This PDF file contains the front matter associated with SPIE Proceedings Volume 10503, including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Incoherent digital holography can be used for several applications, among which are high resolution fluorescence microscopy and imaging through a scattering medium. Historically, an incoherent digital hologram has been usually recorded by self-interference systems in which both interfering beams are originated from the same observed object. The self-interference system enables to read the phase distribution of the wavefronts propagating from an object and consequently to decode the 3D location of the object points. In this presentation, we survey several cases in which 3D holographic imaging can be done without the phase information and without two-wave interference.

We report longitudinal spatial coherence (LSC) gated high-resolution tomography and quantitative phase microscopy of biological cells and tissues with uniform illumination using laser as a light source. To accomplish this a pseudo thermal light source was synthesized by passing laser beams through an optical system, which is basically a speckle reduction system with combined effect of spatial, temporal, angular and polarisation diversity. The longitudinal spatial coherence length of such light was significantly reduced by synthesizing a pseudo thermal source with the combined effect of spatial, angular and temporal diversity. This results in a low spatially coherent (i.e., broad angular frequency spectrum) light source with narrow temporal frequency spectrum. Light from such a pseudo thermal light source was passed through an interference microscope with varying magnification, such as, 10X and 50X. The interference microscope was used for full-field OCT imaging of multilayer objects and topography of industrial objects. Experimental results of optical sectioning of multilayer biological objects with high axial-resolution less than 10μm was achieved which is comparable to broadband white light source. The synthesized light source with reduced speckles having uniform illumination on the sample, which can be very useful for fluorescence microscopy as well as quantitative phase microscopy with less phase noise. The present system does not require any dispersion compensation optical system for biological samples as a highly monochromatic light source is used.

Optical phase changes induced by transient perturbations provide a sensitive measure of material properties. One such measure is associated with the change in refractive index with temperature. Another - with thermal expansion. We demonstrate the high sensitivity and speed of such methods using two interferometric techniques: Quantitative Phase Imaging (QPI) in transmission, and phase-resolved Optical Coherence Tomography (OCT) in reflection. Camera frame rate in QPI varied from 10 to 50 kHz, exposure from 1 to 10 µs, and heating pulse – from 0.02 to 1 ms in duration. The phase-stabilized swept-source OCT was operating at 100 kHz repetition rate. Shot-noise limited QPI can resolve energy deposition of about 3.4 mJ/cm^2 in a single pulse, which corresponds to 0.8 ℃ temperature rise in a single cell. OCT can detect deposition of 24 mJ/cm^2 energy between two scattering interfaces producing about 30 dB SNR signals and 4.7 mJ/cm^2 with 45 dB. Finite element modeling of the phase changes in materials heated by laser and by electric current matched the experimental results very well. These techniques can be used for mapping absorption coefficients, electric current density, doping depth in semiconductors, and many other properties. Integration of the phase changes along the penetrating beam path helps increase sensitivity and reveals the size of the hidden objects by looking at the signal relaxation time. These methods may enable multiple applications, ranging from temperature control in retinal laser therapy and in gene expression to characterization of semiconductor devices.

We present a new interferometric and holographic approach which allows for multiple depth multiplexing in a single acquisition, using an off-axis low-coherence interferometric imaging system. This technique enables quantitative amplitude and phase imaging of multiple slices within a thicker sample, in one single image acquisition. The method is implemented using a compact interferometric module that connects to a regular digital camera, and is useful in a wide range of applications in which multiple z slices of a sample are required simultaneously. The simultaneous acquisition is carried out by using optical multiplexing of the interferograms on the camera, whereby each slice has a distinct off axis interference fringe direction in relation to the other. We demonstrate the proposed technique for quantitative optical thickness imaging of microscopic biological samples.

Optical sectioning plays an important role for in-vivo or 3D optical microscopy. We have developed reflection-mode dynamic speckle-field interferometric microscopy (DSIM) to image targets with high spatial resolution, as well as high depth sectioning. In DSIM, single-shot, wide-field quantitative phase image can be obtained by using broadband, time-varying speckle-field in Linnik interferometer combined with off-axis holography. With broadband laser (center wavelength 900nm, bandwidth ~ 100nm), and high numerical aperture objective lens (1.0NA, water immersion), we could demonstrate up to ~600 nm depth-sectioning. However, there was no concrete theoretical model to describe the system. In this study, we developed a theoretical model to investigate the depth-sectioning effect of DSIM based on the theory of optical diffraction tomography with 1st order Born approximation. We systematically studied the dependence of the depth-sectioning, and image contrast in accordance with the bandwidth, and numerical aperture, respectively, and it shows a good agreement with experiment. We also studied the effect of the aberration, and dispersion induced by the target specimen for better understanding of the system dealing with thick targets. In addition, our model can be easily extended for general optical coherence microscopy, and will help for better designing of such optical systems that requires high depth resolution.

High performance commercial 3D scanners and profilometers are complex and expensive for individual users. On the other hand, there are many onsite applications that require handheld portable devices to reliably measure 3D structures with high accuracy and sensitivity, such as inspecting flat mechanical surfaces and testing water quality. Here, we present a portable quantitative phase microscopy (QPM) system to tackle such onsite applications and laboratory needs. Our portable QPM system integrates digital holography, optical microscopy, and smartphone technologies into one single platform to allow for a broader user accessibility. Unlike the current prevailing smartphone imaging techniques, our method does not require heavy computations and complicated algorithms. Our image retrieval algorithm, particularly phase retrieval based on digital holographic measurements, is simply that allows for achieving real-time imaging on mobile devices. Our system is an effort on miniaturizing a compact QPM system and reducing their cost. With this portable system, we are currently exploring various applications ranging from characterizing microfluidic devices to imaging microscopic structures in water. We envision our QPM unit will be widely available to individual users for more exciting applications in the near future.

Gradient Light Interference Microscopy (GLIM) is a phase-shifting technique that upgrades a conventional differential interference contrast (DIC) microscope to produce quantitative phase images. Benefiting from equally matched beams, fully open microscope condenser, as well as coherence gating due to the removal of non-interferometric terms upon phase-retrieval, GLIM has the potential to enable studies of mesoscopic structure hundreds of microns thick. Nevertheless, many such biological samples are much too big to completely transmit light, requiring the use of reflection microscopy. Here we present our early work on constructing a new reflection mode GLIM microscope designed to image thick biological structures, as well as a detailed comparison between transmitted vs reflected light modalities for a number of calibration samples.

Holography is a tool to record the object wavefront by interference. Complex amplitude of the object wave is coded into a two dimensional hologram. Unfortunately, the conjugate wave and background wave would also appear at the object plane during reconstruction, as noise, which blurs the reconstructed object. From the perspective of wave, we propose a filtering algorithm to get a noise-reduced reconstruction. Due to the fact that the hologram is a kind of amplitude grating, three waves would appear when reconstruction, which are object wave, conjugate wave and background wave. The background is easy to eliminate by frequency domain filtering. The object wave and conjugate wave are signals to be dealt with. These two waves, as a whole, propagate in the space. However, when detected at the original object plane, the object wave would diffract into a sparse pattern while the conjugate wave would diffract into a diffused pattern forming the noise. Hence, the noise can be reduced based on these difference with a filtering algorithm. Both amplitude and phase distributions are truthfully retrieved in our simulation and experimental demonstration.

The Sparsity of the Gradient (SoG) is a robust autofocusing criterion for holography, where the gradient modulus of the complex refocused hologram is calculated, on which a sparsity metric is applied. Here, we compare two different choices of sparsity metrics used in SoG, specifically, the Gini index (GI) and the Tamura coefficient (TC), for holographic autofocusing on dense/connected or sparse samples. We provide a theoretical analysis predicting that for uniformly distributed image data, TC and GI exhibit similar behavior, while for naturally sparse images containing few high-valued signal entries and many low-valued noisy background pixels, TC is more sensitive to distribution changes in the signal and more resistive to background noise. These predictions are also confirmed by experimental results using SoG-based holographic autofocusing on dense and connected samples (such as stained breast tissue sections) as well as highly sparse samples (such as isolated Giardia lamblia cysts). Through these experiments, we found that ToG and GoG offer almost identical autofocusing performance on dense and connected samples, whereas for naturally sparse samples, GoG should be calculated on a relatively small region of interest (ROI) closely surrounding the object, while ToG offers more flexibility in choosing a larger ROI containing more background pixels.

The recent progress in machine learning, a subfield of artificial intelligence (AI) with a focus on learning algorithms, is attracting researchers in quantitative phase imaging (QPI). The fast and label-free nature of QPI is ideal for generating large-scale data to train supervised machine learning algorithms. The algorithms discover important structures in large, multidimensional training data to exploit them for augmenting new QPI measurements. Here, we present two major directions in synergistically combining QPI with AI, with a particular focus on a state-of-the-art machine learning technique called deep learning. One direction is systematic exploitation of QPI data. Employing image classification frameworks, class-dependent characteristics encoded in the images are extracted for rapid diagnosis and screening. This approach has been demonstrated in a wide range of biological systems ranging from microbes to cells to tissues, with various modalities including 2D phase, 3D tomographic, time-lapse, and spectral measurements. In these methods, AI complements limited chemical specificity of QPI by maximally utilizing refractive index information in a data-driven manner. The second direction is an improvement of QPI methods themselves. In computational side, efficient 2D holographic or 3D tomographic reconstruction was demonstrated using neural networks. For an experimental side, reinforcement learning frameworks are employed for efficient measurements in an adaptive fashion. This direction is relatively unexplored and provides a promising frontier. We envision that these approaches would form an indispensable toolbox for QPI and facilitate exciting new applications. As QPI is extensively studied and commercialized, rapidly accumulating data for various biological systems would render the methods increasingly powerful.

Recognizing the value and unmet need for label-free kinetic cell analysis, Phase Holograhic Imaging defines its market segment as automated, easy to use and affordable time-lapse cytometry. The process of developing new technology, meeting customer expectations, sources of corporate funding and R&D adjustments prompted by field experience will be reviewed. Additionally, it is discussed how relevant biological information can be extracted from a sequence of quantitative phase images, with negligible user assistance and parameter tweaking, to simultaneously provide cell culture characteristics such as cell growth rate, viability, division rate, mitosis duration, phagocytosis rate, migration, motility and cell-cell adherence without requiring any artificial cell manipulation.

In this talk, we present correlative approaches in commercial quantitative phase microscopy. By combining both optical diffraction tomography and three-channel 3D fluorescence microscopy, both refractive index tomography and molecular specific imaging of live cells and tissues can be obtained. We will discuss the commercialization of this microscopy (HT-2 series via Tomocube Inc.) and also present potential applications for the study of cell biology and medicine.

Since the establishment of high content imaging for high throughput screening, the evolution of a number of technologies has changed imaging. The emergence of new technologies, both label free and with labels (QPI, SRM, structured Illumination) are enabling the scientist to address ever more complex biological question. In addition, combinations of technologies such as Correlative Light Electron Microscopy are driving more specialized applications. At the same time, the mobile revolution is enabling very user friendly, easy to use instrumentation and user interfaces. Also, the emergence of cloud based data storage and applications is creating an ecosystem of data sharing, as well as standardization of methods. The opportunities and challenges to address this complex applications space from a commercial perspective will be discussed.

Myocardial perfusion imaging (MPI) tests are often used to help diagnose coronary heart disease (CAD). The tests usually involve applying stress, such as hard physical exercise together with administration of vasodilators, to the patients. To date, many of these tests use non-selective A_2A adenosine receptor agonists which, however, can be associated with highly undesirable and life-threatening side effects such as chest pain, dyspnea, severe bronchoconstriction and atrioventricular conduction anomalies. Regadenoson is a relatively new, highly selective A_2A adenosine receptor agonist, suitable for use in MPI tests which exhibits far fewer adverse side effects and, unlike others testing agents, can be used without the necessity of excessive concomitant exercise. Also, the dose of regadenoson required is not dependent upon patient weight or renal impairment, and it can be rapidly administered by i.v. Injection. Regadenoson use in MPI testing thus has the potential as a simplified, relatively safe, time-saving and cost-effective method for helping diagnose CAD. The present study was designed to review several articles on the safety, efficacy, and suitability of regadenoson in MPI testing for CAD. Overall, the combined studies demonstrated that use of regadenoson in conjunction with low-level exercise in MPI is a highly efficient and relatively safe test for CAD, especially for more severe health-compromised patients.

Individual cells within isogenic tumor populations can exhibit distinct cellular morphologies, behaviors, and molecular profiles. Cell state plasticity refers to the propensity of a cell to transition between these different morphologies and behaviors. Elevation of cell state plasticity is thought to contribute to critical stages in tumor evolution, including metastatic dissemination and acquisition of therapeutic resistance. However, methods for quantifying general plasticity in mammalian cells remain limited. Working with a HoloMonitor M4 digital holographic cytometry platform, we have established a machine learning-based pipeline for high accuracy and label-free classification of adherent cells. We use twenty-six morphological and optical density-derived features for label-free identification of cell state in heterogeneous cultures. The system is housed completely within a mammalian cell incubator, permitting the monitoring of changes in cell state over time. Here we present an application of our approach for studying cell state plasticity. Human melanoma cell lines of known metastatic potential were monitored in standard growth conditions. The rate of feature change was quantified for each individual cell in the populations. We observed that cells of higher metastatic potential exhibited more rapid fluctuation of cell state in homeostatic conditions. The approach we demonstrate will be advantageous for further investigations into the factors that influence cell state plasticity.

Main restrictions of using laser light in digital holographic microscopy (DHM) are coherence induced noise and parasitic reflections in the experimental setup which limit resolution and measurement accuracy. We explored, if coherence properties of partial coherent light sources can be generated synthetically utilizing spectrally tunable lasers. The concept of the method is demonstrated by label-free quantitative phase imaging of living pancreatic tumor cells and utilizing an experimental configuration including a commercial microscope and a laser source with a broad tunable spectral range of more than 200 nm.

Assessing tissues’ inhomogeneous optical properties is helpful for label-free diagnosis. In this paper, a method and a setup are proposed to assess the tissues’ inhomogeneous optical properties based on quantitative phase imaging (QPI) and dark-field microscopy using LED illumination. In the bright-field and coherent dark-field imaging, quantitative phase (QP) can be recovered from multi-frame intensity images captured at different axial positions by Transport-intensity-equation (TIE) and multi-height Gerchberg-Saxton(G-S) phase retrieval algorithms. In the incoherent dark-field imaging, some scattering samples can be observed easily without any computation and the resolution can be improved in contrast to bright-field illumination. According to the scattering-phase theorem and the statistical dispersion relation (SDR), tissues’ inhomogeneous optical properties can be assessed. In our experiments, we find, it is a good idea to combine QP maps and dark-field images to assess inhomogeneous optical properties of bio-samples. In contrast to the DHM and interferometers, our setup is cost-effective and use-flexible, thus having the potential to serve as a very powerful tool in biological applications.

Surface plasmon microscopy (SPM) is widely applied for label-free detection of changes of refractive index and concentration, as well as mapping thin films in near field. Traditionally, the SPM systems are based on the detection of light intensity or phase changes. Here, we present two kinds of surface plasmon holographic microscopy (SPHM) systems for amplitude- and phase-contrast imaging simultaneously. Through recording off-axis holograms and numerical reconstruction, the complex amplitude distributions of surface plasmon resonance (SPR) images can be obtained. According to the Fresnel’s formula, in a prism/ gold/ dielectric structure, the reflection phase shift is uniquely decided by refractive index of the dielectric. By measuring the phase shift difference of the reflected light exploiting prism-coupling SPHM system based on common-path interference configuration, monitoring tiny refractive index variation and imaging biological tissue are performed. Furthermore, to characterize the thin film thickness in near field, we employ a four-layer SPR model in which the third film layer is within the evanescent field. The complex reflection coefficient, including the reflectivity and reflection phase shift, is uniquely decided by the film thickness. By measuring the complex amplitude distributions of the SPR images exploiting objective-coupling SPHM system based on common-path interference configuration, the thickness distributions of thin films are mapped with sub-nanometer resolution theoretically. Owing to its high temporal stability, the recommended SPHMs show great potentials for monitoring tiny refractive index variations, imaging biological tissues and mapping thin films in near field with dynamic, nondestructive and full-field measurement capabilities in chemistry, biomedicine field, etc.

Scattering media are an ongoing challenge for all kind of imaging technologies including coherent and incoherent principles. Inspired by new approaches of computational imaging and supported by the availability of powerful computers, spatial light modulators, light sources and detectors, a variety of new methods ranging from holography to time-of-flight imaging, phase conjugation, phase recovery using iterative algorithms and correlation techniques have been introduced and applied to different types of objects. However, considering the obvious progress in this field, several problems are still matter of investigation and their solution could open new doors for the inspection and application of scattering media as well. In particular, these open questions include the possibility of extending the 2d-approach to the inspection of depth-extended objects, the direct use of a scattering media as a simple tool for imaging of complex objects and the improvement of coherent inspection techniques for the dimensional characterization of incoherently radiating spots embedded in scattering media. In this paper we show our recent findings in coping with these challenges. First we describe how to explore depth-extended objects by means of a scattering media. Afterwards, we extend this approach by implementing a new type of microscope making use of a simple scatter plate as a kind of flat and unconventional imaging lens. Finally, we introduce our shearing interferometer in combination with structured illumination for retrieving the axial position of fluorescent light emitting spots embedded in scattering media.

We present an inherently phased synthetic aperture microscopy method to mitigate speckle noise and frame- phasing-related issues affecting existing systems that employ coherent illumination for maximum phase sensitivity. Instead of digital holography, our method uses a vibration-insensitive, common-path, phase-shifting shearing interferometer to detect the phase gradient vector and the amplitude of the optical field at the image plane of the microscope. The stability of the speckle field seen by the sensor allows coherent speckle compensation. For demonstration, we show speckle-free super-resolved reconstructions of polystyrene beads in index matching microscope oil.

For the example of digital holographic microscopy (DHM), it is illustrated how label-free biophysical parameter sets can be extracted from quantitative phase images of adherent and suspended cells, and how the retrieved data can be applied for in-vitro toxicity testing and cell culture quality assessment. This includes results from the quantification of the reactions of cells to toxic substances as well as data from sophisticated monitoring of cell alterations that are related to changes of cell culture conditions.

Histopathology of breast tissue typically involves labelling of a tissue section with the Hematoxylin and Eosin (H&E) counter stain. The contrast generated by the stain allows pathologists to extract both diagnostic and prognostic information (such as malignancy, grade and stage) during microscopic examination. Despite being the most frequent assessment method in use today, this procedure provides limited information and is subject to observer bias due to its qualitative nature. While commercial tissue slide scanners promise to improve the throughput of this method in the coming years, quantitative evaluation of tissue remains a challenge. In this work, we propose a method for simultaneously extracting color bright field and phase images of stained breast tissue biopsies using Spatial Light Interference Microscopy (SLIM) coupled with an RGB camera. The amplitude information allows standard qualitative histopathology while the quantitative phase information can be used, after normalizing for the staining, to extract physical markers characterizing patient health. We demonstrate this by imaging an H&E stained tissue microarray and showing that the normalized phase values can be exploited to classify benign and malignant tissue. Furthermore, we demonstrate that these images allow quantitation of tumor-adjacent collagen structure - an important prognostic marker for breast cancer. Extraction of these biomarkers requires measurement of the tissue optical path-length map which is not available in standard tissue evaluation. Our method, therefore, expands on the current diagnostic pipeline by complementing standard histopathology with quantitative tissue biomarkers, all obtainable in a single scan.

The development of robust non-invasive laboratory screening methods for early diagnosis on the out-patient basis seems quite relevant for practical medicine. It is known, that platelet is an original biosensor, a detector of early changes in hemostasis condition. The aim of this study was to assess a potential of the quantitative phase imaging (QPI) technique for real time evaluation the influence of low-molecular weight and unfractionated heparin on platelets in patients with the end-stage of chronic renal failure, who were treated with program hemodialysis (PHD).

The main group consisted of 21 patients who were administered a low-molecular weight heparin for hypocoagulation during the procedure of hemodialysis. The control group (15 patients) received unfractionated heparin. Morphodensitometric state of living platelets we evaluated by QPI using computer phase-interference microscope MIM (Moscow, Russia). We analyzed the optical–geometrical parameters and the morphological features of living platelets which reflected the degree of their activation at the beginning of PHD (before administration of heparin), in 15 minutes after it and at the end of the procedure. The results allow us to conclude that the use of low-molecular weight heparin provides better ratio of efficacy/safety and causes a reduction of the platelet activation during the hemodialysis procedure. Practical implementation of QPI for clinical monitoring of platelets makes it possible to obtain important information on hemostasis cell. It opens new opportunities to assess the efficacy of treatment, as well as for early diagnosis of complications for disease.

Angiogenesis, the formation of new blood vessels from existing ones, is a biological process that has an essential role in solid tumor growth, development, and progression. Recent advances in Lab-on-a-Chip technology has created an opportunity for scientists to observe endothelial cell (EC) behaviors during the dynamic process of angiogenesis using a simple and economical in vitro platform that recapitulates in vivo blood vessel formation. Here, we use quantitative phase imaging (QPI) microscopy to continuously and non-invasively characterize the dynamic process of tumor cell-induced angiogenic sprout formation on a microfluidic chip. The live tumor cell-induced angiogenic sprouts are generated by multicellular endothelial sprouting into 3 dimensional (3D) Matrigel using human umbilical vein endothelial cells (HUVECs). By using QPI, we quantitatively measure a panel of cellular morphological and behavioral parameters of each individual EC participating in this sprouting. In this proof-of-principle study, we demonstrate that QPI is a powerful tool that can provide real-time quantitative analysis of biological processes in in vitro 3D biomimetic devices, which, in turn, can improve our understanding of the biology underlying functional tissue engineering.

We present a simple and compact phase imaging microscope for long-term observation of non-absorbing biological samples such as unstained cells in nutritive media. The phase image is obtained from a single defocused image taken with a standard wide-field microscope. Using a semi-coherent light source allows us to computationally re-focus image post-acquisition and recover both phase and transmission of the complex specimen. The simplicity of the system reduces both the cost and its physical size and allows a long-term observation of samples directly in a standard biological incubator. The low cost of the system can contribute to the democratization of science by allowing to perform complex long-term biological experiments to the laboratories with constrained budget. In this proceeding we present several results taken with our prototype and discuss the possibilities and limitations of our system.

We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm²). We thereby demonstrate that lens-free video microscopy, with a large field of view (~30 mm²) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x10⁶ cell morphological measurements in a single time-lapse.

Optical diffraction tomography (ODT) can be described using the scattering process through an inhomogeneous media. An inherent nonlinearity exists relating the scattering medium and the scattered field due to multiple scattering. Multiple scattering is often assumed to be negligible in weakly scattering media. This assumption becomes invalid as the sample gets more complex resulting in distorted image reconstructions. This issue becomes very critical when we image a complex sample. Multiple scattering can be simulated using the beam propagation method (BPM) as the forward model of ODT combined with an iterative reconstruction scheme. The iterative error reduction scheme and the multi-layer structure of BPM are similar to neural networks. Therefore we refer to our imaging method as learning tomography (LT). To fairly assess the performance of LT in imaging complex samples, we compared LT with the conventional iterative linear scheme using Mie theory which provides the ground truth. We also demonstrate the capacity of LT to image complex samples using experimental data of a biological cell.

We developed a transportable Linnik-type full-field low-coherent quantitative phase microscope that is able to compensate for optical path length (OPL) disturbance due to environmental mechanical noises. Though two-beam interferometers such as Linnik ones suffer from unstable OPL difference, we overcame this problem with a mechanical feedback system based on digital signal-processing that controls the OPL difference in sub-nanometer resolution precisely with a feedback bandwidth of 4 kHz. The developed setup has a footprint of 200 mm by 200 mm, a height of 500 mm, and a weight of 4.5 kilograms. In the transmission imaging mode, cells were cultured on a reflection-enhanced glass-bottom dish, and we obtained interference images sequentially while performing stepwise quarter-wavelength phase-shifting. Real-time image processing, including retrieval of the unwrapped phase from interference images and its background correction, along with the acquisition of interference images, was performed on a laptop computer. Emulation of the phase contrast (PhC) images and the differential interference contrast (DIC) images was also performed in real time. Moreover, our setup was applied for full-field cell membrane imaging in the reflection mode, where the cells were cultured on an anti-reflection (AR)-coated glass-bottom dish. The phase and intensity of the light reflected by the membrane revealed the outer shape of the cells independent of the refractive index. In this paper, we show imaging results on cultured cells in both transmission and reflection modes.

We developed a compact Mach-Zehnder interferometer module to be used as a replacement of the objective lens in a conventional inverted microscope (Nikon, TS100-F) in order to make them quantitative phase microscopes. The module has a 90-degree-flipped U-shape; the dimensions of the module are 160 mm by 120 mm by 40 mm and the weight is 380 grams. The Mach-Zehnder interferometer equipped with the separate reference and sample arms was implemented in this U-shaped housing and the path-length difference between the two arms was manually adjustable. The sample under test was put on the stage of the microscope and a sample light went through it. Both arms had identical achromatic lenses for image formation and the lateral positions of them were also manually adjustable. Therefore, temporally and spatially low coherent illumination was applicable because the users were able to balance precisely the path length of the two arms and to overlap the two wavefronts. In the experiment, spectrally filtered LED light for illumination (wavelength = 633 nm and bandwidth = 3 nm) was input to the interferometer module via a 50 micrometer core optical fiber. We have successfully captured full-field interference images by a camera put on the trinocular tube of the microscope and constructed quantitative phase images of the cultured cells by means of the quarter-wavelength phase shifting algorithm. The resultant quantitative phase images were speckle-free and halo-free due to spectrally and spatially low coherent illumination.

In this research, phase retrieval using transport of intensity equation (TIE) and transport of phase equation (TPE) have been used for determine the surface of corrosion behavior of electrodeposited Co-Fe alloys, an important component in hard disk drive. Without interferometer as used in digital holography technique, by using TIE and TPE, our observing setup is simple and compact. The experimental results show the capability of TIE and TPE for observing the pitting corrosion. The pitting corrosion of Co-Fe image results have also shown. The dynamic process of microstructure surface system would be conducted and may be applied to hard disk drive industry in the future.

The standard method for cancer diagnosis is the microscopic investigation of tissue biopsies. Because the tissues do not significantly absorb and scatter light, traditionally, the observation is performed using bright-field microscopy after staining. Although this approach has been widely adopted all over the world for 100 years, it generally takes a long preparation time and sometimes the early carcinogenesis is missed due to a variation in a quality of a staining. Quantitative phase imaging (QPI) can access objective information on thickness and refractive index changes from an unstained tissue slice, which cannot be observed by conventional microscopes. This can be an attractive advantage in the field of a medical diagnosis, especially since QPI can access the tissue architecture information with nanoscale sensitivity. In this paper, we used quantitative phase imaging to measure the tissue disorder strength, which is known as one of the effective markers of early carcinogenesis. We retrieved the disorder parameter from the local refractive index fluctuation map obtained by spatial light interference microscopy (SLIM). We show that SLIM imaging combined with the disorder analysis is a valuable approach for screening of benign and malignant breast tissue biopsies.

Quantitative phase imaging (QPI) can access quantitative information on thickness and/or refractive index changes of weakly absorbing and scattering objects, which normally require staining prior to observation. The quantitative phase image itself yields significant information for a medical diagnosis, particularly in cancer biopsies. Previously, several parameters such as a local standard deviation of refractive index have been utilized as a marker of diseases. We focus on the local spatial autocorrelation length, which is calculated at each point in the field of view. The local spatial autocorrelation length is defined as the standard deviation of the local spatial autocorrelation function and reveals the local and directional disorder information of tissues. However, generally, a direct calculation of the local spatial autocorrelation length take an immense amount of time. In this paper, we propose a high-throughput calculation procedure of a local spatial autocorrelation length, by exploiting frequency-domain calculations. After deriving a simple equation to calculate the local spatial autocorrelation length map in a short time, we perform label-free screening of benign and malignant breast tissue biopsies using this parameter as a marker.

Deep learning has been proven to achieve ground-breaking accuracy in various tasks. In this paper, we implemented a deep neural network (DNN) to achieve phase retrieval in a wide-field microscope. Our DNN utilized the residual neural network (ResNet) architecture and was trained using the data generated by a phase SLM. The results showed that our DNN was able to reconstruct the profile of the phase target qualitatively. In the meantime, large error still existed, which indicated that our approach still need to be improved.

Phase shifting white light interferometry (PSWLI) has been widely used for optical metrology applications because of their precision, reliability, and versatility. White light interferometry using monochrome CCD makes the measurement process slow for metrology applications. WLI integrated with Red-Green-Blue (RGB) CCD camera is finding imaging applications in the fields optical metrology and bio-imaging. Wavelength dependent refractive index profiles of biological samples were computed from colour white light interferograms. In recent years, whole-filed refractive index profiles of red blood cells (RBCs), onion skin, fish cornea, etc. were measured from RGB interferograms. In this paper, we discuss the bio-imaging applications of colour CCD based white light interferometry. The approach makes the measurement faster, easier, cost-effective, and even dynamic by using single fringe analysis methods, for industrial applications.

Induced pluripotent stem cells (iPSCs) are reprogrammed cells that can have heterogeneous biological potential. Quality assurance metrics of reprogrammed iPSCs will be critical to ensure reliable use in cell therapies and personalized diagnostic tests. We present a quantitative phase imaging (QPI) workflow which includes acquisition, processing, and stitching multiple adjacent image tiles across a large field of view (LFOV) of a culture vessel.

Low magnification image tiles (10x) were acquired with a Phasics SID4BIO camera on a Zeiss microscope. iPSC cultures were maintained using a custom stage incubator on an automated stage. We implement an image acquisition strategy that compensates for non-flat illumination wavefronts to enable imaging of an entire well plate, including the meniscus region normally obscured in Zernike phase contrast imaging. Polynomial fitting and background mode correction was implemented to enable comparability and stitching between multiple tiles. LFOV imaging of reference materials indicated that image acquisition and processing strategies did not affect quantitative phase measurements across the LFOV. Analysis of iPSC colony images demonstrated mass doubling time was significantly different than area doubling time.

These measurements were benchmarked with prototype microsphere beads and etched-glass gratings with specified spatial dimensions designed to be QPI reference materials with optical pathlength shifts suitable for cell microscopy.

This QPI workflow and the use of reference materials can provide non-destructive traceable imaging method for novel iPSC heterogeneity characterization.

We rely on in vitro cellular cultures to evaluate the effects of the components of multifunctional nano-based formulations under development. We employ an incubator-adapted, label-free holographic imaging cytometer HoloMonitor M4® (Phase Holographic Imaging, Lund, Sweden) to obtain multi-day time-lapse sequences at 5- minute intervals. An automated stage allows hand-free acquisition of multiple fields of view.

Our system is based on the Mach-Zehnder interferometry principle to create interference patterns which are deconvolved to produce images of the optical thickness of the field of view. These images are automatically segmented resulting in a full complement of quantitative morphological features, such as optical volume, thickness, and area amongst many others. Precise XY cell locations and the time of acquisition are also recorded.

Visualization is best achieved by novel 4-Dimensional plots, where XY position is plotted overtime time (Z-directions) and cell-thickness is coded as color or gray scale brightness. Fundamental events of interest, i.e., cells undergoing mitosis or mitotic dysfunction, cell death, cell-to-cell interactions, motility are discernable. We use both 2D and 3D models of the tumor microenvironment.

We report our new analysis method to track feature changes over time based on a 4-sample version of the Kolmogorov-Smirnov test. Feature A is compared to Control A, and Feature B is compared to Control B to give a 2D probability plot of the feature changes over time. As a result, we efficiently obtain vectors quantifying feature changes over time in various sample conditions, i.e., changing compound concentrations or multi-compound combinations.

Digital holographic microscopy (DHM) has become a novel tool with advantages of full field, non-destructive, high-resolution and 3D imaging, which captures the quantitative amplitude and phase information of microscopic specimens. It’s a well-established method for digital recording and numerical reconstructing the full complex field of wavefront of the samples with a diffraction-limited lateral resolution down to 0.3 μm depending on the numerical aperture of microscope objective. Meanwhile, its axial resolution through axial direction is less than 10 nm due to the interferometric nature in phase imaging. Compared with the typical optical configurations such as Mach–Zehnder interferometer and Michelson interferometer, the common-path DHM has the advantages of simple and compact configuration, high stability, and so on. Here, a simple, compact, and low-cost common-path DHM based on a beam displacer unit is proposed for quantitative phase imaging of biological cells. The beam displacer unit is completely compatible with commercial microscope and can be easily set up in the output port of the microscope as a compact independent device. This technique can be used to achieve the quantitative phase measurement of biological cells with an excellent temporal stability of 0.51 nm, which makes it having a good prospect in the fields of biological and medical science. Living mouse osteoblastic cells are quantitatively measured with the system to demonstrate its capability and applicability.

We developed an automated high-resolution full-field spatial coherence tomography (FF-SCT) microscope for quantitative phase imaging that is based on the spatial, rather than the temporal, coherence gating. The Red and Green color laser light was used for finding the quantitative phase images of unstained human red blood cells (RBCs). This study uses morphological parameters of unstained RBCs phase images to distinguish between normal and infected cells. We recorded the single interferogram by a FF-SCT microscope for red and green color wavelength and average the two phase images to further reduced the noise artifacts. In order to characterize anemia infected from normal cells different morphological features were extracted and these features were used to train machine learning ensemble model to classify RBCs with high accuracy.

We propose a new diffraction phase microscope (DPM), in which a photon counting (PC)-based single-pixel imaging (SPI) technique is introduced for obtaining two-dimensional quantitative phase images (QPIs) of transparent objects. The introduction of the SPI technique is promising for alleviating the sensitivity problem in DPM. This is because a highsensitive single-channel photodetector such as a photomultiplier tube can be used and the spatial multiplex advantage in the signal-to-noise ratio (SNR) can be expected. Furthermore, the employment of the PC technique solves the dynamic range problem inherent to the SPI. As a proof-of-principle experiment, we measured the QPI of a 125-nm thickness ITO layer coated on a silica-glass substrate, demonstrating the superiority of the PC-based SPI-DPM over SPI-DPM in SNR.

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

Optical cell stretching provides label-free investigations of cells by measuring their biomechanical properties based on deformability determination in a fiber optical two-beam trap. However, the stretching forces in this two-beam laser trap depend on the optical properties of the investigated specimen. Therefore, we characterized in parallel four cancer cell lines with varying degree of differentiation utilizing quantitative phase imaging (QPI) and optical cell stretching. The QPI data allowed enhanced assessment of the mechanical cell properties measured with the optical cell stretcher and demonstrates the high potential of cell phenotyping when both techniques are combined.

Fourier ptychography (FP) is a promising computational imaging technique that overcomes the physical space-bandwidth product (SBP) limit of a conventional microscope by applying angular diversity illuminations. However, to date, the effective imaging numerical aperture (NA) achievable with a commercial LED board is still limited to the range of 0.3–0.7 with a 4×/0.1NA objective due to the constraint of planar geometry with weak illumination brightness and attenuated signal-to-noise ratio (SNR). Thus the highest achievable half-pitch resolution is usually constrained between 500–1000 nm, which cannot fulfill some needs of high-resolution biomedical imaging applications. Although it is possible to improve the resolution by using a higher magnification objective with larger NA instead of enlarging the illumination NA, the SBP is suppressed to some extent, making the FP technique less appealing, since the reduction of field-of-view (FOV) is much larger than the improvement of resolution in this FP platform. Herein, in this paper, we initially present a subwavelength resolution Fourier ptychography (SRFP) platform with a hemispherical digital condenser to provide high-angle programmable plane-wave illuminations of 0.95NA, attaining a 4×/0.1NA objective with the final effective imaging performance of 1.05NA at a half-pitch resolution of 244 nm with a wavelength of 465 nm across a wide FOV of 14.60 mm² , corresponding to an SBP of 245 megapixels. Our work provides an essential step of FP towards high-NA imaging applications without scarfing the FOV, making it more practical and appealing.

Even though holography is a powerful technique for three-dimensional (3D) imaging, it could not be applied easily to an incoherent imaging system, for instance fluorescence microscopy. 3D imaging capability in fluorescence microscopy becomes more and more important lately because of great research demand on cell behavior changes in 3D environmental conditions triggered by recently developed 3D cell culture techniques. Biological research on contact-based cell-to-cell or membrane-to-membrane interactions need 3D imaging as well. Lately, several incoherent holographic imaging methods such as scanning holography, self-interference incoherent digital holography (SIDH), and Fresnel incoherent correlation holography (FINCH) have been proposed for 3D holographic imaging with incoherent light. Most of these methods commonly use an in-line holographic microscopy configuration, which has fundamental image quality degradation problems associated with the two DC terms and the twin image term in a measured hologram. It can be removed by taking multiple holograms with different phase delays in measurement or by iterative methods in numerical reconstruction algorithms. Even though off-axis holography is a straightforward method to remove the DC terms and the twin image, it is difficult to be used with incoherent light due to large path length difference between two beams in an off-axis holographic microscopy configuration. In this study we propose a new off-axis FINCH system which can overcome the path length mismatch problem by using a wedge plate. The combination of a spatial light modulator and a wedge plate in our proposed off-axis FINCH system makes the path lengths of two beams almost identical.

We propose a simple non-iterative in-line holographic measurement method which can effectively eliminate a twin image in digital holographic 3D imaging. It is shown that a twin image can be effectively eliminated with only two measured holograms by using a simple numerical propagation algorithm and arithmetic calculations.

We perform phase imaging using a non-interferometric approach to measure the complex amplitude of a wavefront. We overcome the limitations in spatial resolution, optical efficiency, and dynamic range that are found in Shack-Hartmann wavefront sensing. To do so, we sample the wavefront with a high-speed spatial light modulator. A single lens forms a time-dependent light distribution on its focal plane, where a position detector is placed. Our approach is lenslet-free and does not rely on any kind of iterative or unwrap algorithm. The validity of our technique is demonstrated by performing both aberration sensing and phase imaging of transparent samples.