We developed a portable and user-friendly prototype of low-coherent reflection-type quantitative phase microscope (QPM). Our setup is based on the full-field Linnik type phase-shifting interference microscope and is optimized for surface profiling of living cell’s membrane. Unlike commonly available transmission-type quantitative phase microscopes which reveal the optical thickness, our reflection-type setup can obtain the geometrical thickness (real shape) of the sample, decoupled with the refractive index. The coherence length of our imaging light source (halogen lamp) was approximately 1 micrometer so that we can selectively obtain the interference of the light reflected from the cell membranes whose reflectivity in culture medium is only on the order of 0.1%. Moreover our setup has a feedback controlled path-length stabilization circuit so that users can implement accurate phase shifting interferometry with one nanometer of reproducibility. The stabilization circuit allows installing our setup even in noisy environments such as biology labs without an optical bench. In this paper, we will also show our studies of recent biomedical applications, including imaging of cell plasma membrane and phase-resolved 3D tomography of living cells.

We present a new technique of three-dimensional tracking by way of incoherent digital holography suitable for non-scanning fluorescence microscopy. By acquiring complex holograms of a sample at different times, a difference hologram can be calculated. This result is another complex hologram representing only the information which has changed throughout the volumetric space during the time interval between one hologram and the next. We first demonstrate the advanced capability of self-interference incoherent digital holography combined with difference holography to track three-dimensional changes in a broadband, unfiltered, sunlit scene containing macroscopic continuous objects. This case is particularly remarkable due to the exceptionally short temporal coherence length and excessive build-up of noninterfering source points. We then demonstrate the ease of adaptation to microscopy, successfully converting a standard two-dimensional commercial microscope into a powerful three-dimensional tool. By overcoming these challenges, we show the feasibility and ease at which this technique may be adapted to the versatile, functional imaging of fluorescence microscopy.

Differential phase contrast (DPC) is a quantitative phase imaging technique which measures the sample’s phase derivative by taking two images from complementary asymmetric illumination patterns. Distinct from coherent techniques, DPC relies on partially coherent illumination, providing 2× better lateral resolution, better optical sectioning, and immunity to speckle noise. In this paper, we derive the weak object transfer function to quantify how sample’s phase is converted into our DPC measurements, then develop quantitative inversion methods. Phase reconstructions from single-axis DPC measurements suffer from missing frequencies along the axis of asymmetry. We measure the missing frequency information by taking DPC measurements from other axes. Our phase reconstruction method provides a unified framework for both single and multi-axis DPC measurements. We implement our DPC measurements in real-time and along arbitrary axes of asymmetry by computational illumination on an LED array microscope.

We present a new approach of optically multiplexing several off-axis interferograms on the same digital camera, each of which encodes a different field of view of the sample. Since the fringes of these interferograms are in different directions, as obtained experimentally by the optical system, we are able to double or even triple the amount of information that can be acquired in a single camera exposure, with the same number of camera pixels, while sharing the camera dynamic range. We show that this method can partially solve the problem of limited off-axis interferometric field of view due to low-coherence illumination. Our experimental demonstrations include quantitative phase imaging of microscopic diatom shells, fast swimming sperm cells and microorganisms, and contracting cardiomyocytes.

Our previous work has proposed an electrochemistry - total internal reflection imaging ellipsometry (EC-TIRIE) technique to observe the dissolved oxygen (DO) reduction on Clark electrode since high interface sensitivity makes TIRIE a useful tool to study redox reactions on the electrode surface. To amplify the optical signal noise ratio (OSNR), a surface tethered weak polyelectrolyte, carboxylated poly(oligo(ethylene glycol) methacrylate-random- 2-hydroxyethylmethacrylate) (abbreviated as carboxylated poly(OEGMA-r-HEMA)), has been introduced on the electrode surface. Since Clark electrode is widely used in biochemical oxygen demand (BOD) detection, we use this technique to measure BOD in the sample. The dynamic range of the system is from 0 ∼ 25 mg/L. Two samples have been measured. Compared with the conventional method, the deviation of both optical and electrical signals are less than 10%.

We propose and experimentally demonstrate a system in which off-axis digital holographic microscopy is realized using a broadband illumination source. Single-shot holographic measurements are enabled, while the coherence noise is removed thanks to the broad bandwidth of the illuminating source. The proposed digital holographic camera is portable and can be attached to the camera port of a conventional optical microscope. This camera is capable of obtaining the complex wavefront i.e the intensity and phase information of the light transmitted or reflected from a sample. A combination of a thick transmission volume grating recorded holographically into thick photosensitive glass and thin transmission phase gratings recorded holographically into thin photopolymers, spatially filters the beam of light containing the sample information in two dimensions through diffraction. This filtered beam creates the reference arm of the interferometer. The untouched transmitted beam creates the sample arm of the interferometer. The spatial filtering performed by the combination of gratings above reduces the alignment spatial sensitivity which is an advantage over conventional spatial filtering done by pinholes. Besides, using a second thin grating, we introduce a desired coherence plane tilt in the reference beam which is sufficient to create high-visibility interference over the entire field of view in off-axis configuration. Full-field off-axis interferograms are thus created from which the phase information can be extracted.

We present a novel technique for coherence engineering of the microscope illumination based on a DLP projector providing fast (millisecond range) switchable both temporal and spatial coherence design. Its performance is experimentally demonstrated for speckle-noise free quantitative phase imaging with different spatial coherence states. Strategies for design and control of the light coherence are discussed.

Our research combines Digital Holographic Microscopy (DHM) and ˛uorescence microscopy to study the basic mechanisms of gold nanoparticle mediated laser manipulation. Herein we describe the technical aspects of the setup and holographic image reconstruction. Furthermore, results pertaining to cell volume change and calcium response of cells in laser manipulation will be presented and discussed. For the reconstruction of phase images from fringe image data, a phase unwrapping algorithm is presented that shows great potential to cope with the vast amount of data that was captured. This algorithm is a hybrid between a tile unwrapping technique and a path following unwrapper. It combines the robustness of a path following algorithm and a parallelizable tile unwrapping preprocessing step. The experimental setup enables simultaneous acquisition of ˛uorescence and phase images. For cell manipulation, a picosecond laser was coupled into the setup and weakly focused on cells incubated with gold nanoparticles. To study the cell volume change in the ˝rst minute, phase images were captured with a frame rate of 33 fps. Fluorescence images yielded the calcium signal of the cells as well as the dynamics of the F-actin cytoskeleton after irradiation. The setup is suitable to study fast changes in biophysical and morphological para

In this paper we present coherence-controlled holographic microscopy (CCHM) and various examples of observations of living cells including combination of CCHM with fluorescence microscopy. CCHM is a novel technique of quantitative phase imaging (QPI). It is based on grating off-axis interferometer, which is fully adapted for the use of incoherent illumination. This enables high-quality QPI free from speckles and parasitic interferences and lateral resolution of classical widefield microscopes. Label-free nature of QPI makes CCHM a useful tool for long-term observations of living cells. Moreover, coherence-gating effect induced by the use of incoherent illumination enables QPI of cells even in scattering media. Combination of CCHM with common imaging techniques brings the possibility to exploit advantages of QPI while simultaneously identifying the observed structures or processes by well-established imaging methods. We used CCHM for investigation of general parameters of cell life cycles and for research of cells reactions to different treatment. Cells were also visualized in 3D collagen gel with the use of CCHM. It was found that both the cell activity and movement of the collagen fibers can be registered. The method of CCHM in combination with fluorescence microscopy was used in order to obtain complementary information about cell morphology and identify typical morphological changes associated with different types of cell death. This combination of CCHM with common imaging technique has a potential to provide new knowledge about various processes and simultaneously their confirmation by comparison with known imaging method.

We provide a quantitative model for image formation in common-path QPI systems under partially coherent illumination. Our model is capable of explaining the phase reduction phenomenon and halo effect in phase measurements. We further show how to fix these phenomena with a novel iterative post-processing algorithm. Halo-free and correct phase images of nanopillars and live cells are used to demonstrate the validity of our method.

We propose a method to recover quantitative phase from a stack of defocused intensity images illuminated with partially coherent light from a source of arbitrary shape in Köhler geometry. The algorithm uses a sparse Kalman filtering approach which is fast, accurate, and robust to noise. The proposed method is able to recover not only the phase, but also the source shape, which defines the spatial coherence of the illumination. We validate our algorithm experimentally in a commercial microscope with biological samples.

A quantitative phase-shifting Differential Interference Contrast (DIC) system is built using a programmable spatial light modulator (SLM). Our system offers halo-free phase gradient images with low illumination coherence and very good axial sectioning. Results are presented for standard polystyrene micro-beads and live cells.

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

Coherence-controlled holographic microscope (CCHM) is an off-axis holographic system. It enables observation of a sample and its quantitative phase imaging with coherent as well as with incoherent illumination. The spatial and temporal coherence can be modified and thus also the quality and type of the image information. The coherent illumination provides numerical refocusing in wide depth range similarly to a classic coherent-light digital holographic microscopy (HM). Incoherent-light HM is characterized by a high quality, coherence-noise-free imaging with up to twice higher resolution compared to coherent illumination. Owing to an independent, free of sample reference arm of the CCHM the low spatial light coherence induces coherence-gating effect. This makes possible to observe specimen also through scattering media. We have described theoretically and simulated numerically imaging of a two dimensional object through a scattering layer by CCHM using the linear systems theory. We have investigated both strongly and weakly scattering media characterized by different amount of ballistic and diffuse light. The influence of a scattering layer on the quality of a phase signal is discussed for both types of the scattering media. A strong dependence of the imaging process on the light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data gained with model samples, as well as real biologic objects particularly then by time-lapse observations of live cells reactions to substances producing optically turbid emulsion.

We review new and efficient algorithms, lately presented by us, for rapid reconstruction of quantitative phase maps from off-axis digital interferograms. These algorithms improve the conventional Fourier-based algorithm by using the Fourier transforms and the phase unwrapping process more efficiently, and thus decrease the calculation complexity required for extracting the sample phase map from the recorded interferograms. Using the new algorithms, on a standard personal computer without using the graphic processing-unit programming or parallel computing, we were able to speed up the processing and reach frame rates of up to 45 frames per second for one megapixel off-axis interferograms. These capabilities allow real-time visualization, calculation and data extraction for dynamic samples and processes, inspected by off-axis digital holography. Specific applications include biological cell imaging without labeling and real-time nondestructive testing.

The multi-shot approach in SLIM requires reliable, synchronous, and parallel operation of three independent hardware devices – not meeting these challenges results in degraded phase and slow acquisition speeds, narrowing applications to holistic statements about complex phenomena. The relative youth of quantitative imaging and the lack of ready-made commercial hardware and tools further compounds the problem as Higher level programming languages result in inflexible, experiment specific instruments limited by ill-fitting computational modules, resulting in a palpable chasm between promised and realized hardware performance. Furthermore, general unfamiliarity with intricacies such as background calibration, objective lens attenuation, along with spatial light modular alignment, makes successful measurements difficult for the inattentive or uninitiated. This poses an immediate challenge for moving our techniques beyond the lab to biologically oriented collaborators and clinical practitioners.

To meet these challenges, we present our new Quantitative Phase Imaging pipeline, with improved instrument performance, friendly user interface and robust data processing features, enabling us to acquire and catalog clinical datasets hundreds of gigapixels in size.

In this work, we experimentally determine the transfer function of our recently reported epi-illumination white light diffraction phase microscopy (epi-wDPM) system. The transfer function identifies how the low frequencies below k₀NA_con are modified due to the limited spatial coherence and how the high frequencies above k₀NA_obj are affected due to the limited objective numerical aperture. Using this transfer function, we perform deconvolution to remove the halo and obtain proper quantitative phase measurements without the need for excessive spatial filtering. The wDPM and epi-wDPM systems are now capable of obtaining halo-free images with proper topography at much higher speeds.

Digital Holography (DH) numerical procedures have been developed to allow imaging through turbid media. A fluid is considered turbid when dispersed particles provoke strong light scattering, thus destroying the image formation by any standard optical system. Here we show that sharp amplitude imaging and phase-contrast mapping of object hidden behind turbid medium and/or occluding objects are possible in harsh noise conditions and with a large field-of view by Multi-Look DH microscopy. In particular, it will be shown that both amplitude imaging and phase-contrast mapping of cells hidden behind a flow of Red Blood Cells can be obtained. This allows, in a noninvasive way, the quantitative evaluation of living processes in Lab on Chip platforms where conventional microscopy techniques fail. The combination of this technique with endoscopic imaging can pave the way for the holographic blood vessel inspection, e.g. to look for settled cholesterol plaques as well as blood clots for a rapid diagnostics of blood diseases.

We present an opto-fluidic measurement system that quantifies cell volume, dry mass and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell’s refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

Most quantitative phase microscopy methods require the use of custom-built or modified microscopic configurations which are not typically available to most bio/pathologists. There are, however, phase retrieval algorithms which utilize defocused bright-field images as input data and are therefore implementable in existing laboratory environments. Among these, deterministic methods such as those based on inverting the transport-of-intensity equation (TIE) or a phase contrast transfer function (PCTF) are particularly attractive due to their compatibility with Köhler illuminated systems and numerical simplicity. Recently, a new method has been proposed, called multi-filter phase imaging with partially coherent light (MFPI-PC), which alleviates the inherent noise/resolution trade-off in solving the TIE by utilizing a large number of defocused bright-field images spaced equally about the focal plane. Despite greatly improving the state-ofthe- art, the method has many shortcomings including the impracticality of high-speed acquisition, inefficient sampling, and attenuated response at high frequencies due to aperture effects. In this report, we present a new method, called bright-field quantitative phase microscopy (BFQPM), which efficiently utilizes a small number of defocused bright-field images and recovers frequencies out to the partially coherent diffraction limit. The method is based on a noiseminimized inversion of a PCTF derived for each finite defocus distance. We present simulation results which indicate nanoscale optical path length sensitivity and improved performance over MFPI-PC. We also provide experimental results imaging live bovine mesenchymal stem cells at sub-second temporal resolution. In all, BFQPM enables fast and accurate phase imaging with unprecedented spatial resolution using widely available bright-field microscopy hardware.

For the analysis of the impact of pharmaceuticals or pathogens on different cellular phenotypes under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of different cell types in a single culture is of particular interest. Digital holographic microscopy (DHM), a var-iant of quantitative phase microscopy (QPM), provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and has been demonstrated in particular to be suitable for long-term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth and motility. Thus, we studied the feasibility of QPM for the analysis of mixed cell cultures and explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with DHM was utilized. Mixed cell cultures with different cell types were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the area covered by the cells, the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability of the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different mor-phology features separately in a single culture.

We report, for the first time, the use of Quantitative Phase Imaging (QPI) images to perform automatic prostate cancer diagnosis. A machine learning algorithm is implemented to learn textural behaviors of prostate samples imaged under QPI and produce labeled maps of different regions for testing biopsies (e.g. gland, stroma, lumen etc.). From these maps, morphological and textural features are calculated to predict outcomes of the testing samples. Current performance is reported on a dataset of more than 300 cores of various diagnosis results.

Even with the recent rapid advances in the field of microscopy, non-laser light sources used for light microscopy have not been developing significantly. Most current optical microscopy systems use halogen bulbs as their light sources to provide a white-light illumination. Due to the confined shapes and finite filament size of the bulbs, little room is available for modification in the light source, which prevents further advances in microscopy.

By contrast, commercial projectors provide a high power output that is comparable to the halogen lamps while allowing for great flexibility in patterning the illumination. In addition to their high brightness, the illumination can be patterned to have arbitrary spatial and spectral distributions. Therefore, commercial projectors can be adopted as a flexible light source to an optical microscope by careful alignment to the existing optical path.

In this study, we employed a commercial projector source to a quantitative phase imaging system called spatial light interference microscopy (SLIM), which is an outside module for an existing phase contrast (PC) microscope. By replacing the ring illumination of PC with a ring-shaped pattern projected onto the condenser plane, we were able to recover the same result as the original SLIM. Furthermore, the ring illumination is replaced with multiple dots aligned along the same ring to minimize the overlap between the scattered and unscattered fields. This new method minimizes the halo artifact of the imaging system, which allows for a halo-free high-resolution quantitative phase microscopy system.

A highly sensitive laser-based quantitative phase imaging tool, using an epi-illumination diffraction phase microscope, has been developed for silicon wafer defect inspection. The first system used a 532 nm solid-state laser and detected 20 nm by 100 nm by 110 nm defects in a 22 nm node patterned silicon wafer. The second system, using a 405 nm diode laser, is more sensitive and has enabled detection of 15 nm by 90 nm by 35 nm defects in a 9 nm node densely patterned silicon wafer. In addition to imaging, wafer scanning and image-post processing are also crucial for defect detection.

In this work, we present recent results on several novel applications including optically monitoring the dissolution of biodegradable materials proposed for use in biological electronic implants, the self-assembly of microtubes during semiconductor etching, and the expansion and deformation of palladium structures for use in hydrogen sensing applications. The measurements are done using diffraction phase microscopy (DPM), a quantitative phase imaging (QPI) technique, which uses the phase of the imaging field to reconstruct a map of the sample’s surface. It combines off-axis and common-path geometries allowing for single-shot, high-speed dynamics with sub-nanometer noise levels.

Various cellular activities such as motility, division, and endocytosis involve a change in the cell shape. The mechanical interactions between the cell membrane and cytoskeleton play an important role in regulating changes in the cell shape. Tether formation from cell membranes provides a technique to characterize the mechanical properties of cell membranes and membrane-cytoskeleton interactions. Accurate measurement of the nano-scale tether diameter is relevant to quantification of membrane tension, bending modulus, and adhesion energy of the membrane-cytoskeleton structure. We have integrated optical tweezers with quantitative phase imaging, based on spatial light interference microscopy (SLIM), to simultaneously form tethers from HEK-293 cells and measure their diameters. Tether thickness along the illumination axis was measured using the quantitative phase map of the sample, and the refractive index (RI) mismatch between the sample and the surrounding media. The RI of the tethers ranged from 1.354 to 1.368 (cell culture medium RI=1.337). Our SLIM imaging system provided a 38 nm resolution in tether thickness measurements. Tether diameter fluctuations of <100 nm were resolved on tethers that ranged between 600-900 nm in diameter. Our integrated platform also provides the ability to simultaneously manipulate and image cell organelles in a non-contact and marker-free manner at nanometer spatial resolution.

We used a new quantitative high spatiotemporal resolution phase imaging tool to explore the nuclear structure and dynamics of individual cells. We used a novel analysis tool to quantify the diffusion outside and inside the nucleus of live cells. We also obtained information about the nuclear spatio temporal mass density in metastatic cells. The results indicate that in the cytoplasm, the intracellular transport is mainly active (direct, deterministic), while inside the nucleus it is both active and passive (diffusive, random). We calculated the standard deviation of velocities in active transport and the diffusion coefficient for passive transport.

The standard practice in the histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope. The pathologist looks at certain morphological features, visible under the stain, to diagnose whether a tumor is benign or malignant. This determination is made based on qualitative inspection making it subject to investigator bias. Furthermore, since this method requires a microscopic examination by the pathologist it suffers from low throughput. A quantitative, label-free and high throughput method for detection of these morphological features from images of tissue biopsies is, hence, highly desirable as it would assist the pathologist in making a quicker and more accurate diagnosis of cancers. We present here preliminary results showing the potential of using quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated optical path length maps of unstained breast tissue biopsies using Spatial Light Interference Microscopy (SLIM). As a first step towards diagnosis based on quantitative phase imaging, we carried out a qualitative evaluation of the imaging resolution and contrast of our label-free phase images. These images were shown to two pathologists who marked the tumors present in tissue as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on H&E stained tissue images and the number of agreements were counted. In our experiment, the agreement between SLIM and H&E based diagnosis was measured to be 88%. Our preliminary results demonstrate the potential and promise of SLIM for a push in the future towards quantitative, label-free and high throughput diagnosis.

1 in 7 men receive a diagnosis of prostate cancer in their lifetime. The aggressiveness of the treatment plan adopted by the patient is strongly influenced by Gleason grade. Gleason grade is determined by the pathologist based on the level of glandular formation and complexity seen in the patient’s biopsy. However, studies have shown that the disagreement rate between pathologists on Gleason grades 3 and 4 is high and this affects treatment options. We used quantitative phase imaging to develop an objective method for Gleason grading. Using the glandular solidity, which is the ratio of the area of the gland to a convex hull fit around it, and anisotropy of light scattered from the stroma immediately adjoining the gland, we were able to quantitatively separate Gleason grades 3 and 4 with 81% accuracy in 43 cases marked as difficult by pathologists.

White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique.

Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead.

WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.

Digital inline holographic microscopy was used to record holograms of mammalian cells (HEK293, B16, and E0771) in culture. The holograms have been reconstructed using Octopus software (4Deep inwater imaging) and phase shift maps were unwrapped using the FFT-based phase unwrapping algorithm. The unwrapped phase shifts were used to determine the maximum phase shifts in individual cells. Addition of 0.5 mM H₂O₂ to cell media produced rapid rounding of cultured cells, followed by cell membrane rupture. The cell morphology changes and cell membrane ruptures were detected in real time and were apparent in the unwrapped phase shift images. The results indicate that quantitative phase contrast imaging produced by the digital inline holographic microscope can be used for the label-free real time automated determination of cell viability and confluence in mammalian cell cultures.

While automated blood cell counters have made great progress in detecting abnormalities in blood, the lack of specificity for a particular disease, limited information on single cell morphology and intrinsic uncertainly due to high throughput in these instruments often necessitates detailed inspection in the form of a peripheral blood smear. Such tests are relatively time consuming and frequently rely on medical professionals tally counting specific cell types. These assays rely on the contrast generated by chemical stains, with the signal intensity strongly related to staining and preparation techniques, frustrating machine learning algorithms that require consistent quantities to denote the features in question. Instead we opt to use quantitative phase imaging, understanding that the resulting image is entirely due to the structure (intrinsic contrast) rather than the complex interplay of stain and sample. We present here our first steps to automate peripheral blood smear scanning, in particular a method to generate the quantitative phase image of an entire blood smear at high throughput using white light diffraction phase microscopy (wDPM), a single shot and common path interferometric imaging technique.

Quantitative imaging and three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes at Lab on Chip scale. Diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. In recent years digital holography (DH) has been improved to be considered as suitable diagnostic method in several research field. In this paper we demonstrate that DH can be used for retrieving 3D morphometric data for sorting and diagnosis aims. Several techniques exist for 3D morphological study as optical coherent tomography and confocal microscopy, but they are not the best choice in case of dynamic events as flowing samples. Recently, a DH approach, based on shape from silhouette algorithm (SFS), has been developed for 3D shape display and calculation of cells biovolume. Such approach, adopted in combination with holographic optical tweezers (HOT) was successfully applied to cells with convex shape. Unfortunately, it’s limited to cells with convex surface as sperm cells or diatoms. Here, we demonstrate an improvement of such procedure. By decoupling thickness information from refractive index ones and combining this with SFS analysis, 3D shape of concave cells is obtained. Specifically, the topography contour map is computed and used to adjust the 3D shape retrieved by the SFS algorithm. We prove the new procedure for healthy red blood cells having a concave surface in their central region. Experimental results are compared with theoretical model.

Common-path diffraction optical tomography (cDOT) is a non-invasive and label-free optical holographic technique for measuring both the three-dimensional refractive index (RI) tomograms and two-dimensional dynamic phase images of a sample. Due to common-path geometry, cDOT provides quantitative phase imaging with high phase sensitivity. However, the image quality of the cDOT suffers from speckle noise; the use of a monochromatic laser inevitably results in the formation of parasitic fringe patterns in measured quantitative phase images. Here, we present a technique to reduce speckle noise in the cDOT using a low-coherence illumination source. Utilizing a Ti-sapphire pulsed laser in the cDOT, we achieved the reduction of speckle noise in both the three-dimensional RI tomograms and two-dimensional dynamic phase images.

A new microscopy method for observing phase objects without halos and directional shadows is proposed. The key optical element is an annular aperture at the front focal plane of a condenser with a larger diameter than those used in standard phase contrast microscopy. The light flux passing through the annular aperture is changed by the specimen's surface profile and then passes through an objective and contributes to image formation. This paper presents essential conditions for realizing the method.

In this paper, images of colonies formed by induced pluripotent stem (iPS) cells using this method are compared with the conventional phase contrast method and the bright-field method when the NA of the illumination is small to identify differences among these techniques. The outlines of the iPS cells are clearly visible with this method, whereas they are not clearly visible due to halos when using the phase contrast method or due to weak contrast when using the bright-field method. Other images using this method are also presented to demonstrate a capacity of this method: a mouse ovum and superimposition of several different images of mouse iPS cells.

The objective of the present study is to increase the quality of the early diagnosis using cytological differential-diagnostic criteria for reactive changes in the nuclear structures of the immunocompetent cells. The morphofunctional status of living cells were estimated in the real time using new technologic platform of the hardware-software complex for phase cell imaging. The level of functional activity for lymphocyte subpopulations was determined on the base of modification of nuclear structures and decreasing of nuclear phase thickness. The dynamics of nuclear parameters was used as the quantitative measuring for cell activating level and increasing of proliferative potential.

Brightfield, darkfield and phase contrast are three of the most popular imaging modalities in biological microscopy. Each provides visualization of different sample properties, without fluorescent labels or staining. We demonstrate here a single-camera imaging system that can simultaneously acquire brightfield, darkfield and phase contrast images in real-time. Our method uses a programmable LED array as the illumination source, which provides flexible patterning of illumination angles. We achieve a frame rate of 50 Hz with 2560×2160 pixels of lateral resolution, with speed being limited by the camera.

White-light imaging systems are free of laser-speckle. Thus, they offer high sensitivity for optical defect metrology, especially when used with interferometry based quantitative phase imaging. This can be a potential solution for wafer inspection beyond the 9 nm node. Recently, we built a white-light epi-illumination diffraction phase microscopy (epi-wDPM) for wafer defect inspection. The system is also equipped with an XYZ scanning stage and real-time processing. Preliminary results have demonstrated detection of 15 nm by 90 nm in a 9 nm node densely patterned wafer with bright-field imaging. Currently, we are implementing phase imaging with epi-wDPM for additional sensitivity.