.

Micro-plastics dispersion in water is one of the major global threats due to the potential of plastic items to affect the food chain and reproduction of marine organisms. However, reliable and automatic recognition of micro-plastic in water is still an unmatched goal. Here we identify micro-plastics in water samples through digital holography microscopy combined to machine learning. We exploit the rich content of information of the holographic signature to design new distinctive features that specifically characterize micro-plastics and allow distinguishing them from marine plankton of comparable size. We use these features to train a plain support vector machine, remarkably improving its performance. Thus, we obtain a very accurate classifier using a simple machine learning approach, which does not require a large amount of training data and identifies micro-plastics of various morphology and optical properties over a wide range of characteristic scales. This is a first mandatory step to develop sensor networks to map the distribution of micro-plastics in water and their flows.

Pulse holographic imaging along with time-domain spectroscopy scan and tomographic techniques are of great interest. Since the advantages of holography are the lack of focusing optics and high spatial resolution, and, comparing with tomography, less computation cost for numerical reconstruction, this technique is preferable for the analysis of thin histological samples. In this work we have created the experimental scheme that involves measurement of diffraction pattern of the collimated THz pulse field spatial distribution at some distance behind the object in the time-domain mode, thus allowing reconstruction of amplitude and phase distribution at the object plane by numerical backpropagation of the wavefront in the spectral domain. In our experiment, we used a breast biopsy sample containing cancer tissues, we also performed numerical simulations accounting for experimental conditions to confirm the conceptual applicability of the reconstruction method.

We describe a novel snapshot imaging polarimeter based on off-axis digital holographic scheme. The proposed digital holographic imaging polarimeter is based on a compact interferometric module and it requires neither moving parts nor time dependent modulation. From a snapshot polarizing digital hologram, we can reconstruct a spatially resolved polarimetric phase image with moderate precision and accuracy. The real time capability of the proposed digital holographic imaging polarimeter is demonstrated by using a nano-patterned transmissive object.

We propose a novel technique for volumetric spectroscopy of scattering objects. The concept can be considered as a combination of optical coherence tomography (OCT) and Fourier transform spectroscopy. The 3D imaging capability is obtained with the use of coherence gating, but the detection spectrometer is based on 2D CMOS camera that allows for single-shot acquisition of spectral data corresponding to different depths in the object. The latter is possible as the beams from reference and object arms of the OCT interferometer are incident on the diffraction grating at relative angle (α) in the plane determined by the grating lines and the optical axis. In the perpendicular plane the diffraction angles are the same for both beams. The resultant sequence of spectra is subject to 2D Fourier transformation that provides the representation of the OCT signal in form of depth dependent distribution of spectrum of light scattered from sample and the envelope corresponding to the spectrum of the light coming back from particular depth in the object can be extracted and used for calculation of light extinction in the sample. The feasibility of the spectroscopic analysis using acquired 2D interferograms was proved by realization of the differential measurement of the glass cuvette filled with water in one case and with the water solution of the indocyanine green dye in the other. The proposed measurement scheme can show great potential in spectroscopic measurement of biomedical objects, in particular in vivo, due to resistance of the signal on the motion of the object.

The notions of spatial resolution and signal-to-noise are central to most forms of imaging. However, it appears that rigorous definitions of these quantities, which would be general enough to be useful in a broad range of imaging problems, while being also sufficiently specific to enable precise quantitative evaluation of the relevant properties of imaging systems, have been somewhat lacking. This is particularly true in respect to many modern forms of imaging that include digital processing of the acquired imaging data as an integral step leading to final images presented to the enduser. In the present paper, both the well-known historical definitions of spatial resolution and some more recent approaches suitable for many modern computational imaging techniques are discussed. An intrinsic duality of the spatial resolution and signal-to-noise exists in almost all types of imaging, with the related uncertainty relationship determining the inevitable trade-offs between the two quantities. Examples are presented with applications to some forms of X-ray imaging. Relations to Shannon and Fisher information capacity of imaging systems and super-resolution are also briefly discussed.

Quantitative phase imaging (QPI) has become an important imaging modality providing rapid, label-free measurements of a biological structure’s morphology and permittivity. In particular, reflection QPI systems are advantageous for their improved sensitivity to high-resolution axial structures and their ability to image thin and thick tissues alike. Existing reflection modalities often utilize interferometric setups requiring specialized system designs that limit their application in widespread biological research. We developed reflection intensity diffraction phase microscopy (rIDPM) to provide an easily accessible reflection QPI system for biological imaging applications. This new modality recovers a biological structure’s phase from intensity-only measurements using a standard reflection microscope modified with a translatable light source. We derived inverse scattering models for rIDPM addressing the common imaging condition of biological cells on a glass sample slide. Our models utilize the first Born approximation with a semi-infinite, partially reflective boundary condition accounting for reflections from the glass slide interface. The resulting volumetric model is linear and easily implementable providing fast, computationally efficient recovery of the object’s complex permittivity. Under these imaging conditions, we show forward-scattered fields primarily contribute to the final intensity image for objects taller than half the illumination wavelength. We show this rIDPM modality provides improved contrast of subcellular features from unstained HeLa cell samples compared to existing QPI transmission systems. We also demonstrate our model’s flexibility in recovering high-frequency features with improved contrast from designed annular illumination patterns.

The ultraviolet region of the spectrum offers unique capabilities for label-free molecular imaging of biological samples by providing highly-specific, quantitative information of many important endogenous biomolecules. However, the application of UV spectral imaging to biomedicine has been limited. To this end, we have recently introduced ultraviolet hyperspectral interferometric (UHI) microscopy, which applies interferometry to overcome significant challenges associated with UV spectroscopy when applied to molecular imaging. Here we present an alternative approach for UV multi-spectral microscopy which enables faster wide-field imaging at the expense of fewer spectral data points. Instead of line-scanning to recover high-resolution spectral information with an imaging spectrometer, we detect a wide field-of-view using a UV-sensitive camera and recover the spectral information using several (>5) UV-filters. Moreover, rather than using interferometry to recover the phase to correct for chromatic aberrations, we leverage the chromatic aberrations themselves to obtain a stack of through-focus intensity images (at various wavelengths) and then apply an iterative solution of the Transport of Intensity (TIE) equation to recover the phase and produce in-focus images at all wavelengths without moving the sample or objective. This configuration greatly simplifies the instrumentation, reducing its footprint and making it less expensive, while enabling fast, wide area imaging with better photon efficiency. We assess the capabilities of this technique through a series of simulations and experiments on red blood cells, which show good quantitative agreement with UHI and tabulated hemoglobin absorption properties. Potential biomedical applications are also discussed.

We demonstrate a three-dimensional (3D) optical diffraction tomographic technique with multi-frequency combination (MFC-ODT) for the 3D quantitative phase imaging of unlabeled specimens. Three sets of through-focus intensity images are captured under an annular aperture and two circular apertures with different coherence parameters. The 3D phase optical transfer functions (POTF) corresponding to different illumination apertures are combined to obtain a synthesized frequency response, achieving high-quality, low-noise 3D reconstructions with imaging resolution up to the incoherent diffraction limit. Besides, the expression of 3D POTF for arbitrary illumination pupils is derived and analyzed, and the 3D imaging performance of annular illumination is explored. It is shown that the phase-contrast washout effect in high-NA circular apertures can be effectively addressed by introducing a complementary annular aperture, which strongly boosts the phase contrast and improves the imaging resolution. By incorporating high-NA illumination as well as high-NA detection, MFC-ODT can achieve a theoretical transverse resolution up to 200 nm and an axial resolution of 645 nm. To test the feasibility of the proposed MFC-ODT technique, the 3D refractive index reconstruction results are based on a simulated 3D resolution target and experimental investigations of micro polystyrene bead and unstained biological samples are presented. Due to its capability for high-resolution 3D phase imaging as well as the compatibility with a widely available commercial microscope, the MFC-ODT is expected to find versatile applications in biological and biomedical research.

Deficient myelination in the internal capsule of the brain is associated with neurodevelopmental delays, particularly in high-risk infants such as those born small for gestational age (SGA). New methods are needed to further study this condition and assess how it relates to early life nourishment. MRI technology has been effective at measuring brain growth and composition but lacks myelin specificity and is low resolution. The development of new quantitative approaches that are rapid and precise may complement MRI results with insight into the pathology of deficient myelination and efficacy of nutritional interventions. Color Spatial Light Interference Microscopy (cSLIM) uses a brightfield objective and RGB camera to generate phase map images in conjunction with a regular brightfield image. Using paraffin embedded brain tissue sections, stained myelin was segmented from a brightfield image and, with a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. This technique was therefore sensitive to subtle variations in myelin density. The results of this study indicate a positive correlation between an experimental diet, rich in critical nutrients such as iron, and dry mass levels of myelin in the internal capsules of both appropriate (AGA) and SGA piglets. In summary, neonatal dietary treatments affect the degree of myelination in certain regions of the brain, irrespective of gestational size, and may therefore impact cognitive health.

The retina is composed of several transparent layers of neuronal and glial cells active during the vision process. Several studies showed that their structure and density is impacted by numerous eye diseases even at early stages. Understanding their role and decline during diseases has therefore become an important research field. The widespread method to observe the retina ex-vivo at a cellular level is fluorescent confocal microscopy. This technique requires cumbersome staining protocols to prepare the retina samples before observing them. Moreover, due to the selectivity of the staining process, only specific and targeted features can be observed at a time, making the intricate network of cells more difficult to understand. The classical unlabeled phase contrast imaging methods such as differential interference contrast (DIC) and digital holographic microscopy (DHM) are not well suited for whole retina samples as they usually require a transmission configuration or are limited to one or two cell layers. Here we present phase image comparisons of healthy and diseased ex-vivo retinas obtained using oblique phase imaging (OPI). OPI was previously developed for a transmission configuration and here we apply it to a reflection configuration on thick samples. We show that OPI is able to obtain quantitative phase images that matches images obtained by DHM. This technique enables the observation of whole retina samples still attached to the choroid without the need of any preparation. The different cellular layers are clearly visible and the effect of diseases evident. We will also show preliminary results of phase images obtained in-vivo. We believe that using OPI to visualize retina samples will greatly facilitate and accelerate the understanding of this complex tissue in the future.

We discuss recently emerging applications of the state-of-art deep learning methods on optical microscopy and microscopic image reconstruction, which enable new transformations among different modes and modalities of microscopic imaging, driven entirely by image data. We believe that deep learning will fundamentally change both the hardware and image reconstruction methods used in optical microscopy in a holistic manner.

In a recent paper [Goy et al., Phys. Rev. Lett. 121, 243902, 2018], we showed that deep neural networks (DNNs) are very efficient solvers for phase retrieval problems, especially when the photon budget is limited. However, the performance of the DNN is strongly conditioned by a preprocessing step that consists in producing a proper initial guess. In this paper, we study the influence of the preprocessing in more details, in particular the choice of the preprocessing operator. We also empirically demonstrate that, for a DenseNet architecture, the performance of the DNN increases with the number of layers up to a point after which it saturates.

PhENN is a convolutional deep neural network that reconstructs quantitative phase images from diffracted intensity measurements some distance away from the phase objects. PhENN is trained on known phase-intensity pairs created from a particular database (e.g. ImageNet) but then found to perform well on objects created from other databases (e.g. Faces-LFW, MNIST, etc.). In this paper, we analyze the dependence of quantitative phase measurement quality on PhENN's architecture and the layout of the lensless imaging system, in particular, the number of layers (depth), the size of the innermost layer (waist size), the presence or absence of skip connections, the choice of training loss function and the free space propagation distance.

Ptychography, a relatively new form of phase retrieval, can reconstruct both intensity and phase images of a sample from a group of diffraction patterns, which are recorded as the sample is translated through a grid of positions¹. To recover the phase information lost in the recording of these diffraction patterns, iterative algorithms must optimise an objective function full of local minima, in a huge multidimensional space². Many such algorithms have been developed, each aiming to converge rapidly whilst avoiding stagnation. Our research aims to compare some of the more popular algorithms, to determine their advantages and disadvantages under a range of different conditions, and hence to suggest guidelines for choosing suitable algorithms for any given data set.

We have implemented and tested several well-known phase retrieval algorithms: the ‘PIE’ family of algorithms^{3 4}, the difference map (DM)^{5 6}, hybrid projection/reflection (HPR)⁷ and relaxed averaged alternating reflections (RAAR)⁸. The PIE-type algorithms are based on the stochastic gradient descent concept⁴, whilst the rests are based on the set projection and reflection concept², and hence named the ‘PR’ algorithms in this paper. We began our tests by tuning algorithm parameters using multiple sets of simulated calibration data. Then, these tuned algorithms were tested on simulated data generated from a range of scenarios using either a randomised illumination function or convergent beam illumination, combined with either a weakly- or a strongly-scattering sample. Because ptychographic reconstructions are subject to certain pathological ambiguities, we then used an ambiguity-invariant error measure to evaluate the differences between the resulting images⁴.

I will discuss the emerging trend in computational imaging to train deep neural networks (DNNs) for image formation. The DNNs are trained from examples consisting of pairs of known objects and their corresponding raw images drawn from databases such as ImageNet, Faces-LFW and MNIST. The raw images are converted to complex amplitude maps and displayed on a Spatial Light Modulator (SLM.) After training, the DNNs are capable of recovering unknown objects, i.e. objects not previously included in the training sets, from the raw images in several scenarios: (1) phase objects retrieved from intensity after lensless propagation; (2) phase objects retrieved from intensity after lensless propagation at extremely low photon counts; and (3) amplitude objects retrieved from intensity in-focus after propagation through a strong scatterer. Recovery is robust to disturbances in the optical system, such as additional defocus or various misalignments. This suggests that DNNs may form robust internal models of the physics of light propagation and detection and generalize priors from the training set. In the talk I will discuss in more detail various methods to incorporate the physics into DNN training, and how DNN architecture and “hyper-parameters” (i.e., depth, number of units in each depth, presence or absence of skip connections, etc.) influence the quality of image recovery.

We investigate quantitative phase imaging techniques based on oblique illumination including differential phase contrast microscopy (DPC) and Fourier Ptychography Microscopy (FPM). DPC uses partially coherent, asymmetric illumination to achieve 2X resolution improvement but has small field of view (FOV). FPM achieves both wide FOV and high resolution but requires a large number of measurements. Achieving high space-bandwidth product (SBP) imaging in real-time remains challenging. Our goal is to develop a data-driven approach to enable highly multiplexed illumination to substantially improve the acquisition speed for high-SBP quantitative phase imaging. To do so, we abandon the traditional sampling strategy and phase retrieval algorithms. Instead we design a convolutional neural network (CNN) that uses only 4 brightfield and 3 darkfield images under asymmetrically coded illuminations as input and predicts high-SBP phase images. Particularly, instead of restoring a deterministic image, our CNN predicts pixel-wise probability distributions (Laplace) that is characterized by the location and scale. The predicted location map corresponds to the desired high-resolution phase image; in addition, the scale map provides per-pixel confidence of the prediction. Additionally, we show the potential of transfer learning that with minor extra training, the CNN can be optimized for different cell types. Experimental results demonstrate that the proposed method is robust against experimental imperfections, e.g. aberrations, misalignment, and reconstructs high-SBP phase images with significantly reduced acquisition and processing times.

We report an effective approach to analyze the effect of different amplitude patterns on image quality of asymmetric illumination-based differential phase contrast (AIDPC) microscope. Specifically, we investigate how the amplitude pattern can enhance the imaging quality. Two types of amplitude patterns are tested by the pseudospectral time-domain (PSTD) simulation. Preliminary simulation show promising results. Furthermore, the reported simulation may enhance the AIDPC system by helping researchers to optimize the optical system and facilitate new optical imaging strategies on AIDPC imaging.

In overview, for the example of digital holographic microscopy (DHM), we present quantitative phase imaging (QPI)- based characterization of inflammation in colonic tissue sections. Tissue density alterations are determined by the refractive index retrieval from quantitative DHM phase images. We show that the average tissue refractive index represents a marker that allows to distinguish between different layers of the intestinal wall and that the revealed tissue refractive index data correlated well with the severity of inflammation. In summary, QPI provides biophysical parameters for the quantification of inflammation in intestinal tissues and paves the way for future applications in terms of ‘‘digital pathology’’.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have promise for elucidating basic biological processes, drug testing, and regenerative medicine, yet are known to be heterogenous and immature. Methods to analyze cardiomyocytes are typically destructive or require labeling that alters the cells’ performance. Thus, we have developed a non-invasive image-based method for analyzing and classifying cardiomyocytes based on their morphology and contractile properties, and applied this method to analyze the effects of controlling cell shape. We optimized a diffraction phase microscope (DPM) to yield low noise optical thickness measurements at over 100 frames per second. We extracted contraction and relaxation motion cycles of single hiPSC-CMs and analyzed beat frequency and regularity. DPM also enabled comparisons of morphological characteristics by measuring the optical thickness of the cells. We compared populations of hiPSC-CMs with controlled (patterned) and uncontrolled (unpatterned) shape and we observed the following: 1) patterning effectively controls the shape of the cells, while cells with the desired mature-like shape rarely appear in the unpatterned population, 2) patterned cells are more likely to beat with consistent and lower beat frequency compared to unpatterned cells, and 3) the patterns tend to select for larger (more mature-like) cells. Finally, we identified a cutoff point under which cells of a certain dry mass do not adhere to the patterns. These results indicate that controlling the shape of hiPSC-CMs improves their characteristics, which can be analyzed using DPM, and has the potential to yield more consistent research results and homogenous populations of cells for clinical applications.

Refractive index tomography as an emerging technique enables the 3D morphological investigation of cells with no marker. Here, refractive index tomographic imaging of myelinating glial cells is presented. Myelin as a signal insulation layer around an axon is formed by the wrapping of Schwann cells or oligodendrocytes. Microscopic investigation of myelination traditionally requires fluorescent markers. Glial cells generally wrap the axon for more than ten layers. This multilayer formation has alternating and uniform layers of protein and lipid. Earlier studies on the structure of the myelin sheath have shown that the thickness period is lower than 20nm including the thickness of the extracellular medium after each layer. Direct observation of an individual layer is not possible (using classical microscopy techniques) due to dimensions being very small compared to the wavelength of the illumination light. However, periodic nature of the layers enables the differentiation of a myelinated axon from an unmyelinated one. Rapid change of the integrated refractive index and the Bragg fiber like structure alters the transmission behavior as a function of wavelength and incidence angle. With the 3D sectioning capability of refractive index tomography, these features can be easily identified.

Label-free microscopy enables the possibility of measuring biological samples noninvasively and purely based on endogenous contrast. In particular, quantitative phase microscopy (QPM) can provide signals proportional to the intracellular refractive index with high-throughput. To improve the specificity of these measurements, we coupled QPM with Raman spectroscopy, another label-free modality that provides a signal related to the molecular content of the sample. We then developed a hybrid imaging approach where imaging is restricted to QPM to maintain a high-throughput despite the inherent slow acquisition time of Raman signals, while ensuring that the measured spectrum is representative of the whole cellular content.

This approach provides signals for both the morphology, related to the phenotype, and the intracellular molecular content at single-cell level, that we employed to study cell populations under different stimuli. In particular, we studied macrophage cells and their response to a simulated bacterial infection upon exposure to lipopolysaccharide, and show how this approach is able to noninvasively detect the activation state at single-cell level by coupling it with multivariate analysis and machine learning algorithms.

Movements of the cell membrane accompanying action potentials have been detected by various methods, including reflection of a laser beam, atomic force microscopy and even bright-field microscopy. However, imaging of the entire cell dynamics during action potential has not been achieved, and the mechanism behind this phenomenon is still actively debated. Here we report full-field interferometric imaging of cellular movements during action potential by simultaneous quantitative phase microscopy (QPM) and multi-electrode array (MEA) recordings. Using spike-triggered averaging of the movies synchronized to electrical recording, we demonstrate deformations of up to 3 nm (0.9 mrad) during the action potential in spiking HEK-293 cells, with a rise time of 4 ms. The time course of the optically-recorded action potential is very similar to intracellular potential recorded with a whole-cell patch clamp, while the time derivative of the rising edge of the optical spike matches the timing and duration of the extracellular electrical recording on MEA. In some cells, phase increases at the center and decreases along the cell boundaries, while in others it increases on one side and decreases on the other. These findings suggest that optical phase changes during an action potential are due to cellular deformation, likely associated with changes in the membrane tension, rather than refractive index change due to ion influx or cell swelling. High-speed QPM may enable all-optical, label-free, full-field imaging of electrical activity in mammalian cells.

High content screening consists in acquiring a large number of samples to obtain statistically significant information on cell populations and their changes over time. It is also used to compare different growth conditions. Quantitative phase imaging enables to image semi-transparent samples without any label. This technology has the advantage of not modifying samples so as not to disturb them and to study them over a long period of time (> 3 days) by providing relevant quantitative phase information. We develop a solution to follow a very large number of cells (> 1000) over a long period of time (> 72 h). We set up a semi-automated imaging station using QuadriWave Lateral Shearing Interferometry. This station solves at least four challenges: - Scan an entire multi-well plate within minutes - Keep focus over time and distance at high magnification - Correct for meniscus effect at the well edges - Process data as fast as it is acquired To image wide fields, sample is scanned thanks to the microscope stage. Work on synchronization and analysis’ speed with GPU made it possible to perform such a scan at a speed making possible the cell follow-up between two acquisitions. As scanned surface is large and time lapse can be performed over long periods of time, a numerical refocusing algorithm processes phase images even after acquisition. Performances and limitations of this approach will be presented. Different algorithms developed to stitch phase images together enable to get quantitative phase information over the whole field of view. With this system, we perform scans of entire surface of tissue samples with high resolution. We are also able to image entire wells of multiwell plates with phase modality. It is then possible to study the evolution of quantitative phase and morphological features for long time periods at the individual cell level for a large number of cells evolving in different wells according to different conditions. We will present cell growth comparison for different experimental culture conditions.

We have fabricated constructs from erythrocytes that contain the near-infrared (NIR) dye, indocyanine green (ICG). We refer to these constructs as NIR erythrocyte mimicking transducers (NETs). Mechanical properties of NETs can play an important role in the circulation kinetics and biodistribution of these particles. We characterize the mechanical properties of erythrocytes, hemoglobin-depleted erythrocytes ghosts (EGs), and micron-sized NETs (μNETs) through analysis of membrane fluctuations measured by quantitative phase imaging, and forces associated with membrane tethers pulled by optical tweezers. EGs were prepared from erythrocytes by hypotonic treatment. μNETs were prepared through hypotonic loading of 25 μM ICG into EGs. Quantitative phase images were obtained by a common-path interferometric phaseshifting system. Approximating the membrane as a sheet of springs, we estimated the stiffness of the membrane of erythrocytes, EGs, and µNETs as 3.0 ± 0.6 pN/μm, 6.5 ± 2.1 pN/μm, and 8.0 ± 2.1 pN/μm. Optical tweezers experiments yielded a similar trend. Differences in membrane stiffness suggest that the circulation dynamics of μNETs may be altered as compared to native erythrocytes.

Nanometer-scale movements of the cell membrane associated with changes in cell potential can reveal the underlying electrical activity. Using wide-field quantitative phase imaging, we observed deformations of up to 3 nm (0.9 mrad) during the action potential in spiking HEK cells, and about 0.3 nm in neurons. The time course of the optically-recorded action potential is similar to intracellular recordings based on patch clamp, while time derivative of the rising edge of the optical spike matches the timing and duration of the extracellular electrical recording. Sufficiently fast QPI may enable non-invasive and label-free monitoring of cellular physiology. Imaging of the optical phase changes induced by transient heating provides a sensitive measure of material properties associated with refractive index dependence on temperature and thermal expansion. Using fast (50 kHz) QPI, we demonstrate the shot-noise limited sensitivity of about 3.4 mJ/cm2 in a single pulse. Phase-resolved OCT can detect energy deposition of 4.7 mJ/cm2 between two scattering interfaces producing signals with about 45 dB SNR. Integration of the phase changes along the beam path helps increase temperature sensitivity during perturbation. For example, temperature rise of about 0.8 C can be detected in a single cell layer, while hundred times lower heating produces the same phase change in 100-fold thicker tissue layer. Time course of thermal relaxation in QPI can reveal the size and shape of the hidden objects. Methods based on fast phase imaging may enable multiple applications, ranging from temperature control in retinal laser therapy to subsurface characterization of semiconductor devices.

Quantitative differential interference contrast (qDIC) microscopy is applied to the study of the main phase transition of dipentadecanoylphosphatidylcholine (DC₁₅PC) supported lipid bilayers. We measure thickness changes of about 1nm occurring in the bilayer with sub-nanometre resolution and show how the presence of fluorescently labelled lipids, even at small concentrations, can broaden the phase transition.

According to the International agency for Research on Cancer, cadmium (Cd) is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. Nowadays, the cytotoxic potential of heavy metals is commonly evaluated by different cellular endpoints as reactive oxygen species formation, cell viability or cell death. Heavy metals cytotoxicity testing is based on in-vitro methods such as MTT assay, for the colorimetric detection of mitochondrial activity, propidium iodide-staining of DNA, as cell death marker, fluorometric detection of ROS generation to evaluate the stress response and colorimetric detection of cytokine secretion for the inflammatory reaction by ELISA method. In this work, we present a label-free digital holography (DH) based technique as an in-vitro cytotoxicity assay, which overcomes the limitations of conventional in vitro test based on color or fluorescence read outs. In particular, we show how DH is able to quantify the evolution of key biophysical parameters of cells during the exposure to cadmium. Murine embryonic fibroblasts NIH 3T3 are chosen here as cellular model for studying the cadmium effects. The results demonstrate that DH is able to retrieve the temporal evolution of different key parameters such as cell volume, projected area, cell thickness and dry mass, thus providing a full quantitative characterization of the cell physical behaviour during cadmium exposure. This demonstrates DH as an elegant label-free tool for heavy metals toxicity analysis.

Fluorescence-based cell sorting instruments are capable of classifying sperm for desired characteristics such as gender. It is not surprising that, as these techniques are approaching clinical use, closer scrutiny has revealed that the sorting process introduces a signification increase in oxidative stress in addition to an undesired selection bias due to fragmented DNA. Although some of the damage can be attributed to rough mechanical handling, from studies on adherent cells, it is well known that these defects are also associated with the intrinsic toxicity of the fluorescence labels. In effect, fluorescence-based markers reduce viability, motivating the use of intrinsic contrast methods. Here we present recent progress on evaluating reproductive outcomes using Spatial Light Interference Microscopy (SLIM) for high sensitivity phase imaging. We compared our label-free markers to existing staining techniques. We show how the compatibility of our system with fluorescence/color image acquisition allows our techniques to be used concurrently rather than as an alternative, to conventional methods.

The robustness of Quantitative Phase Imaging (QPI) has enabled QPI to be used in applications to answer both research and clinical questions. QPI requires no labels, is non-destructive, and has nanoscale sensitivity to 3-d morphology. Various applications have included recording cellular force dynamics, identifying parasite-infected red blood cells, detecting cancer prognosis from colon cancer samples, and most recently predicting therapeutic sensitivity from live cell biomass accumulation measurements in patient derived xenograft (PDX) mice. However, challenges remain for clinical adoption, as QPI-based methods must first be proven more effective than current standards of care and patient inconvenience and costs must be minimized. Here we applied basic upgrades to previously described High Speed Live Cell Interferometry (HSLCI) to predict in vivo and in vitro PDX mouse tumor sensitivity to a range of cancer drugs from only Fine Needle Biopsy of the tumor. As demonstrated by our group and many others, the applications of QPI are not limited to the clinical realm. Using HSLCI, we revealed the growth dynamics of senescent and control H460 lung large cell carcinoma cells treated with cancer chemotherapy. The continued improvements in optics and throughput of QPI promise to answer many more clinical and basic science questions.

We applied quantitative phase imaging (QPI) for quantification of nanocapsule-induced morphology and migration changes in single cell layers. In time-lapse observations, cells were monitored with a Mach-Zehnder interferometerbased off-axis digital holographic microscopy (DHM) setup. For quantification of cell migration, single cells were tracked in the recorded series of quantitative DHM phase images. Moreover, QPI images were evaluated as novel stainfree assay to quantify the temporal course of global cellular morphology changes. The label-free acquired data shows that capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, significantly influence the direction of cell migration and cellular motility.

Confocal reflectance quantitative phase microscopy system is developed in our lab to quantify nuclear membrane fluctuations. This system able to provide the µm level depth resolved phase information of the back scattered signal from nucleic membrane at ms temporal resolution. The phase information quantify the height fluctuations of nucleic membranes, which are subject to thermal fluctuations around the stable equilibrium in viscoelastic mediums. We further combined this system with Brillouin spectroscopic system, which measures longitudinal modulus of the nuclear material in the gigahertz (GHz) frequency range. Combining the information from confocal phase and Brillouin spectroscopy provides the nuclear membrane and material mechanical properties. Studies of anti-cancer drug effect on nuclear stiffness is performed on human lung cancer cells. Chemotherapeutic agent Doxorubicin (Dox) were used to treat these cancer cells and mechanical properties of nucleus were studied using the combined confocal and Brillouin spectroscopic system, as discussed above. The combined study of membrane fluctuations and stiffness measurements represent the positive correlation and indicate the softening the nuclei of tumor cells after treating with chemotherapeutic drug.

Biological applications of quantitative phase imaging (QPI) seem to lag behind technique development, and the purpose of this paper is to sketch the existing or anticipated cell biological problems that could possibly be resolved with the help of QPI. The phase delay reported by QPI is directly related to the amount of intracellular macromolecules, such as proteins and nucleic acids. Some QPI methods additionally allow the measurement of cell volume or are compatible with volume measurements by other techniques; the combination of phase and volume gives the local or cell-averaged concentration of macromolecules. This opens unique possibilities to study processes that are hardly amenable to other methods. The examples include: discrimination between anabolic growth and water accumulation or, conversely, between cell fragmentation and water loss; long-term maintenance of osmotic balance; studying metabolism on the organelle level; investigation of the effects of macromolecular crowding; detection of intracellular water gradients during cell chemotaxis. The need to relate observations to quantitative physiological models is emphasized.

Complete blood count and differentiation of leukocytes belong to the most frequently performed laboratory diagnostic tests. Here, a flow cytometry-based method for label-free differentiation of untouched leukocytes by digital holographic microscopy on the rich phase contrast of peripheral leukocyte images, using highly controlled 2D hydrodynamic focusing conditions is reported. Principal component analysis of morphological characteristics of the reconstructed images allows to classify not only nine leukocyte types, but also different types of leukemia and demonstrate disappearance of acute myeloid leukemia cells in remission. To exclude confounding effects, the classification strategy is tested by the analysis of 20 blinded clinical samples. Here, 70% of the specimens are correctly classified with further 20% classifications close to a correct diagnosis. Taken together, the findings indicate a broad clinical applicability of the cytometry method for automated and reagent-free diagnosis of hematological disorders.

It is now known that interaction between cells and their environment or between intracellular compartments is based on complex vesicular transport processes [1]. This transport consists in material internalization from the external environment and compartment exchanges inside the cell itself. Understanding the mechanisms and regulation of this intracellular trafficking is an intense object of study in the field of cellular biology. The goal is to understand how vesicles transporting proteins and lipids are targeted to specific cellular compartments and fused with the membrane. Progress about intracellular trafficking is currently essentially made by constant innovation in fluorescence based techniques. They now reach single molecule resolution in living cells. It is possible to follow molecules all along their travel inside the cell. In this case, the main limitation is fluorescent probes could bias the vesicle behaviors and alter their transport inside the cell. Quantitative phase imaging techniques are conventionally used in microscopy, enhancing the contrast for imaging semi-transparent samples with a non-invasive (i.e. label free) and fast approach. For instance, phase correlation imaging introduced by [2] showed that global statistics on the whole cell or a population can give insight of motions and dynamics. We propose to study in details the behavior of vesicles at the intracellular scale and their interaction with the cytoskeleton. The resolution and the high sensitivity brought by a High Definition wave front sensor allows following all individual vesicles through time follow up, while imaging the whole cell. We can study their interaction with the surrounding environment (Brownian or guided motion along the cytoskeleton). We will show how we can extract information from quantitative phase images about the intracellular transport and the influence of the intracellular order/disorder. [1] Tokarev AA, Alfonso A, Segev N. Overview of Intracellular Compartments and Trafficking Pathways. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. [2] Phase correlation imaging of unlabeled cell dynamics, Lihong Ma and al., Scientific Reports volume 6, Article number: 32702 (2016)

We have explored strategies for the analysis of confluent cell layers utilizing histogram based-evaluation of quantitative phase images for example of digital holographic microscopy (DHM), a variant of quantitative phase microscopy (QPM). The applicability of the proposed numerical procedures is illustrated by the DHM-based quantification of drug induced cell morphology changes. The achieved results show that histogram-based evaluation of quantitative phase images allows a highly reliable detection and continuous observation of global cellular morphology changes in confluent cell layers.

In this study, we investigate polarization effect of quantitative phase imaging (QPI) using digital holography (DH) technique. By using different polarization state of writing beam the reconstructed images of different kinds of samples including biological cells from DH interferometer have been analyzed and shown their difference of quantitative measurement with reconstructed images of unpolarized writing fields. In addition, the property changes of some samples have been studied using the property analysis of the polarization image fields.

The field of microfluidics provides a robust toolkit for biomedical applications such as disease diagnosis and drug discovery, especially when combined with advanced microscopy techniques. An important challenge facing the combination of microfluidic devices with quantitative microscopy techniques, such as quantitative phase imaging (QPI), is the mismatch in refractive index between channel structures and aqueous media. This mismatch can introduce artifacts at material interfaces due to scattering and, in the case of QPI, phase unwrapping. We will show that these issues can be addressed through the use of MY133-V2000, a UV-curable, fluorinated polymer with a low refractive index similar to water (n = 1.33). MY133-V2000 can be fabricated into microfluidic devices using standard soft lithography techniques based on an SU-8 or polydimethylsiloxane (PDMS) mold. The addition of fluorine reduces the overall polarizability of the material, lowering refractive index. However, this introduces a new challenge due to the typically low adhesion of fluorinated polymers. We will discuss device integration and packaging strategies to overcome this limitation. Using QPI, we will demonstrate measurement of the distribution of cell biomass in live, adherent cells, both in the center of the channel and at the interface with microchannel structures, to demonstrate the dramatic reduction in artifacts due to the matching indices of refraction. We will also discuss applications to other microscopy techniques, including fluorescence. MY133-V2000 therefore provides QPI researchers with the opportunity to leverage the advantages of microfluidics for a diverse range of biomedical applications.

Gold standard methods for anaemia diagnosis are the complete blood count and the peripheral smear observation. However, they do not allow for a complete differential diagnosis, which requires biochemical assays, thus being labeldependent techniques. On the other hand, recent studies focus on label-free quantitative phase imaging (QPI) of blood samples to investigate blood diseases by using video-based morphological methods. However, when sick cells are very similar to healthy ones in terms of morphometric features, identification of a blood disease becomes challenging even by morphometric analysis as well as QPI. Here we exploit in-flow tomographic phase microscopy to retrieve the exact 3D rendering of Red Blood Cells (RBCs) from anaemic patients and to identify the pathology, distinguishing it from healthy samples. Moreover, we introduce a Label-free Optical Marker (LOM) to detect RBC phenotypes demonstrating that a single set of all-optical parameters can clearly identify a signature directly related to the erythrocytes disease by modelling each RBC as a biolens. We tested this novel bio-photonic analysis by proofing that several inherited anaemias, specifically Iron-deficiency Anaemia, Thalassemia, Hereditary Spherocytosis and Congenital Dyserythropoietic Anaemia, can be identified and sorted thus opening a novel route for blood diagnosis on a completely different concept based on LOMs.

Multiple-wavelength interferometric techniques have been successfully used for large step-height and large deformation measurements. Also it could resolve the step height between smooth and rough surfaces which is not possible with single wavelength interferometry. Temporal phase shifting algorithm, which requires a phase shifter such as PZT, has been widely used for accurate phase evolution in interferometry. The phase shifter needs to be calibrated at every wavelength if multiple wavelengths are used for measurement, it is a time consuming process. If phase shifter is not calibrated accurately, it can introduce phase shift errors. In this work, we will discuss various phase shifting algorithms, and their tolerance for phase shift error. And the applications of higher-order phased shifting algorithms will be presented. The study is useful for multiple-wavelength and white light interferometry where more than one wavelength is used for optical phase measurements.

Endometrial cancer is one of the most common gynecological malignancies. In endometrial cancer treatment, drug resistance test plays the vital role since different patients have different reactions to chemotherapy. Traditional methods of drug resistance test usually take a few days to obtain results, which will be quite a long time for patients waiting for cancer treatment. In this research, in order to quickly quantify the drug resistance of cancer cells, we managed to find some relationships between the dynamic changing processes and drug resistance of endometrial cancer cells. To accurately obtain and quantitatively analyze the dynamic processes, we utilized digital holographic microscopy (DHM) to retrieve phase maps of living cancer cells. Based on the real-time reconstructed phase maps, we reestablished the dynamic process of both the cisplatin-resistant cell (Ishikawa, ISK) and non-cisplatin-resistant cell (Ishikawa/CisR, ISKC). ISK and ISK-C were separately treated with cisplatin (0ug/ml, control; 5ug/ml, low concentration, LC; and 100ug/ml, high concentration, HC), and holograms of cells in each group were recorded by a DHM setup for 30min before and 150min after cisplatin treatment with a frame rate of one record every five second. Several morphological parameters, including cell height, cell projected area, and cell volume, were calculated from the retrieved phase maps and membrane fluctuations were analyzed both in temporal and spatial domains. Statistically significant differences in the changing processes were found between the two kinds of cells.

Non-motile, polarized epithelial cells are embedded via cell-cell junctions in a tissue environment. The tumor induced conversion to into individual, non-polarized, motile and invasive mesenchymal cancer cells dissolves cell-cell junctions. The metastatic potential of a tumor cell can therefore be characterized by morphological changes as well as the cell motility. Quantitative phase microscopy (QPM) provides label-free investigation of living cells by minimized interaction with the sample. The analysis of QPM images enables the determination of morphological cell parameters like cell thickness and elongation and facilitates single cell tracking for migration analysis. To demonstrate the potential of QPM in determining the metastatic potential of cells, we analyzed pancreatic tumor cell lines utilizing digital holographic microscopy (DHM) in an off-axis Mach-Zehnder configuration. Since the cell lines were supposed to differ in their metastatic potential, complementary investigations with a common transwell migration assay (Boyden chamber assay) were performed. The morphological analysis of the different cell lines via QPM in combination with image segmentation-based evaluation of the retrieved quantitative phase images showed that cells with a high metastatic potential had a lower cell thickness and a higher elongation than cells with a low metastatic potential. Moreover, computer assisted tracking of single cells over a period of 12 hours showed that highly metastatic cells covered a longer distance and had a higher motility compared to cells with a low metastatic potential. In summary, we demonstrate the multi-functional potential of QPM in cancer cell research applications and in quantifying the metastatic potential of tumor cells.

Revealing and characterization of circulating tumor cells (CTCs) is one of the most actively investigated field of oncology. It was established by the past time studies that some of these cells were in condition of apoptosis and so couldn’t initiate formation of a clinically significant metastasis. The novel opportunities associated with development of computer technologies and interferential microscopes facilitate the solution of many medico-biological problems. One of them is a method of determination of CTCs functional condition based on the phase-interferential characteristics of their nuclear structures which reflect cellular metabolic and proliferative activity and are the markers of their malignant transformation.

The analysis was carried out of the morphodensitometric biomarkers of the metastatic activity of circulating tumorous cells including conformational alterations of their nuclear structures. The reference collection of phase images of metastatic cells was created as well as the data of the morphodensitometric parameters of their metabolic activity was received.

A new information was received about heterogeneity of the circulating tumorous cells with different metastatic activity which allows the broadening of fundamental scientific knowledge concerning cancerogenesis, individualization of the observation and treatment of patients, improvement of the quality of treatment, and optimization of its cost.

Acoustophoresis devices are popular tools for manipulation and diagnostic in microfluidic environments. They offer the opportunity for contactless manipulation of cells. We demonstrate that the combination of acoustic manipulation and holographic imaging provides a suitable system for the simultaneous handling and of biological matter. We employ an acoustofluidic device with a transparent piezo element, to enable optical investigation through the channel. The holographic imaging is thus employed to observe and analyze the behavior of Red Blood Cells during the application of ultrasound radiation. The flexible refocusing, and quantitative phase imaging of single cells and RBCs clusters is reported.

Axially-offset differential interference contrast (ADIC) microscopy was developed for quantitative phase contrast imaging (QPI) by using polarization wavefront shaping approach with a matched pair of micro-retarder arrays. In ADIC microscopy, wavefront shaping with a micro-retarder array (μRA) produces a pattern of half-wave retardance varying spatially in the azimuthal orientation of the fast-axis. For a linearly polarized input beam, the polarization pattern induced from the linearly polarized plane wave through the μRA is identical to the interference between a slightly diverging right circularly polarized (RCP) and a slightly converging left circularly polarized (LCP) plane wave. Using a 10× objective, two axially offset foci separated by 70 μm are consequently generated from the patterned wavefront with orthogonal polarization states, serving as the sample and reference focal planes respectively for QPI. A paired μRA in transmission coherently recombines the two orthogonal components to recover the incident polarization state in the absence of sample. The large spatial offset (roughly 1/10 of the field of view) between the two foci provides a stable and uniform reference. Quantitative phase contrast images are directly recovered from sample-scan measurements with a single-channel detector and lock-in amplification with fast polarization modulation. This method has been successfully used for bio-sample imaging, nanoparticle detection and refractive index calculation of silica microbeads.

Information about the refractive index difference between a biological material and its surrounding medium is of key importance in various research fields in biophotonics. However, the optical properties of many physiological buffer solutions are not well characterized over a broad spectral range. In this study, we measure the refractive index of mainly transparent liquids commonly used for live cell imaging and tissue embedding in the wavelength range from 500 nm to 1100 nm utilizing a modified and calibrated Abbe refractometer. The presented approach for obtaining multi-spectral refractive index data builds the basis to retrieve the dispersion coefficients of various physiological buffer solutions.

Optical cryptography has attracted extensive interest because of the inherent nature of parallel and multidimensional capability of optical information processing compared with computer cryptography. However, the linear space-invariant (LSI) cryptosystems are easy to be simulated and may be vulnerable to different attacks. To resist attacks, several phasetruncated Fourier transforms based asymmetric cryptosystem are proposed to utilize the nonlinear operations in the LSI system, but they are proved to be vulnerable due to the inherent nature of LSI system. Notice that several works misunderstand the concept of nonlinear operations to the nonlinear systems. But the nonlinear systems are not easy to be achieved. Herein, an optical cryptosystem based on Fourier ptychography (FP) with double random phase masks is proposed. The encryption process cannot be precisely simulated but only by optical experiment due to the vignetting effect, which is linear space-variant (LSV) and can act as an one-way function from the perspective of optics purely and guarantee the security of our system. In addition, the encryption for a high resolution, large field-of-view and complexvalued image is achievable. Optical experiments are presented to prove the validity and the security of the proposed system. Our method would give more insights to separate the optical cryptography from computer cryptography in nature.

Interferometry has been widely used for surface metrology because of their precision, reliability, and versatility. Although monochromatic-light interferometery can provide high sensitivity and resolution, but it fails to quantify largediscontinuities. Multiple-wavelength techniques have been successfully used to extend the unambiguous step height measurement rage of single wavelength interferometer. The use of RGB CCD camera allows simultaneous acquisition of fringes generated at different wavelengths. In this work, we discuss details about the fringe analysis of white light interferograms acquired using colour CCD camera. The colour image acquired using RGB camera is decomposed in to red, green, blue components and corresponding interference phase is measured using phase evaluation algorithms. The approach makes the 3D surface measurements faster, cost-effective for industrial applications.

Quantitative phase imaging (QPI) measurement is achieved by interference, e.g., in digital holographic microscopy and interference microscopy, where the fringe pattern (hologram/interferogram) phase distribution stores information about the refractive index structure of studied transparent biological samples. In this contribution we report the base for new endto- end QPI computational technique named the Variational Hilbert Imaging (VHI). It can be divided into two steps: hologram filtration using modified variational image decomposition (mVID) approach and phase map (sample-induced optical path delay) extraction using the Hilbert spiral transform (HST). The mVID employs new denoising approach and reliable criterion for determination of the end of calculations with careful investigation of proper parameter values. Quality of obtained results is therefore significantly increased ensuring acceleration and automation of calculations combined with remarkable robustness to different strongly varying hologram characteristics, i.e., local fringe period and orientation, background intensity, contrast deteriorations and noise. Additionally the HST makes it possible to retrieve phase from single hologram, even in case of closed fringes, providing efficient means for biological events characterization in dynamic regime. The VHI algorithm enables analysis of variety of biological samples without user’s meddling and loss of the accuracy. It is an important step to simplify optical measurement of complicated and fragile biological samples. Investigated VHI algorithm is tested on simulated and experimental data (i.e., swine spermatozoon). Phase decoding results are compared with reference algorithms, i.e., the Hilbert-Huang and Fourier transforms. Versatility of the proposed method and its potentially ubiquitous applications in full-field optical metrology are highlighted.

In this research, the mapping of the path length difference that involves the specimen is proposed for improving qualitative phase imaging (QPI) techniques. Phase-quality images are developed using the principle of phase- shift in digital holography and then this system has been applied to investigate biological cells and tissues. In our setup a microscopic digital holographic interferometer with polarizers and a quarter wave plate has been designed for detecting cells specimen. The resulting image contains information of the thickness in any area and the refractive index of the specimen. Moreover, a quadrature – phase shifting holography (QPSH) technique is purposed. Experimental results are shown improving QPI. Merits and limitations of this method are also described.

Optical diffraction tomography (ODT), which is one of the 3-D imaging techniques, enables non-invasive, label-free, and quantitative imaging of microscopic particles by retrieving multiple optical fields and reconstructing the 3-D hologram image of a sample. However, ODT employing the illumination scanning method suffers from poor axial resolution. In order to remedy this problem, we introduce TOmographic MOuld for optical TRAPping (TOMOTRAP) in ODT, which can control the orientation and shape of arbitrarily shaped dielectric objects. From optical field images of a sample rotated by several angles via TOMOTRAP, we reconstruct the 3-D RI distributions of samples with isotropic spatial resolution.