Deep-learning-assisted snapshot optical tomography for microscopic volume prediction: a simulation study.

In this simulation study, we demonstrate fast-yet-accurate volume measurement of microscopic objects by combining snapshot optical tomography and deep learning. Snapshot optical tomography simultaneously collects a multitude of projection images and thus can perform 3D imaging in a single snapshot. However, as with other wide-field microscopy techniques, it suffers from the missing-cone problem, which can seriously degrade the quality of 3D reconstruction. We use deep learning to generate a volume prediction from 2D projection images bypassing the 3D reconstruction.

Optical projection tomography of fluorescent microscopic specimens using lateral translation of tube lens.

Optical projection tomography (OPT) is a three-dimensional (3D) fluorescence imaging technique, in which projection images are acquired for varying orientations of a sample using a large depth of field. OPT is typically applied to a millimeter-sized specimen, because the rotation of a microscopic specimen is challenging and not compatible with live cell imaging. In this Letter, we demonstrate fluorescence optical tomography of a microscopic specimen by laterally translating the tube lens of a wide-field optical microscope, which allows for high-resolution OPT without rotating the sample. The cost is the reduction of the field of view to about halfway along the direction of the tube lens translation. Using bovine pulmonary artery endothelial cells and 0.1 µm beads, we compare the 3D imaging performance of the proposed method with that of the conventional objective-focus scan method.

Hyperspectral confocal microscopy in the short-wave infrared range.

We demonstrate hyperspectral confocal microscopy in the short-wave infrared (SWIR) range of 1100-1600 nm using a wavelength-scanning laser in tandem with laser scanning confocal microscopy. Confocal microscopy in the SWIR range allows for high-resolution inspection of an integrated circuit (IC) chip, while hyperspectral imaging, together with a chemometric analysis, enables us to identify functional circuit block groups in the acquired image. With the extended capability, the developed instrument can be potentially used for inline inspection and non-invasive failure analysis of IC chips.

Refractive index dispersion measurement in the short-wave infrared range using synthetic phase microscopy.

Refractive index is an optical property explored in the light scattering measurement of micro- and nano-particles as well as in label-free imaging of cells and tissues. Because the refractive index value is a major input to the characterization and quantification of the analyzed specimens, various methods have been developed targeting at different sample types. In this paper, we demonstrate a technique for the refractive index measurement of homogeneous microspheres and liquids in the short-wave infrared (SWIR) range. We use synthetic phase microscopy (SPM), which records a scattering-corrected projection of the 3D refractive index distribution, in combination with a least-squares fitting to a theoretical model of a sphere. Using the method, we determine the refractive index dispersion of two polymer microspheres (polymethyl methacrylate and polystyrene), two glass microspheres (silica and soda lime), and three microscopy mounting media (glycerol, FluorSave, and Eukitt) in the SWIR range of 1100-1650 nm.

Spectroscopic Microtomography in the Short-Wave Infrared Wavelength Range.

Spectroscopic microtomography provides the ability to perform 4D (3D structural and 1D chemical) imaging of a thick microscopic specimen. Here, we demonstrate spectroscopic microtomography in the short-wave infrared (SWIR) wavelength using digital holographic tomography, which captures both the absorption coefficient and refractive index. A broadband laser in tandem with a tunable optical filter allows us to scan the wavelength from 1100 to 1650 nm. Using the developed system, we measure human hair and sea urchin embryo samples. The resolution estimated with gold nanoparticles is 1.51 µm (transverse) and 1.57 µm (axial) for the field of view of 307 × 246 µm2. The developed technique will enable accurate and efficient analyses of microscopic specimens that have a distinctive absorption or refractive index contrast in the SWIR range.

Hyperspectral Three-Dimensional Fluorescence Imaging Using Snapshot Optical Tomography.

Hyperspectral three-dimensional (3D) imaging can provide both 3D structural and functional information of a specimen. The imaging throughput is typically very low due to the requirement of scanning mechanisms for different depths and wavelengths. Here we demonstrate hyperspectral 3D imaging using Snapshot projection optical tomography (SPOT) and Fourier-transform spectroscopy (FTS). SPOT allows us to instantaneously acquire the projection images corresponding to different viewing angles, while FTS allows us to perform hyperspectral imaging at high spectral resolution. Using fluorescent beads and sunflower pollens, we demonstrate the imaging performance of the developed system.

Forward model for propagation-based x-ray phase contrast imaging in parallel- and cone-beam geometry.

We demonstrate a fast, flexible, and accurate paraxial wave propagation model to serve as a forward model for propagation-based X-ray phase contrast imaging (XPCI) in parallel-beam or cone-beam geometry. This model incorporates geometric cone-beam effects into the multi-slice beam propagation method. It enables rapid prototyping and is well suited to serve as a forward model for propagation-based X-ray phase contrast tomographic reconstructions. Furthermore, it is capable of modeling arbitrary objects, including those that are strongly or multi-scattering. Simulation studies were conducted to compare our model to other forward models in the X-ray regime, such as the Mie and full-wave Rytov solutions.

Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions.

The wide diversity of skeletal proportions in mammals is evident upon a survey of any natural history museum's collections and allows us to distinguish between species even when reduced to their calcified components. Similarly, each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation. However, the mechanisms of chondrocyte volume enlargement have remained a mystery. Here we use quantitative phase microscopy to show that mammalian chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types, this is a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through a mechanism dependent on insulin-like growth factor. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify and establish a volume set point.

Large population cell characterization using quantitative phase cytometer.

A major challenge in cellular analysis is the phenotypic characterization of large cell populations within a short period of time. Among various parameters for cell characterization, the cell dry mass is often used to describe cell size but is difficult to be measured directly with traditional techniques. Here, we propose an interferometric approach based on line-focused beam illumination for high-content precision dry mass measurements of adherent cells in a non-invasive fashion-we call it quantitative phase cytometry (QPC). Besides dry mass, abundant cellular morphological features such as projected area, sphericity, and phase skewness can be readily extracted from the QPC interferometric data. To validate the utility of our technique, we demonstrate characterizing a large population of ∼104 HeLa cells. Our reported QPC system is envisioned as a promising quantitative tool for label-free characterization of a large cell count at single cell resolution. © 2017 International Society for Advancement of Cytometry.

Size homeostasis in adherent cells studied by synthetic phase microscopy.

The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins-Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and size-dependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.

Investigating Effects of Proteasome Inhibitor on Multiple Myeloma Cells Using Confocal Raman Microscopy.

Due to its label-free and non-destructive nature, applications of Raman spectroscopic imaging in monitoring therapeutic responses at the cellular level are growing. We have recently developed a high-speed confocal Raman microscopy system to image living biological specimens with high spatial resolution and sensitivity. In the present study, we have applied this system to monitor the effects of Bortezomib, a proteasome inhibitor drug, on multiple myeloma cells. Cluster imaging followed by spectral profiling suggest major differences in the nuclear and cytoplasmic contents of cells due to drug treatment that can be monitored with Raman spectroscopy. Spectra were also acquired from group of cells and feasibility of discrimination among treated and untreated cells using principal component analysis (PCA) was accessed. Findings support the feasibility of Raman technologies as an alternate, novel method for monitoring live cell dynamics with minimal external perturbation.

Scanning color optical tomography (SCOT).

We have developed an interferometric optical microscope that provides three-dimensional refractive index map of a specimen by scanning the color of three illumination beams. Our design of the interferometer allows for simultaneous measurement of the scattered fields (both amplitude and phase) of such a complex input beam. By obviating the need for mechanical scanning of the illumination beam or detection objective lens; the proposed method can increase the speed of the optical tomography by orders of magnitude. We demonstrate our method using polystyrene beads of known refractive index value and live cells.

Crystal analyser-based X-ray phase contrast imaging in the dark field: implementation and evaluation using excised tissue specimens.

OBJECTIVES: We demonstrate the soft tissue discrimination capability of X-ray dark-field imaging (XDFI) using a variety of human tissue specimens. METHODS: The experimental setup for XDFI comprises an X-ray source, an asymmetrically cut Bragg-type monochromator-collimator (MC), a Laue-case angle analyser (LAA) and a CCD camera. The specimen is placed between the MC and the LAA. For the light source, we used the beamline BL14C on a 2.5-GeV storage ring in the KEK Photon Factory, Tsukuba, Japan. RESULTS: In the eye specimen, phase contrast images from XDFI were able to discriminate soft-tissue structures, such as the iris, separated by aqueous humour on both sides, which have nearly equal absorption. Superiority of XDFI in imaging soft tissue was further demonstrated with a diseased iliac artery containing atherosclerotic plaque and breast samples with benign and malignant tumours. XDFI on breast tumours discriminated between the normal and diseased terminal duct lobular unit and between invasive and in-situ cancer. CONCLUSIONS: X-ray phase, as detected by XDFI, has superior contrast over absorption for soft tissue processes such as atherosclerotic plaque and breast cancer. KEY POINTS: • X-ray dark field imaging (XDFI) can dramatically increase sensitivity of phase detection. • XDFI can provide enhanced soft tissue discrimination. • With XDFI, abnormal anatomy can be visualised with high spatial/contrast resolution.

Realistic wave-optics simulation of X-ray dark-field imaging at a human scale.

BACKGROUND: X-ray dark-field imaging (XDFI) has been explored to provide superior performance over the conventional X-ray imaging for the diagnosis of many pathologic conditions. A simulation tool to reliably predict clinical XDFI images at a human scale, however, is currently missing. PURPOSE: In this paper, we demonstrate XDFI simulation at a human scale for the first time to the best of our knowledge. Using the developed simulation tool, we demonstrate the strengths and limitations of XDFI for the diagnosis of emphysema, fibrosis, atelectasis, edema, and pneumonia. METHODS: We augment the XCAT phantom with Voronoi grids to simulate alveolar substructure, responsible for the dark-field signal from lungs, assign material properties to each tissue type, and simulate X-ray wave propagation through the augmented XCAT phantom using a multi-layer wave-optics propagation. Altering the density and thickness of the Voronoi grids as well as the material properties, we simulate XDFI images of normal and diseased lungs. RESULTS: Our simulation framework can generate realistic XDFI images of a human chest with normal or diseased lungs. The simulation confirms that the normal, emphysematous, and fibrotic lungs show clearly distinct dark-field signals. It also shows that alveolar fluid accumulation in pneumonia, wall thickening in interstitial edema, and deflation in atelectasis result in a similar reduction in dark-field signal. CONCLUSIONS: It is feasible to augment XCAT with pulmonary substructure and generate realistic XDFI images using multi-layer wave optics. By providing the most realistic XDFI images of lung pathologies, the developed simulation framework will enable in-silico clinical trials and the optimization of both hardware and software for XDFI.

Homeostasis of cytoplasmic crowding by cell wall fluidization and ribosomal counterions.

In bacteria, algae, fungi, and plant cells, the wall must expand in concert with cytoplasmic biomass production, otherwise cells would experience toxic molecular crowding1,2 or lyse. But how cells achieve expansion of this complex biomaterial in coordination with biosynthesis of macromolecules in the cytoplasm remains unexplained3, although recent works have revealed that these processes are indeed coupled4,5. Here, we report a striking increase of turgor pressure with growth rate in E. coli, suggesting that the speed of cell wall expansion is controlled via turgor. Remarkably, despite this increase in turgor pressure, cellular biomass density remains constant across a wide range of growth rates. By contrast, perturbations of turgor pressure that deviate from this scaling directly alter biomass density. A mathematical model based on cell wall fluidization by cell wall endopeptidases not only explains these apparently confounding observations but makes surprising quantitative predictions that we validated experimentally. The picture that emerges is that turgor pressure is directly controlled via counterions of ribosomal RNA. Elegantly, the coupling between rRNA and turgor pressure simultaneously coordinates cell wall expansion across a wide range of growth rates and exerts homeostatic feedback control on biomass density. This mechanism may regulate cell wall biosynthesis from microbes to plants and has important implications for the mechanism of action of antibiotics6.

X-ray phase tomography of a moving object.

We propose an algorithm for tomographic reconstruction of the refractive index map of an object translated across a fan-shaped X-ray beam. We adopt a forward image model valid under the non-paraxial condition, and use a unique mapping of the acquired projection images to reduce the computational cost. Even though the imaging setup affords only a limited angular coverage, our algorithm provides accurate refractive index values by employing the positivity and piecewise-smoothness constraints.

Rytov approximation for x-ray phase imaging.

In this study, we check the accuracy of the first-order Rytov approximation with a homogeneous sphere as a candidate for application in x-ray phase imaging of large objects e.g., luggage at the airport, or a human patient. Specifically, we propose a validity condition for the Rytov approximation in terms of a parameter V that depends on the complex refractive index of the sphere and the Fresnel number, for Fresnel numbers larger than 1000. In comparison with the exact Mie solution, we provide the accuracy of the Rytov approximation in predicting the intensity and phase profiles after the sphere. For large objects, where the Mie solution becomes numerically impractical, we use the principle of similarity to predict the accuracy of the Rytov approximation without explicit calculation of the Mie solution. Finally, we provide the maximum radius of the sphere for which the first order Rytov approximation remains valid within 1% accuracy.

Full-wave approach for x-ray phase imaging.

We present a rigorous forward model for phase imaging of a 3-D object illuminated by a cone-shaped x-ray beam. Our model is based on a full-wave approach valid under the first Rytov approximation, and thus can be used with large and thick objects, e.g., luggage and human patients. We unify light-matter interaction and free-space propagation into an integrated wave optics framework. Therefore, our model can accurately calculate x-ray phase images formed with sources of arbitrary shape, and it can be effectively incorporated into x-ray phase tomography as a forward model. Within the best of our knowledge, this is the first non-paraxial, full-wave model for X-ray phase imaging.

A universal mechanism of biomass density homeostasis via ribosomal counterions.

In all growing cells, the cell envelope must expand in concert with cytoplasmic biomass to prevent lysis or molecular crowding. The complex cell wall of microbes and plants makes this challenge especially daunting and it unclear how cells achieve this coordination. Here, we uncover a striking linear increase of cytoplasmic pressure with growth rate in E. coli. Remarkably, despite this increase in turgor pressure with growth rate, cellular biomass density was constant across a wide range of growth rates. In contrast, perturbing pressure away from this scaling directly affected biomass density. A mathematical model, in which endopeptidase-mediated cell wall fluidization enables turgor pressure to set the pace of cellular volume expansion, not only explains these confounding observations, but makes several surprising quantitative predictions that we validated experimentally. The picture that emerges is that changes in turgor pressure across growth rates are mediated by counterions of ribosomal RNA. Profoundly, the coupling between rRNA and cytoplasmic pressure simultaneously coordinates cell wall expansion across growth rates and exerts homeostatic feedback control on biomass density. Because ribosome content universally scales with growth rate in fast growing cells, this universal mechanism may control cell wall biosynthesis in microbes and plants and drive the expansion of ribosome-addicted tumors that can exert substantial mechanical forces on their environment.

Characterizing dry mass and volume changes in human multiple myeloma cells upon treatment with proteotoxic and genotoxic drugs.

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients; however, limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 h for sensitive cells and 4 h for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.