This PDF file contains the front matter associated with SPIE Proceedings Volume 9718, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Interferometry is a powerful tool often used for the metrology of surfaces with many applications in industries such as optical fabrication, data storage, machine tool, and semiconductor. For many years interferometers have been built into microscopes so surface microstructure can be measured. Phase-shifting interferometric techniques have provided an extremely accurate rapid way of getting the interferogram data into the computer and the inherent noise in the data taking process is so low that in a good environment angstrom or sub-angstrom surface height or thickness measurements can be performed. The recent development of single-shot phase-shifting techniques has made it possible to perform accurate phase measurement techniques in less than ideal environments and to make movies showing how surface shape or optical thickness is varying with time. These same interferometric techniques can be applied to biomedical applications. This paper will trace the history of the development of these interferometric techniques that led to the application of these techniques to looking at cells and tissues.

Quantitative phase imaging (QPI) aims at studying weakly scattering and absorbing biological specimens with subwavelength accuracy without any external staining mechanisms. Use of a reference beam at an angle is one of the necessary criteria for recording of high resolution holograms in most of the interferometric methods used for quantitative phase imaging. The spatial separation of the dc and twin images is decided by the reference beam angle and Fourier-filtered reconstructed image will have a very poor resolution if hologram is recorded below a minimum reference angle condition. However, it is always inconvenient to have a large reference beam angle while performing high resolution microscopy of live cells and biological specimens with nanometric features. In this paper, we treat reconstruction of digital holographic microscopy images as a constrained optimization problem with smoothness constraint in order to recover only complex object field in hologram plane even with overlapping dc and twin image terms. We solve this optimization problem by gradient descent approach iteratively and the smoothness constraint is implemented by spatial averaging with appropriate size. This approach will give excellent high resolution image recovery compared to Fourier filtering while keeping a very small reference angle. We demonstrate this approach on digital holographic microscopy of live cells by recovering the quantitative phase of live cells from a hologram recorded with nearly zero reference angle.

Our latest methods for non-invasive label-free acquisition of the three-dimensional (3-D) refractive-index maps of live cells in suspension are reviewed. These methods are based on the acquisition of off-axis interferograms of single or multiple cells in suspension from different angles using an external interferometric module, while fully rotating each cell using micro-manipulations. The interferometric projections are processed via computed tomographic phase microscopy reconstruction technique, which considers optical diffraction effects, into the 3-D refractive-index structure of the suspended cell. Till now, tomographic phase microscopy was obtained by acquiring a series of interferograms of the light transmitted through the sample in different angles by either using an entire sample rotation, or patch clamping a single cell, which is invasive to the cells, or alternatively, using various angles of illumination, which causes a limited acceptance angle, and an incomplete 3-D Fourier spectrum. In contrast, our methods allow fast acquisition with full angular range, and thus obtain an accurate 3-D refractive-index map of the imaged cell. By inspection of the 3-D refractive-index distribution of cells in suspension, the proposed methods can be useful for high-throughput, label-free characterization of biological processes and cellular transformations from healthy to pathological conditions.

This paper will review the development of wide-field and confocal microscopes with wavefront sensing and adaptive optics for correcting refractive aberrations and compensating scattering when imaging through thick tissues (Drosophila embryos and mouse brain tissue). To make wavefront measurements in biological specimens we have modified the laser guide-star techniques used in astronomy for measuring wavefront aberrations that occur as star light passes through Earth’s turbulent atmosphere. Here sodium atoms in Earth’s mesosphere, at an altitude of 95 km, are excited to fluoresce at resonance by a high-power sodium laser. The fluorescent light creates a guide-star reference beacon at the top of the atmosphere that can be used for measuring wavefront aberrations that occur as the light passes through the atmosphere. We have developed a related approach for making wavefront measurements in biological specimens using cellular structures labeled with fluorescent proteins as laser guide-stars. An example is a fluorescently labeled centrosome in a fruit fly embryo or neurons and dendrites in mouse brains. Using adaptive optical microscopy we show that the Strehl ratio, the ratio of the peak intensity of an aberrated point source relative to the diffraction limited image, can be improved by an order of magnitude when imaging deeply into live dynamic specimens, enabling near diffraction limited deep tissue imaging.

Low coherence of the illumination substantially improves the quality of holographic and quantitative phase imaging (QPI) by elimination of the coherence noise and various artefacts and by improving the lateral resolution compared to the coherent holographic microscopy. Attributes of coherence-controlled holographic microscope (CCHM) designed and built as an off-axis holographic system allowing QPI within the range from complete coherent to incoherent illumination confirmed these expected advantages. Low coherence illumination also furnishes the coherence gating which constraints imaging of some spatial frequencies of an object axially thus forming an optical section in the wide sense. In this way the depth discrimination capability of the microscope is introduced at the price of restricting the axial interval of possible numerical refocusing. We describe theoretically these effects for the whole range of illumination coherence. We also show that the axial refocusing constraints can be overcome using advanced mode of imaging based on mutual lateral shift of reference and object image fields in CCHM. Lowering the spatial coherence of illumination means increasing its numerical aperture. We study how this change of the illumination geometry influences 3D objects QPI and especially the interpretation of live cells QPI in terms of the dry mass density measurement. In this way a strong dependence of the imaging process on the light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data including a chance of time-lapse watching of live cells even in optically turbid milieu.

A challenging issue has to be faced in microscopy whenever samples are immersed inside a dynamic turbid medium, as occluding objects provoke severe light scattering or unpredictable time-variable phase delays that scramble the object information. In these cases, the transmission matrix of the medium cannot be fully characterized. Here we show a simple technique, named Multi-Look Digital Holography (MLDH), able to fully recover the useful signal of biological specimens dipped inside a turbid liquid phase. Multiple hologram recordings are incoherently combined to synthesize the whole complex field diffused by the sample, which is revealed through turbidity providing quantitative phase-contrast information. Moreover, we show that the presence of a turbid medium can have a positive effect on a coherent imaging system, helping to reduce the effect of speckle artifacts. In other words, occluding biological elements, like e.g. Red Blood Cells (RBCs), can be thought as useful optical components providing denoising capabilities.

This talk will describe new source coding methods for rapid acquisition of high-resolution phase images across a very large field of view, using a modified commercial microscope with LED array illumination. Our methods are inspired by a hybrid combination of Fourier Ptychography and differential phase contrast imaging, combined with source multiplexing strategies for fast capture and significantly reduced data requirements. Such computational approaches to optical microscopy add significant new capabilities to commercial microscopes without much cost or hardware modification. We demonstrate our system on live samples in vivo, validating our results for unstained, label-free samples against other popular phase retrieval methods.

Imaging in a bright field or phase contrast microscope is partially coherent. We have found that the image can be conveniently considered and modeled in terms of the Wigner distribution function (WDF) of the object transmission. The WDF of the object has a simple physical interpretation for the case of a slowly varying object. Basically, the image intensity is the spatial marginal of the spatial convolution of the object WDF with the phase space imager kernel (PSIkernel), a rotated version of the transmission cross-coefficient. The PSI-kernel can be regarded as a partially-coherent generalization of the point spread function.

This approach can be extended to consider the partial coherence of the image itself. In particular, we can consider the mutual intensity, WDF or ambiguity function of the image. It is important to note that the spatial convolution of the object WDF with the PSI-kernel is not a WDF, and not the WDF of the image. The phase space representations of the image have relevance to phase reconstruction methods such as phase space tomography, or the transport of intensity equation approach, and to the three-dimensional image properties.

By means of numerical solutions of Maxwell’s equations, we model the complex light scattering phenomenon. Light propagation through scattering medium is a deterministic process; with specific amplitude and phase, light can propagate to the target position via multiple scattering. By means of numerical solutions of Maxwell’s equations, the complex light scattering phenomenon can be accurately analyzed. The reported simulation enables qualitative and quantitative analyses of the effectiveness of directing light through turbid media to a targeted position

In recent years, tremendous efforts have been spent on deep tissue imaging using phase conjugation, a technique used to undo the effects of light scattering in a thick tissue. Despite the early debates between Yariv and Wolf, it is still not well understood physically how deep can a field propagate into biological tissue and still be phase conjugated. In order to answer this question, we developed a light scattering theory to describe the evolution of the phase associated with a field scattered by a thick tissue block. The multiple scattering through the sample is simplified to a series of single scattering through consecutive thin tissue slices. With this theory, we identify the limits of the phase conjugation operation and recover the previous results by Yariv and Wolf, which asserts that phase conjugation is rooted in small angle approximation. Importantly, we discover the fundamental principle that rules phase conjugation: the mean axial wavenumber of a field progressively decreases to zero as it scatters multiple times. At this point, phase becomes a spatially random variable and phase conjugation becomes impossible. This result describes a fundamental phenomenon: the interaction between a deterministic object and a deterministic field can result in a random scattered field. We show that this phenomenon is rooted into Heisenberg’s uncertainty principle.

We present highly parallel and efficient algorithms for real-time reconstruction of the quantitative three-dimensional (3-D) refractive-index maps of biological cells without labeling, as obtained from the interferometric projections acquired by tomographic phase microscopy (TPM). The new algorithms are implemented on the graphic processing unit (GPU) of the computer using CUDA programming environment. The reconstruction process includes two main parts. First, we used parallel complex wave-front reconstruction of the TPM-based interferometric projections acquired at various angles. The complex wave front reconstructions are done on the GPU in parallel, while minimizing the calculation time of the Fourier transforms and phase unwrapping needed. Next, we implemented on the GPU in parallel the 3-D refractive index map retrieval using the TPM filtered-back projection algorithm. The incorporation of algorithms that are inherently parallel with a programming environment such as Nvidia’s CUDA makes it possible to obtain real-time processing rate, and enables high-throughput platform for label-free, 3-D cell visualization and diagnosis.

Quantitative Phase Imaging based on the Transport of Intensity Equation (TIE) has shown to be a practical tool for retrieving the phase information of biological and technical samples. When recovering the phase information with the TIE, the maximum lateral resolution that can be obtained is limited by the numerical aperture (NA) of the optical system. In order to overcome this limitation, a system that combines structured illumination and TIE-like techniques have been proposed. These methodologies enlarge synthetically the NA of the optical system, and thus, the lateral resolution of the retrieved phase can be improved. However, the employment of structured illumination may bring error amplifications in the retrieved phase due to its sensitiveness to phase discontinuities and shot noise. In this work, we propose a new methodology that improves the lateral resolution of the retrieved phase beyond the diffraction limit avoiding the problems related with the structured illumination. The methodology presented here uses tilted illumination and a TIE solver. We show that when using this configuration, we can extend the set of recovered frequencies by adjusting the angle of the tilted wave-front. Further, our methodology is designed to extend the NA by employing less tilted angles than other similar techniques. Hence, the final retrieved phase will have an enhanced lateral resolution without amplifying the numerical errors and employing a few tilted angles. Moreover, we show that the algorithm presented here can be combined with other TIE algorithms that are used for suppressing the Low Frequency Artifacts (LFAs) usually present when using TIE based techniques.

Morphological assessment of spermatozoa is of critical importance for in vitro fertilization (IVF), especially intracytoplasmic sperm injection (ICSI)-based IVF. In ICSI, a single sperm cell is selected and injected into an egg to achieve fertilization. The quality of the sperm cell is found to be highly correlated to IVF success. Sperm morphology, such as shape, head birefringence and motility, among others, are typically evaluated under a microscope. Current observation relies on conventional techniques such as differential interference contrast microscopy and polarized light microscopy. Their qualitative nature, however, limits the ability to provide accurate quantitative analysis. Here, we demonstrate quantitative morphological measurement of sperm cells using two types of spectral interferometric techniques, namely spectral modulation interferometry and spectral multiplexing interferometry. Both are based on spectral-domain low coherence interferometry, which is known for its exquisite phase determination ability. While spectral modulation interferometry encodes sample phase in a single spectrum, spectral multiplexing interferometry does so for sample birefringence. Therefore they are capable of highly sensitive phase and birefringence imaging. These features suit well in the imaging of live sperm cells, which are small, dynamic objects with only low to moderate levels of phase and birefringence contrast. We will introduce the operation of both techniques and demonstrate their application to measuring the phase and birefringence morphology of sperm cells.

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Parkinson’s disease (PD) is a common neurodegenerative disease that causes symptoms of postural instability and slowness of movement. Neurodegeneration in dopaminergic neurons at the substantia nigra has been reported as pathologic features, however, detailed mechanisms underlying neurodegeneration are still remain unclear. To investigate a neurodegenerative process, various imaging tools including phase contrast microscopy, electron microscopy, and fluorescence microscopy are utilized. However, these imaging methods provide qualitative information and require invasive approaches such as the use of fluorescence agents or chemical fixation procedures that disturb normal physiological conditions of neuron cells. In order to quantify the neurodegenerative process in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a 3D quantitative phase imaging method that measures 3D refractive index (RI) distributions of a sample which provide quantitative structural (volume, surface area, sphericity) and biochemical (protein concentration, total cellular dry mass) information. We investigated neurotoxic effects of MPP+ on SH-SY5Y cells by using quantitative information obtained from 3D RI distributions. We also performed temporal measurements of 3D RI distributions of an individual SH-SY5Y cell to analyze neurotoxic effects on intracellular vesicle dynamics.

Conventional label-based contrast enhancement techniques (e.g., fluorescence) frequently modify the genetic makeup of tagged cells, making them poor candidates for use in in-vitro fertilization applications. Instead, we choose a label-free form of contrast, based on interferometric imaging, sensitive to optical path length differences. Compared to, single HeLa cells, typical mammalian ova and embryos are more than an order of magnitude thicker. As a result, regions of large phase variation lead to phase wrapping and an overall reduction in signal intensity occurs due to multiple scattering. These effects manifest themselves in low-spatial frequencies (blurs), with the desired details buried in the background. We present a phase shifting interferometer that yields the derivative of the phase, a quantity whose value is particularly sensitive to local variations and fine details. We demonstrate that our new real-time imaging platform is valuable in measuring the multiday development of bovine embryos. Reconstructing the derivative of the image phase and amplitude, we characterize the motion of previously low-contrast structures, which are relevant for embryo viability tests.

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Most living cells are optically transparent which makes it difficult to visualize them under bright field microscopy. Use of contrast agents or markers and staining procedures are often followed to observe these cells. However, most of these staining agents are toxic and not applicable for live cell imaging. In the last decade, quantitative phase imaging has become an indispensable tool for morphological characterization of the phase objects without any markers. In this paper, we report noninterferometric quantitative phase imaging of live sperm cells by solving transport of intensity equations with recorded intensity measurements along optical axis on a commercial bright field microscope.

We present a new method, referred to as phase correlation imaging (PCI), to study cell dynamics and function through temporal phase correlation analysis. PCI offers label-free, high-performance, simple-design, as well as suitability for operation in a conventional microscopy setting. PCI works without the need for controlled or synchronized photoactivation and sophisticated acquisition schemes, and only involves taking a sequence of phase images. The PCI image incorporates information on the phase fluctuations induced by both Brownian motion and deterministic motion of intracellular transport across large scales. We employed spatial light interference microscopy (SLIM) recently developed in our laboratory to experimentally measure quantitative phase information which renders the thickness and refractive index of cellular components without adding contrast agents. The acquisition process is repeated to obtain time-lapse phase images. We calculate the correlation time at each pixel for acquired time-lapse phase images and obtain the correlation time map in space. By temporal correlation analysis, PCI reveals cell dynamics information, which is complementary to quantitative phase imaging itself.

We present a new analysis tool for studying texture changes in the quantitative phase maps of live cells acquired by wide-field interferometry. The sensitivity of wide-field interferometry systems to small changes in refractive index enables visualizing cells and inner cell organelles without the using fluorescent dyes or other cell-invasive approaches, which may affect the measurement and require external labeling. Our label-free texture-analysis tool is based directly on the optical path delay profile of the sample and does not necessitate decoupling refractive index and thickness in the cell quantitative phase profile; thus, relevant parameters can be calculated using a single-frame acquisition. Our experimental system includes low-coherence wide-field interferometer, combined with simultaneous florescence microscopy system for validation. We used this system and analysis tool for studying lipid droplets formation in adipocytes. The latter demonstration is relevant for various cellular functions such as lipid metabolism, protein storage and degradation to viral replication. These processes are functionally linked to several physiological and pathological conditions, including obesity and metabolic diseases. Quantification of these biological phenomena based on the texture changes in the cell phase map has a potential as a new cellular diagnosis tool.

Active neurons tends to have a different dynamical behavior compared to resting ones. Non-exhaustively, vesicular transport towards the synapses is increased, since axonal growth becomes slower. Previous studies also reported small phase variations occurring simultaneously with the action potential. Such changes exhibit times scales ranging from milliseconds to several seconds on spatial scales smaller than the optical diffraction limit. Therefore, QPI systems are of particular interest to measure neuronal activity without labels. Here, we report the development of two new QPI systems that should enable the detection of such activity. Both systems can acquire full field phase images with a sub nanometer sensitivity at a few hundreds of frames per second. The first setup is a synchronous combination of Full Field Optical Coherence Tomography (FF-OCT) and Fluorescence wide field imaging. The latter modality enables the measurement of neurons electrical activity using calcium indicators. In cultures, FF-OCT exhibits similar features to Digital Holographic Microscopy (DHM), except from complex computational reconstruction. However, FF-OCT is of particular interest in order to measure phase variations in tissues. The second setup is based on a Quantitative Differential Interference Contrast setup mounted in an epi-illumination configuration with a spectrally incoherent illumination. Such a common path interferometer exhibits a very good mechanical stability, and thus enables the measurement of phase images during hours. Additionally, such setup can not only measure a height change, but also an optical index change for both polarization. Hence, one can measure simultaneously a phase change and a birefringence change.

We provide an experimental demonstration of Spatial Light Interference Microscopy (SLIM) as a tool for measuring the motion of 25 nm tubulin structures without the use of florescence labels. Compared to intensity imaging methods such as phase contrast or DIC, our imaging technique relies on the ratios of images associated with optically introduced phase shifts, thus implicitly removing background illumination. To demonstrate our new found capabilities, we characterize kinesin-based motility continuously over periods of time where fluorescence would typically photobleach. We exploit this new method to compare the motility of microtubules at low ATP concentrations, with and without the tagging proteins formerly required to perform these studies. Our preliminary results show that the tags have a non-negligible effect on the microtubule motility, slowing the process down by more than 10%.

We have explored the utilization of quantitative phase imaging with digital holographic microscopy (DHM) as a novel tool for quantifying the dynamics of morphologic parameters (morphodynamics) of confluent endothelial cell layers under fluid shear stress conditions. Human umbilical vein endothelial cells (HUVECs) were exposed to fluid shear stress in a transparent cone/plate flow device (BioTech-Flow-System) and imaged with a modular setup for quantitative DHM phase imaging for up to 48 h. The resulting series of quantitative phase image sequences were analyzed for the average surface roughness of the cell layers and cell alignment. Our results demonstrate that quantitative phase imaging is a powerful and reliable tool to quantify the dynamics of morphological adaptation of endothelial cells to fluid shear stress.

White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions. In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.

We demonstrate quantitative assessment of sperm cell morphology, primarily acrosomal volume, using quantitative interference phase microscopy (IPM). Normally, the area of the acrosome is assessed using dyes that stain the acrosomal part of the cell. We have imaged fixed individual sperm cells using IPM. Following, the sample was stained and the same cells were imaged using bright field microscopy (BFM). We identified the acrosome using the stained BFM image, and used it to define a quantitative corresponding area in the IPM image and determine a quantitative threshold for evaluating the volume of the acrosome.

We systematically measure the morphological, biochemical, and biomechanical properties of individual human red blood cells (RBCs) from patients with diabetes mellitus using quantitative phase imaging technique to characterize the diabetic red cells with respect to those of the healthy. The 3-D refractive index tomograms and 2-D dynamic membrane fluctuation maps of individual RBCs are reconstructed from a set of the retrieved complex optical fields at various laser incidence angles using the Common-path diffraction optical tomography, from which volume, surface area, sphericity, hemoglobin (Hb) concentration, Hb content, and membrane fluctuation are obtained simultaneously. The correlative relations among the retrieved red cell indices of diabetic and healthy RBCs are also investigated with capabilities of individual cell measurement. As expected, there are no significant alterations in morphologies (cellular volumes, surface area, and sphericity) between diabetic and healthy RBCs. However, despite the minute mean corpuscular Hb differences in cell blood count datasheet, the measured Hb concentrations and Hb contents of diabetic RBCs are statistically higher than those of healthy RBCs, which might be related to the glycation of Hb molecules by hyperglycemia. Meanwhile, the membrane fluctuations of diabetic RBCs are clearly diminished compared to healthy red cells, implying the significantly decreased RBC deformability. In particular, it seems that the membrane fluctuations have mild negative relationships with the reported HbA1c levels.

In this paper, we present an updated automatic diagnostic procedure for prostate cancer using quantitative phase imaging (QPI). In a recent report [1], we demonstrated the use of Random Forest for image segmentation on prostate cores imaged using QPI. Based on these label maps, we developed an algorithm to discriminate between regions with Gleason grade 3 and 4 prostate cancer in prostatectomy tissue. The Area-Under-Curve (AUC) of 0.79 for the Receiver Operating Curve (ROC) can be obtained for Gleason grade 4 detection in a binary classification between Grade 3 and Grade 4. Our dataset includes 280 benign cases and 141 malignant cases. We show that textural features in phase maps have strong diagnostic values since they can be used in combination with the label map to detect presence or absence of basal cells, which is a strong indicator for prostate carcinoma. A support vector machine (SVM) classifier trained on this new feature vector can classify cancer/non-cancer with an error rate of 0.23 and an AUC value of 0.83.

Optical microscopy of live cells and tissues provides the main insight for life science researchers in academia and bio-pharma. The cells have very small features, are transparent, and require long term observations (hours to days) to measure the effects of drugs and diseases. New technologies - under the umbrella term of Quantitative Phase Imaging (QPI) - have come to light in the past decade to challenge and complement the current state of the art solutions that use fluorophores. Phi Optics talk will outline their lessons learned in the process of bringing an academic idea to the commercial space.

We describe several examples of technology transfer from academic laboratories to PHASICS. PHASICS was created in 2003 as a spin-off of LULI an academic laboratory working on plasma physics and developing high power lasers to create such objects which temperature and pressure conditions are close to those at the center of stars. In order to optimize the intensity at laser focus, several thesis treated the subject of adaptive optics for lasers. LULI decided to collaborate with ONERA who just invented a technique for wave front sensing called multiwave lateral shearing interferometry. Though developed at first for infrared metrology applications, this technique proved to be very efficient with lasers because it was able to analyze wave front of modulated beams with sharp edges. Before being industrialized the technique was further improved to a compact version called quadriwave lateral shearing interferometry. As soon as PHASICS was created, we felt the potential of making wave front images from transparent objects because of QWLSI high spatial resolution. PHASICS and Institut Fresnel started a collaboration to study applications in microscopy imaging. Research subjects include biological imaging, CARS microscopy, anisotropy imaging, or laser damage testing. The results of research were then included in PHASICS products but sometimes only a tool developed during the project became a product. We will present research works that led to transfers as well as the method we used to ensure fruitful collaboration and transfer.

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures.

The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system – hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks.

We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

Optical diffraction tomography (ODT) is an interferometric microscopy technique capable of measuring 3-D refractive index (RI) distribution of transparent samples. Multiple 2-D holograms of a sample illuminated with various angles are measured, from which 3-D RI map of the sample is reconstructed via the diffraction theory. ODT has been proved as a powerful tool for the study of biological cells, due to its non-invasiveness, label-free and quantitative imaging capability. Recently, our group has demonstrated that a digital micromirror device (DMD) can be exploited for fast and precise control of illumination beams for ODT. In this work, we systematically study the precision and stability of the ODT system equipped with a DMD and present measurements of 3-D and 4-D RI maps of various types of live cells including human red blood cells, white blood cells, hepatocytes, and HeLa cells. Furthermore, we also demonstrate the effective visualization of 3-D RI maps of live cells utilizing the measured information about the values and gradient of RI tomograms.

Ovizio Imaging Systems, a quantitative microscopic imaging spin-off of the Université Libre de Bruxelles, Belgium, was founded in the beginning of 2010 by Philip Mathuis, Serge Jooris, Prof. Frank Dubois and Dr. Catherine Yourassowky. The company has launched a range of specialized microscopy instruments for quantitative imaging mainly focused on the bioprocessing and diagnostics fields within the life sciences market. During my talk I will present the story of how an idea, emerged from the research labs of the University made it to a manufactured and sold product. The talk will look at many aspects of entrepreneurship and setting up a company, finding the funding for the project, attracting people, industrialization and product design and commercialization. It will also be focused on choices one has to make during the start-up phase and methodologies that can be applied in many different settings.

Curiously, the coherence-controlled holographic microscopy (CCHM) was brought into the world owing to the endeavor of Chmelik's team at Brno University of Technology (BUT) to avoid scanning in confocal microscopy. As coherence gating seemed to be the way, the Leith & Upatnieks proposal of incoherent holography had been considered attractive. Their method made interference system free from strict dependence on both spatial and temporal coherence. Off axis holographic system proposed on such basis has been proved capable of coherence based depth discrimination in single wide-field shot in reflected-light arrangement. Consequently, extremely low-coherence holographic imaging had been found highly contributive also to the image quality depriving it from coherence artefacts and improving its transversal resolution. This is why CCHM promised high precision of quantitative phase imaging (QPI) in transmitted light set up that was realized for cell biology. However the cost of necessarily complicated optical design and need of very precise mechanics forced the team of prof Chmelik at BUT to search for a company capable of mastering the instrument.

It was TESCAN ORSAY the highly successful scanning electron microscopes producer that finally took charge of the commercial design. Long-term collaboration of the company with BUT made possible both the CCHM technology successful transfer up to Q-PHASE microscope production as well as the company Light microscopy division reinforcement. This contribution merges views of CCHM technology author and the TESCAN development team.

The current tissue evaluation method for breast cancer would greatly benefit from higher throughput and less inter-observer variation. Since quantitative phase imaging (QPI) measures physical parameters of tissue, it can be used to find quantitative markers, eliminating observer subjectivity. Furthermore, since the pixel values in QPI remain the same regardless of the instrument used, classifiers can be built to segment various tissue components without need for color calibration. In this work we use a texton-based approach to segment QPI images of breast tissue into various tissue components (epithelium, stroma or lumen). A tissue microarray comprising of 900 unstained cores from 400 different patients was imaged using Spatial Light Interference Microscopy. The training data were generated by manually segmenting the images for 36 cores and labelling each pixel (epithelium, stroma or lumen.). For each pixel in the data, a response vector was generated by the Leung-Malik (LM) filter bank and these responses were clustered using the k-means algorithm to find the centers (called textons). A random forest classifier was then trained to find the relationship between a pixel’s label and the histogram of these textons in that pixel’s neighborhood. The segmentation was carried out on the validation set by calculating the texton histogram in a pixel’s neighborhood and generating a label based on the model learnt during training. Segmentation of the tissue into various components is an important step toward efficiently computing parameters that are markers of disease. Automated segmentation, followed by diagnosis, can improve the accuracy and speed of analysis leading to better health outcomes.

It is commonly known that alteration of erythrocyte deformability lead to serious microcirculatory diseases such as retinopathy, nephropathy, etc. Various methods and technologies have been developed to diagnose such membrane properties of erythrocytes. In this study, we developed an innovative method to measure hemorheological characteristics of the erythrocyte membrane using a light scattering analysis with simplified optic setting and multi-cell analysis as well. Light scattering intensity through multiple erythrocytes and its power density spectrum were obtained. The results of light scattering analyses were compared in healthy control and artificially hardened sample which was treated with glutaraldehyde. These results were further compared with conventional assays to measure deformable property in hemorheology. We found that light scattering information would reflect the disturbance of membrane fluctuation in artificially damaged erythrocytes. Therefore, measuring fluctuation of erythrocyte membrane using light scattering signal could facilitate simple and precise diagnose of pathological state on erythrocyte as well as related complications.

We have studied the RBC membrane properties between a normal RBC and a RBC in Paroxysrnal nocturnal hemoglobinuria (PNH) patient using common path interferometric quantitative phase microscopy (CPIQPM). CPIQPM system has provided the subnanometer optical path length sensitivity on a millisecond. We have measured the dynamic thickness fluctuations of a normal RBC membrane and a RBC membrane in PNH patient over the whole cell surface with CPIQPM. PNH is a rare and serious disease of blood featured by destruction of red blood cells (RBCs). This destruction happens since RBCs show the defect of protein which protects RBCs from the immune system. We have applied CPIQPM to study the characteristic of RBC membrane in PNH patient. We have shown the morphological shape, volume, and projected surface for both different RBC types. The results have showed both RBCs had the similar shape with donut, but membrane fluctuations in PNH patient was shown to reveal the difference of temporal properties compared with a normal RBC. In order to demonstrate the practical tool of the CPIQPM technique, we have also obtained the time series thickness fluctuation outside a cell.

Recently great progress has been made in biological and biomedical imaging by combining non-invasive optical methods, novel adaptive light manipulation and computational techniques for intensity-based phase recovery and three dimensional image reconstruction. In particular and in relation to the work presented here, Optical Projection Tomography (OPT) is a well-established technique for imaging mostly transparent absorbing biological models such as C. Elegans and Danio Rerio. On the contrary, scattering layers like the cocoon surrounding the Drosophila during the pupae stage constitutes a challenge for three dimensional imaging through such a complex structure. However, recent studies enabled image reconstruction through scattering curtains up to few transport mean free paths via phase retrieval iterative algorithms allowing to uncover objects hidden behind complex layers. By combining these two techniques we explore the possibility to perform a three dimensional image reconstruction of fluorescent objects embedded between scattering layers without compromising its structural integrity. Dynamical cross correlation registration was implemented for the registration process due to translational and flipping ambiguity of the phase retrieval problem, in order to provide the correct aligned set of data to perform the back-projection reconstruction. We have thus managed to reconstruct a hidden complex object between static scattering curtains and compared with the effective reconstruction to fully understand the process before the in-vivo biological implementation.

The aim of this study is to determine the displacement profile due to shrinkage in acrylamide-based photopolymer layer during holographic recording. Using phase shifting electronic speckle pattern interferometry the displacement at each pixel in the image of the object is measured by phase shifting technique so that a complete displacement profile of the object can be obtained. It was observed that the displacement profile is Gaussian and resembles to the profile of the recording beam. We observed an increase in shrinkage from 2 μm at 20 seconds of recording to 7.5 μm after 120 seconds of recording. The technique allows for real time measurement of the shrinkage profile.

We report white light phase shifting interferometry in conjunction with color fringe analysis for the detection of contaminants in water such as Escherichia coli (E.coli), Campylobacter coli and Bacillus cereus. The experimental setup is based on a common path interferometer using Mirau interferometric objective lens. White light interferograms are recorded using a 3-chip color CCD camera based on prism technology. The 3-chip color camera have lesser color cross talk and better spatial resolution in comparison to single chip CCD camera. A piezo-electric transducer (PZT) phase shifter is fixed with the Mirau objective and they are attached with a conventional microscope. Five phase shifted white light interferograms are recorded by the 3-chip color CCD camera and each phase shifted interferogram is decomposed into the red, green and blue constituent colors, thus making three sets of five phase shifted intererograms for three different colors from a single set of white light interferogram. This makes the system less time consuming and have lesser effect due to surrounding environment. Initially 3D phase maps of the bacteria are reconstructed for red, green and blue wavelengths from these interferograms using MATLAB, from these phase maps we determines the refractive index (RI) of the bacteria. Experimental results of 3D shape measurement and RI at multiple wavelengths will be presented. These results might find applications for detection of contaminants in water without using any chemical processing and fluorescent dyes.

For defect detection or undesired object in commercial products, it is required to develop a fast measurement system that can obtain three-dimensional distribution of surface of the opaque medium such as metal or inside of the transparent medium. For this purpose, we fabricated a digital holographic microscope using a fast image sensor when the phase object is put on a fast movable stage. In the fabricated system, an image sensor operated at maximum frame rate of 2000 fps and a movable stage operated at maximum speed of 300 mm/s are introduced. Under the continuous wave illumination, motion-blurred phase object is reconstructed. By using numerical processing such as deconvolution filter, the reconstructed phase distribution is much improved. Numerical results are presented.

A study in porcine femoral bones with and without the presence of cortical drilling is presented. An out of plane digital holographic interferometer is used to retrieve the optical phase during the controlled compression tests. These tests try to simulate physiological deformations in postmortem healthy bones and compare their mechanical response with those having a cortical hole. The cortical drilling technique is widely used in medical procedures to fix plaques and metallic frames to a bone recovering from a fracture. Several materials and drilling techniques are used for this purpose. In this work we analyze the superficial variations of the bone when different drilling diameters are used. By means of the optical phase it is possible to recover the superficial deformation of the tissue during a controlled deformation with high resolution. This information could give a better understand about the micro structural variations of the bone instead of a bulk response. As proof of principle, several tests were performed to register the modes and ranges of the displacements for compressive loads. From these tests notorious differences are observed between both groups of bones, having less structural stiffness the drilled ones as expected. However, the bone's characteristic to absorb and adjust itself due the load is also highly affected according to the number of holes. Results from different kind of samples (undrilled and drilled) are presented and discussed in this work.

Industrial applications of embedded materials have been increased in the recent years as the study of their mechanical properties. A particular interest is their homogeneousness which will determine a significant improvement or decay in the possible application. The optical system proposed here can show the internal micro structure and the internal displacements along a scanned volume through consecutives 2D tomographic and optical phase images. The volumetric information is retrieved by means of a liner stage which avoids the use of expensive tilting devices. Results show the response of homogeneous and in-homogeneous PMMA samples during controlled tests in order to find the simplest one which determines the sample’s condition.

In this work we present the coherence controlled holographic microscopy (CCHM)1 and its ability to image the living cells in turbid media².

The CCHM method and its advantages are introduced. A 'coherence gate effect'³, that enables imaging in turbid media, occurs owing to the low coherence illumination in our setup. The coherence gate effect is briefly theoretically explained and comparison of images with different illumination sources is shown. After that, the possibility of imaging in turbid media is applied to investigation of cell reactions to cytopathic turbid emulsions. In our experiments we used human cancer cells treated by biologically active phospholipids (BAPs). Cellular events leading to cell death, that would otherwise remain hidden in turbid media, are clearly observable and according to them cell fate can be deduced.

An optical tomography system measures the light scattered by an object as a function of spatial coordinates and as a function of the illumination angle. The measured signals are digitally processed to produce a 3D image of the object. In this paper we describe how we can learn the shape of an object by constructing a neural network that models the optical system and training the network to match the experimentally measured data. The variables of the trained network yield the image of the unknown object at the end of training phase. [1] Ulugbek, Papadopoulos, Shoreh, Goy, Vonesh, Unser, Psaltis, “A Learning Approach to Optical Tomography” Optica, May 2015.

We present a high speed, phase-sensitive, line-scanning reflectance confocal interference microscope. We achieved rapid confocal imaging using a fast line-scan camera and quantitative phase imaging using off-axis digital holography on a 1D, line-by-line basis. In our prototype system, a He-Ne laser (~1.2 mW) was used to demonstrate the principle of operation. Using a 20 kHz line scan rate (1024 pixels per line scan), we achieved a video-rate frame rate of 20 Hz for 1024x500 pixel en-face confocal images (20 MHz total pixel rate). By using an objective lens of a NA 0.65, we achieved an axial and lateral resolution of ~3.5 micrometers and ~0.8 micrometers, respectively. By z-stack imaging of a custom silicon target with a stepped structure, we confirmed that the axial sectioning of the interference microscope is similar to that of a traditional line-scan confocal microscope (our microscope with the reference arm blocked). The utility of phase-sensitive holographic detection in line-scan confocal was demonstrated in two ways. First, using a custom axial height phantom fabricated using chrome deposition, we demonstrated variations in phase corresponding to heights in the 100 nm range with a contrast-to-noise ratio of ~31 dB. Second, we demonstrate digital refocusing of an out-of-focus holographic image. The mechanism of confocality in our line-scan system is 1D physical pinholing. Our ongoing work aims to add an additional mechanism of confocality by using low spatial coherence sources to impose interferometric pinholing.

Multi-modal digital holographic microscopy is a combination of epifluorescence microscopy and digital holographic microscopy, the main function of which is to obtain images from fluorescence intensity and quantified phase contrasts, simultaneously. The proposed system is mostly beneficial to biological studies, with the reason that often the studies are depending on fluorescent labeling techniques to detect certain intracellular molecules, while phase information reflecting properties of unstained transparent elements. This paper is presenting our latest researches on applications such as randomly moving micro-fluorescent beads and living cells of Physcomitrella patens. The experiments are succeeded on obtaining a succession of wide-field fluorescent images and holograms from micro-beads, and different depths focusing is realized via numerical reconstruction. Living cells of Physcomitrella patens are recorded in the static manner, the reconstruction distance indicates thickness of cellular structure. These results are implementing practical applications toward many biomedical science researches.

The sizing of individual nanoparticles and the recovery of the distributions of sizes from populations of nanoparticles provide valuable information in virology, exosome analysis, air and water quality monitoring, and nanomaterials synthesis. Conventional approaches for nanoparticle sizing include those based on costly or low-throughput laboratory-scale equipment such as transmission electron microscopy or nanoparticle tracking analysis, as well as those approaches that only provide population-averaged quantities, such as dynamic light scattering. Some of these limitations can be overcome using a new family of alternative approaches based on quantitative phase imaging that combines lensfree holographic on-chip microscopy with self-assembled liquid nanolenses. In these approaches, the particles of interest are deposited onto a glass coverslip and the sample is coated with either pure liquid polyethylene glycol (PEG) or aqueous solutions of PEG. Due to surface tension, the PEG self-assembles into nano-scale lenses around the particles of interest. These nanolenses enhance the scattering signatures of the embedded particles such that individual nanoparticles as small as 40 nm are clearly visible in phase images reconstructed from captured holograms. The magnitude of the phase quantitatively corresponds to particle size with an accuracy of +/-11 nm. This family of approaches can individually size more than 10^5 particles in parallel, can handle a large dynamic range of particle sizes (40 nm – 100s of microns), and can accurately size multi-modal distributions of particles. Furthermore, the entire approach has been implemented in a compact and cost-effective device suitable for use in the field or in low-resource settings.

Optical imaging of objects embedded within scattering media such as biological tissues suffers from the strong background noise due to multiple light scattering. The signal strength from the target objects decays exponentially at the length scale of the scattering mean free path, which is typically on the order of 100 micron for biological tissues. As a consequence, targets located at a depth of just a few scattering mean free paths lose their fine details. In this work, we performed synthetic aperture imaging of targets embedded within a scattering medium and demonstrated that the aperture synthesis process can suppress multiple scattering background better than conventional incoherent imaging. In the reflection geometry, we sent planar waves of various incidence angles and recorded the phase and amplitude maps of the reflected waves using off-axis digital holographic microscopy. A He-Ne laser was used as a light source and target objects were sandwiched between scattering layers made of PDMS mixed with polystyrene beads. We converted each reflected images taken at specific incidence angles into the maps of in-plane momentum difference between reflected and incidence waves. We then synthesized the maps in such a way that the scattered waves with the same momentum differences were added together. In this way, single-scattered waves from the targets were added coherently, which made them outgrow the incoherently added multiple-scattered waves. We achieved 1 micron lateral resolution for a target located deeper than four times the scattering mean free path in which conventional incoherent imaging fails to work.

Optical diffraction tomography has been steadily proving its potential to study one of the hot topics in modern cell biology — 3D dynamic changes in cells' morphology represented with refractive index values. In this technique digital holography is combined with tomographic reconstruction and thus it is necessary to provide projections acquired at different viewing directions. Usually the Mach-Zehnder interferometer configuration is used and while the object beam performs scanning, the reference beam is in most cases stationary. This approach either limits possible scanning strategies or requires additional mechanical movement to be introduced in the reference beam. On the other hand, spiral or grid scanning is possible in alternative common-path or Michelson configurations. However, in this case there is no guarantee that a specimen is sparse enough for the object to interfere with an object-free part of the beam. In this paper we present a modified version of Mach-Zehnder interferometer-based tomographic microscope, in which both object and reference beam are subject to scanning using one scanning device only thus making any scanning scenario possible. This concept is realized with a custom-built optical system in the reference beam and is appropriate for mechanical as well as optical scanning. Usually, the tomographic reconstruction setups and algorithms are verified using a microsphere phantom, which is not enough to test the influence of the distribution of the projections. In this work we propose a more complex calibration object created using two-photon polymerization.

We demonstrate three-dimensional (3D) optical phase and amplitude reconstruction based on coded source illumination using a programmable LED array. Multiple stacks of images along the optical axis are computed from recorded intensities captured by multiple images under off-axis illumination. Based on the first Born approximation, a linear differential phase contrast (DPC) model is built between 3D complex index of refraction and the intensity stacks. Therefore, 3D volume reconstruction can be achieved via a fast inversion method, without the intermediate 2D phase retrieval step. Our system employs spatially partially coherent illumination, so the transverse resolution achieves twice the NA of coherent systems, while axial resolution is also improved 2× as compared to holographic imaging.

Digital holographic phase microscopy is a well-established quantitative phase imaging technique. However, interference artifacts from inside the system, typically induced by elements whose optical thickness are within the source coherence length, limit the imaging quality as well as sensitivity. In this paper, a swept laser source based technique is presented. Spectra acquired at a number of wavelengths, after Fourier Transform, can be used to identify the sources of the interference artifacts. With proper tuning of the optical pathlength difference between sample and reference arms, it is possible to avoid these artifacts and achieve sensitivity below 0.3nm. Performance of the proposed technique is examined in live cell imaging.

We present a single-shot white light interference microscopy for the quantitative phase imaging (QPI) of biological cells and tissues. A common path white light interference microscope is developed and colorful white light interferogram is recorded by three-chip color CCD camera. The recorded white light interferogram is decomposed into the red, green and blue color wavelength component interferograms and processed it to find out the RI for different color wavelengths. The decomposed interferograms are analyzed using local model fitting (LMF)" algorithm developed for reconstructing the phase map from single interferogram. LMF is slightly off-axis interferometric QPI method which is a single-shot method that employs only a single image, so it is fast and accurate. The present method is very useful for dynamic process where path-length changes at millisecond level. From the single interferogram a wavelength-dependent quantitative phase imaging of human red blood cells (RBCs) are reconstructed and refractive index is determined. The LMF algorithm is simple to implement and is efficient in computation. The results are compared with the conventional phase shifting interferometry and Hilbert transform techniques.

Quantitative phase imaging has been used in the past to study the dry mass of cells and study cell growth under various treatment conditions. However, the relationship between cellular redox and growth rates has not yet been studied in this context. This study employed the recombinant Glrx-roGFP2 redox biosensor targeted to the mitochondrial matrix or cytosolic compartments of A549 lung epithelial carcinoma cells. The Glrx-roGFP2s biosensor consists of a modified GFP protein containing internal cysteine residues sensitive to the local redox environment. The formation/dissolution of sulfide bridges contorts the internal chromophore, dictating corresponding changes in florescence emission that provide direct measures of the local redox potential. Combining 2-channel florescent imaging of the redox sensor with quantitative phase imaging allowed observation of redox homeostasis alongside measurements of cellular mass during full cycles of cellular division. The results indicate that mitochondrial redox showed a stronger inverse correlation with cell growth than cytoplasmic redox states; although redox changes are restricted to a 5% range. We are now studying the relationship between mitochondrial redox and cell growth in an isogenic series of breast cell lines built upon the MCF-10A genetic background that vary both in malignancy and metastatic potential.

We describe the use of spatially incoherent illumination combined with quantitative phase imaging (QPI) [1] to make tridimensional reconstruction of semi-transparent biological samples. Quantitative phase imaging is commonly used with coherent illumination for the relatively simple interpretation of the phase measurement. We propose to use spatially incoherent illumination which is known to increase lateral and axial resolution compared to classical coherent illumination. The goal is to image thick samples with intracellular resolution [2]. The 3D volume is imaged by axially scanning the sample with a quadri-wave lateral shearing interferometer used as a conventional camera while using spatially incoherent white-light illumination (native microscope halogen source) or NIR light. We use a non-modified inverted microscope equipped with a Z-axis piezo stage. A z-stack is recorded by objective translation along the optical axis. The main advantages of this approach are its easy implementation, compared to the other state-of-the-art diffraction tomographic setups, and its speed which makes even label-free 3D living sample imaging possible. A deconvolution algorithm is used to compensate for the loss in contrast due to spatially incoherent illumination. This makes the tomographic volume phase values quantitative. Hence refractive index could be recovered from the optical slices. We will present tomographic reconstruction of cells, thick fixed tissue of few tens of micrometers using white light, and the use of NIR light to reach deeper planes in the tissue.

Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug “Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

Quantitative phase measurements at multiple wavelengths has created an opportunity for exploring new avenues in phase microscopy such as enhancing imaging-depth (1), measuring hemoglobin concentrations in erythrocytes (2), and more recently in tomographic mapping of the refractive index of live cells (3). To this end, quantitative phase imaging has been demonstrated both at few selected spectral points as well as with high spectral resolution (4,5). However, most of these developed techniques compromise imaging speed, field of view, or the spectral resolution to perform interferometric measurements at multiple colors. In the specific application of quantitative phase in studying blood diseases and red blood cells, current techniques lack the required sensitivity to quantify biological properties of interest at individual cell level. Recently, we have set out to develop a stable quantitative interferometric microscope allowing for measurements of such properties for red cells without compromising field of view or speed of the measurements. The feasibility of the approach will be initially demonstrated in measuring dispersion curves of known solutions, followed by measuring biological properties of red cells in sickle cell anemia. References: 1. Mann CJ, Bingham PR, Paquit VC, Tobin KW. Quantitative phase imaging by three-wavelength digital holography. Opt Express. 2008;16(13):9753–64. 2. Park Y, Yamauchi T, Choi W, Dasari R, Feld MS. Spectroscopic phase microscopy for quantifying hemoglobin concentrations in intact red blood cells. Opt Lett. 2009;34(23):3668–70. 3. Hosseini P, Sung Y, Choi Y, Lue N, Yaqoob Z, So P. Scanning color optical tomography (SCOT). Opt Express. 2015;23(15):19752–62. 4. Jung J-H, Jang J, Park Y. Spectro-refractometry of individual microscopic objects using swept-source quantitative phase imaging. Anal Chem. 2013;85(21):10519–25. 5. Rinehart M, Zhu Y, Wax A. Quantitative phase spectroscopy. Biomed Opt Express. 2012;3(5):958–65.

Traction force microscopy is the most widely used technique for studying the forces exerted by cells on deformable substrates. However, the method is computationally intense and cells have to be detached from the substrate prior to measuring the displacement map. We have developed a new method, referred to as Hilbert phase dynamometry (HPD), which yields real-time force fields and, simultaneously, cell dry mass and growth information. HPD operates by imaging cells on a deformable substrate that is patterned with a grid of fluorescent proteins. A Hilbert transform is used to extract the phase map associated with the grid deformation, which provides the displacement field. By combining this information with substrate stiffness, an elasticity model was developed to measure forces exerted by cells with high spatial resolution. In our study, we prepared 10kPa gels and them with a 2-D grid of FITC-conjugated fibrinogen/fibronectin mixture, an extracellular matrix protein to which cells adhere. We cultured undifferentiated mesenchymal stem cells (MSC), and MSCs that were in the process of undergoing adipogenesis and osteogenesis. The cells were measured over the course of 24 hours using Spatial Light Interference Microscopy (SLIM) and wide-field epi-fluorescence microscopy allowing us to simultaneously measure cell growth and the forces exerted by the cells on the substrate.

A method for the depth-sensitive detection of fluorescent light is presented. It relies on a structured illumination restricting the excitation volume and on an interferometric detection of the wave front curvature. The illumination with two intersecting beams of a white-light laser separated in a Sagnac interferometer coupled to the microscope provides a coarse confinement in lateral and axial direction. The depth reconstruction is carried out by evaluating shearing interferograms produced with a Michelson interferometer. This setup can also be used with spatially and temporally incoherent light as emitted by fluorophores.

A simulation workflow of the method was developed using a combination of a solution of Maxwell's equations with the Monte Carlo method. These simulations showed the principal feasibility of the method.

The method is validated by measurements at reference samples with characterized material properties, locations and sizes of fluorescent regions. It is demonstrated that sufficient signal quality can be obtained for materials with scattering properties comparable to dental enamel while maintaining moderate illumination powers in the milliwatt range. The depth reconstruction is demonstrated for a range of distances and penetration depths of several hundred micrometers.

We present a Nomarski microscope with variable shear distance that allows for determining the full complex modulation function including the quantitative phase contrast that a specimen imposes on incident light, rather than only its differential phase contrast. The system preserves all beneficial properties of the Nomarski setup with respect to coherence and stability. Hence, we can measure the quantitative phase contrast under Köhler illumination with full condenser aperture and without vibration isolation. We will explain the system, give an analysis of the coherence requirements and provide an example from the field of MEMS inspection.

We present a three-dimensional microscopic technique based on simultaneous dual wavelength digital holography. In digital holographic microscopy, interference patterns produced by an object and reference waves are recorded by a camera. The computationally reconstructed holographic images contain the information about both amplitude and phase of the light reflected from the object. Phase is then mapped across the sample and converted into height information for each pixel. This technique was applied to imaging of electrodes embedded into glass substrates, which allowed three-dimensional reconstruction of their structure. Holographic imaging of the embedded layered structures, where each layer can be separated from the others by axial distances exceeding multiple wavelengths of imaging light, is difficult, because software phase unwrapping is practically impossible. The use of two wavelengths enables accurate axial measurements of multiple layers by comparing the phase maps produced by each individual wavelength. We demonstrated that the correct choice of wavelengths maximizes the axial range, at which an unambiguous 3D imaging can be performed. This provides not just three-dimensional structure of each layer, but also allows for height differentiation of layers. By employing wavelength cutoff filters, we were able to obtain the phase maps simultaneously, enabling fast measurements. We also developed a background removal technique, based on the quality of interference fringe pattern, which suppresses low intensity signal when no reliable phase information can be extracted. We showed that this is especially useful for multilayered embedded electrode structures, where each sample consists of both high and low reflectivity features.

Here we show a novel imaging modality, named Space-Time Scanning Interferometry (STSI), which synthesizes interferograms mapped in a hybrid space-time domain. A single linear sensor array is sufficient to create hybrid interferograms with unlimited Field of View (FoV) along the scanning direction, and allowing quantitative phase retrieval by Phase Shifting (PS) interferometry algorithms. We applied the STSI method to microfluidic imaging of biological samples, where the required phase shift between interferograms is intrinsically offered due to the sample movement. Besides, out-of-focus recordings are performed using a single line detector, in order to synthesize an unlimited FoV Space-Time Digital Hologram (STDH) yielding full-field, 3D information. Experimental proofs have been carried out to demonstrate the useful capability of STDH to overcome the trade-off existing between FoV and sample magnification, thus providing a high-throughput, label/free, quantitative, diagnostic tool to study biological elements onboard LoC platforms.

We describe the use of polarized spatially coherent illumination to perform linear retardance imaging and measurements of semi-transparent biological samples using a quantitative phase imaging technique [1]. Quantitative phase imaging techniques [2-5] are used in microscopy for the imaging of semi-transparent samples and gives information about the optical path difference (OPD). The strength of those techniques is their non-invasive (the sample is not labelled) and fast approach. However, this high contrast is non-specific and cannot be linked to specific properties of the sample. To overcome this limitation, we propose to use polarized light in combination with QPI. Indeed, anisotropy has been used to reveal ordered fibrous structures in biological samples without any staining or labelling with polarized light microscopy [6-8]. Recent studies have shown polarimetry as a potential diagnostic tool for various dermatological diseases on thick tissue samples [9]. Particularly, specific collagen fibers spatial distribution has been demonstrated to be a signature for the optical diagnosis and prognosis of cancer in tissues [10]. In this paper, we describe a technical improvement of our technique based on high-resolution quadri-wave lateral shearing interferometry (QWLSI) and liquid crystal retarder to perform quantitative linear birefringence measurements on biological samples. The system combines a set of quantitative phase images with different excitation polarizations to create birefringence images. These give information about the local retardance and orientation of biological anisotropic components. We propose using a commercial QWLSI [11] (SID4Bio, Phasics SA, Saint Aubin, France) directly plugged onto a lateral video port of an inverted microscope (TE2000-U, Nikon, Japan). We are able to take retardance images in less than 1 second which allows us to record dynamic phenomena (living cells study) and make high speed acquisitions to reconstruct tissues virtual slides. Results on living cells and comparisons between healthy and tumoral 10 µm thick skin tissues will be presented.

We present an efficient and less time consuming implementation of Computational Shear Interferometry (CoSI) by applying a smoothness on the assigned wave field. The method is used to recover the three dimensional form of a micro injection molded part from a set of shear measurements. For this purpose, six shear experiments have been recorded with the shears varying in orientation and magnitude. Varying the shears orientation and magnitude is applied using a setup consisting of a 4f filter with a reflective phase-only spatial light modulator (SLM) in the corresponding Fourier plane. The SLM is used as an electronic diffraction grating with a blazed structure. Based on the birefringent properties of the SLM two orthogonally polarized images, one diffracted and one reflected, appear on the camera sensor and produce shear interferograms. The shift between the images depends on the period and the orientation of the blazed grating. From shear interferograms, the phase of light diffracted by the object is reconstructed by CoSI. Thus the height map of the object is determined.

In the past decade, various quantitative phase microscopy (QPM) techniques have emerged, driven by the need to study biological samples non-invasively. However, the fundamental limit for phase noise is scarcely discussed in the literature. In a typically off-axis phase microscope system, the phase noise is limited to a few milliradians using a moderate camera. Common-path QPMs offer much reduced phase noise compared to typical Mach-Zehnder-based systems. However, further scaling down the phase noise becomes difficult. Here we propose a high-sensitivity common-path QPM that promises to reduce the phase noise by a factor of 10 (assuming the mechanical noise is negligible). This is achieved by a specifically designed signal filter, leaving only the subtle phase fluctuations coming from the dynamics sample scattering. By working at photon shot-noise limited detection, we can magnify the subtle phase contrast which is proportional to the camera well depth. We expect this system to have the height sensitivity similar to an atomic force microcopy, while measuring biological structures with a full field of view in a single-shot. We plan to use this system to study cell dynamics, particularly lamellipodial height fluctuations as well as stiffer cell membrane fluctuations.

Generalized Phase Contrast (GPC) is a light efficient method for generating speckle-free contiguous optical distributions using binary-only or analog phase levels. It has been used in applications such as optical trapping and manipulation, active microscopy, structured illumination, optical security, parallel laser marking and labelling and recently in contemporary biophotonics applications such as for adaptive and parallel two-photon optogenetics and neurophotonics. We will present our most recent GPC developments geared towards these applications. We first show a very compact static light shaper followed by the potential of GPC for biomedical and multispectral applications where we experimentally demonstrate the active light shaping of a supercontinuum laser over most of the visible wavelength range. Finally, we discuss how GPC can be advantageously applied for Quantitative Phase Imaging (QPI).

We present a technique of visible spectrum quantitative phase imaging with ten millimeter range without phase ambiguity while achieving better than ten micrometer precision. A suitable synthetic wavelength for absolute phase measurement, without unwrapping, is implemented by two-wavelength digital holography. This results in amplified phase noise which is then reduced by an iterative optical and algorithm based process involving one or two additional source wavelengths. We demonstrate that these source wavelengths can be acquired from a single laser diode source by realizing the wavelength output response to fine temperature control.

Compared to the Phase Contrast, Differential Interference Contrast (DIC) has been known to give higher depth sectioning as well as a halo-free images when investigating transparent specimens. Thanks to relying on generating two slightly shifted replicas with a small amount of shift, within the coherence area, DIC is able to operate with very low coherence light. More importantly, the method is able to work with very large numerical aperture of the illumination, which offer comparable sectioning capability to bright field microscopy. However, DIC is still a qualitative method, which limits potential applications of the technique. In this paper, we introduce a method that extends the capability of DIC by combining it with a phase shifting module to extract the phase gradient information. A theoretical model of the image formation is developed and the possibility of integrating the gradient function is analyzed.. Our method is benchmarked on imaging embryos during their 7-day development, HeLa cells during mitosis, and control samples.

The phase retrieval problem arises in various fields ranging from physics and astronomy to biology and microscopy. Computational reconstruction of the Fourier phase from a single diffraction pattern is typically achieved using iterative alternating projections algorithms imposing a non-convex computational challenge. A different approach is holography, relying on a known reference field. Here we present a conceptually new approach for the reconstruction of two (or more) sufficiently separated objects. In our approach we combine the constraint the objects are finite as well as the information in the interference between them to construct an overdetermined set of linear equations. We show that this set of equations is guaranteed to yield the correct solution almost always and that it can be solved efficiently by standard numerical algebra tools. Essentially, our method combine commonly used constraint (that the object is finite) with a holographic approach (interference information). It differs from holographic methods in the fact that a known reference field is not required, instead the unknown objects serve as reference to one another (hence blind holography). Our method can be applied in a single-shot for two (or more) separated objects or with several measurements with a single object. It can benefit phase imaging techniques such as Fourier phytography microscopy, as well as coherent diffractive X-ray imaging in which the generation of a well-characterized, high resolution reference beam imposes a major challenge. We demonstrate our method experimentally both in the optical domain and in the X-ray domain using XFEL pulses.

Diffraction Phase Microscopy (DPM) is a common-path, single shot QPI technique that has found applications in studies of red blood cell morphology and dynamics, cell growth measurement, as well as in Fourier Transform Light Scattering. In DPM, the phase is retrieved by interfering two orders of diffraction from a grating placed at the image plane. The reference field has been, in the past, generated by low pass filtering the zero order via a pinhole placed in the Fourier plane. For achieving the desired spatial coherence, the pinhole is often only 5-10 µm in diameter, making the system difficult to align every time an imaging study is performed. In this work, we eliminated the pinhole from the DPM optical path and generated instead the reference field by magnifying strongly the zero order. We show that a gradient-index (GRIN) lens (effective focal length of 300 µm) can be used to magnify the Fourier transform of the zero order to the point where the DC component fills the camera sensor. We show that the resulting Magnified Object Spectrum Interference Microscopy (MOSIM) system can successfully reconstruct quantitative phase images, without the need for tedious alignment. Because it conserves the common path geometry, MOSIM is characterized by 1.1 nm spatiotemporal pathlength noise. Since it is single shot, we demonstrated 400 frames/s acquisition. We anticipate that this new method can potentially lead to a more robust and less vibration sensitive QPI instrument for carrying out biological studies at various spatiotemporal scales.

Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

We built and tested a Holographic Surface Plasmon Resonance (HoloSPR) objective-based microscope for simultaneous amplitude-contrast and phase-contrast Surface Plasmon Resonance imaging (SPRi). SPRi is a widely spread tool for label-free detection of changes in refractive index and concentration, as well as mapping of thin films. However, to obtain quantitative data of thin film thickness, usually scanning techniques have to be employed. Thanks to the simultaneous detection of amplitude and phase, we show that HoloSPRi provides a versatile imaging tool for high-throughput SPR detection, which yields, moreover, the possibility of non-scanning quantitative measurements of thin film thickness.

Lorenz-Mie scattering theory allows to predict the field scattered by spherical objects illuminated by coherent light. By fitting the fringe pattern resulting from the interference of incident and scattered light, it is possible to track and size colloidal particles with a few nanometer precision. Using digital holographic microscopy (DHM) we extend the applications of Lorenz-Mie theory to hollow spherical structures and to extremely high pressure conditions. On the one hand, we geometrically and optically characterize complex colloids as polymer-shelled microbubbles, with high precision, low costs and short acquisition time. These microbubbles are likely to be unique tools for targeted drug delivery and are currently used as contrast agents for sonography. We measured size, shell thickness and refractive index for hundreds of polymeric microbubbles showing that shell thickness displays a large variation that is strongly correlated with its refractive index and thus with its composition. On the other hand we demonstrate that DHM can be used for accurate 3D tracking and sizing of a holographically trapped colloidal probe in a diamond anvil cell (DAC). Polystyrene beads were trapped in water up to Gigapascal pressures while simultaneously recording in-line holograms at 1 KHz frame rate. This technique may potentially provide a new method for spatially resolved pressure measurements inside a DAC.