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.

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.

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.

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.

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.

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.

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.

Deep-learning-assisted Fourier transform imaging spectroscopy for hyperspectral fluorescence imaging.

Hyperspectral fluorescence imaging is widely used when multiple fluorescent probes with close emission peaks are required. In particular, Fourier transform imaging spectroscopy (FTIS) provides unrivaled spectral resolution; however, the imaging throughput is very low due to the amount of interferogram sampling required. In this work, we apply deep learning to FTIS and show that the interferogram sampling can be drastically reduced by an order of magnitude without noticeable degradation in the image quality. For the demonstration, we use bovine pulmonary artery endothelial cells stained with three fluorescent dyes and 10 types of fluorescent beads with close emission peaks. Further, we show that the deep learning approach is more robust to the translation stage error and environmental vibrations. Thereby, the He-Ne correction, which is typically required for FTIS, can be bypassed, thus reducing the cost, size, and complexity of the FTIS system. Finally, we construct neural network models using Hyperband, an automatic hyperparameter selection algorithm, and compare the performance with our manually-optimized model.

Hyperspectral three-dimensional absorption imaging using snapshot optical tomography.

Hyperspectral imaging (HSI) records a series of two-dimensional (2D) images for different wavelengths to provide the chemical fingerprint at each pixel. Combining HSI with a tomographic data acquisition method, we can obtain the chemical fingerprint of a sample at each point in three-dimensional (3D) space. The so-called 3D HSI typically suffers from low imaging throughput due to the requirement of scanning the wavelength and rotating the beam or sample. In this paper we present an optical system which captures the entire four-dimensional (4D), i.e., 3D structure and 1D spectrum, dataset of a sample with the same throughput of conventional HSI systems. Our system works by combining snapshot projection optical tomography (SPOT) which collects multiple projection images with a single snapshot, and Fourier-transform spectroscopy (FTS) which results in superior spectral resolution by collecting and processing a series of interferogram images. Using this hyperspectral SPOT system we imaged the volumetric absorbance of dyed polystyrene microbeads, oxygenated red blood cells (RBCs), and deoxygenated RBCs. The 4D optical system demonstrated in this paper provides a tool for high-throughput chemical imaging of complex microscopic specimens.

Tomography of dark-field scatter including single-exposure Moiré fringe analysis with X-ray biprism interferometry-A simulation study.

PURPOSE: In this work, we present tomographic simulations of a new hardware concept for X-ray phase-contrast interferometry wherein the phase gratings are replaced with an array of Fresnel biprisms, and Moiré fringe analysis is used instead of "phase stepping" popular with grating-based setups. METHODS: Projections of a phantom consisting of four layers of parallel carbon microfibers is simulated using wave optics representation of X-ray electromagnetic waves. Simulated projections of a phantom with preferential scatter perpendicular to the direction of the fibers are performed to analyze the extraction of small-angle scatter from dark-field projections for the following: (1) biprism interferometry using Moiré fringe analysis; (2) grating interferometry using phase stepping with eight grating steps; and (3) grating interferometry using Moiré fringe analysis. Dark-field projections are modeled as projections of voxel intensities represented by a fixed finite vector basis set of scattering directions. An iterative MLEM algorithm is used to reconstruct, from simulated projection data, the coefficients of a fixed set of seven basis vectors at each voxel representing the small-angle scatter distribution. RESULTS: Results of reconstructed vector coefficients are shown comparing the three simulated imaging configurations. The single-exposure Moiré fringe analysis shows not only an increase in noise because of one-eighth the number of projection samples but also is obtained with less dose and faster acquisition times. Furthermore, replacing grating interferometry with biprism interferometry provides better contrast-to-noise. CONCLUSION: The simulations demonstrate the feasibility of the reconstruction of dark-field data acquired with a biprism interferometry system. With the potential of higher fringe visibility, biprism interferometry with Moiré fringe analysis might provide equal or better image quality to that of phase stepping methods with less imaging time and lower dose.

Snapshot three-dimensional absorption imaging of microscopic specimens.

Snapshot projection optical tomography (SPOT) uses a micro-lens array (MLA) to simultaneously capture the projection images of a three-dimensional (3D) specimen corresponding to different viewing directions. Compared to other light-field imaging techniques using an MLA, SPOT is dual telecentric and can block high-angle stray rays without sacrificing the light collection efficiency. Using SPOT, we recently demonstrated snapshot 3D fluorescence imaging. Here we demonstrate snapshot 3D absorption imaging of microscopic specimens. For the illumination, we focus the incoherent light from a light-emitting diode onto a pinhole, which is placed at a conjugate plane to the sample plane. SPOT allows us to capture the ray bundles passing through the specimen along different directions. The images recorded by an array of lenslets can be related to the projections of 3D absorption coefficient along the viewing directions of lenslets. Using a tomographic reconstruction algorithm, we obtain the 3D map of absorption coefficient. We apply the developed system to different types of samples, which demonstrates the optical sectioning capability. The transverse and axial resolutions measured with gold nanoparticles are 1.3 µm and 2.3 µm, respectively.

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.

Wave optics simulation of grating-based X-ray phase-contrast imaging using 4D Mouse Whole Body (MOBY) phantom.

PURPOSE: Demonstrate realistic simulation of grating-based x-ray phase-contrast imaging (GB-XPCI) using wave optics and the four-dimensional Mouse Whole Body (MOBY) phantom defined with non-uniform rational B-splines (NURBS). METHODS: We use a full-wave approach, which uses wave optics for x-ray wave propagation from the source to the detector. This forward imaging model can be directly applied to NURBS-defined numerical phantoms such as MOBY. We assign the material properties (attenuation coefficient and electron density) of each model part using the data for adult human tissues. The Poisson noise is added to the simulated images based on the calculated photon flux at each pixel. RESULTS: We simulate the intensity images of the MOBY phantom for eight different grating positions. From the simulated images, we calculate the absorption, differential phase, and normalized visibility contrast images. We also predict how the image quality is affected by different exposure times. CONCLUSIONS: GB-XPCI can be simulated with the full-wave approach and a realistic numerical phantom defined with NURBS.

Improving contrast and spatial resolution in crystal analyzer-based x-ray dark-field imaging: Theoretical considerations and experimental demonstration.

PURPOSE: This paper describes and experimentally validates a methodology for improving contrast and spatial resolution of the x-ray dark-field imaging (XDFI) by cutting the monochromator-collimator asymmetrically and thinning the Laue angle analyzer. METHODS: We measure the spatial resolution of our XDFI setup using a test object consisting of wolfram tungsten meshes and compare it with the theoretical prediction. Using x-ray dark-field computed tomography of breast cancer specimens (lobular carcinoma in situ), we demonstrate that the resolution of XDFI is sufficient for histopathologic analysis. RESULTS: Our experimental results show that the overall spatial resolution of XDFI can be improved by approximately a factor of 2 when these modifications are implemented. The reconstructed images of breast cancer specimens provide sufficient details for radiologic histopathology. CONCLUSIONS: By cutting the monochromator-collimator and thinning the Laue angle analyzer, XDFI can achieve the resolution sufficient for radiologic histopathology.

Dependence of fluorodeoxyglucose (FDG) uptake on cell cycle and dry mass: a single-cell study using a multi-modal radiography platform.

High glucose uptake by cancer compared to normal tissues has long been utilized in fluorodeoxyglucose-based positron emission tomography (FDG-PET) as a contrast mechanism. The FDG uptake rate has been further related to the proliferative potential of cancer, specifically the proliferation index (PI) - the proportion of cells in S, G2 or M phases. The underlying hypothesis was that the cells preparing for cell division would consume more energy and metabolites as building blocks for biosynthesis. Despite the wide clinical use, mixed reports exist in the literature on the relationship between FDG uptake and PI. This may be due to the large variation in cancer types or methods adopted for the measurements. Of note, the existing methods can only measure the average properties of a tumor mass or cell population with highly-heterogeneous constituents. In this study, we have built a multi-modal live-cell radiography system and measured the [18F]FDG uptake by single HeLa cells together with their dry mass and cell cycle phase. The results show that HeLa cells take up twice more [18F]FDG in S, G2 or M phases than in G1 phase, which confirms the association between FDG uptake and PI at a single-cell level. Importantly, we show that [18F]FDG uptake and cell dry mass have a positive correlation in HeLa cells, which suggests that high [18F]FDG uptake in S, G2 or M phases can be largely attributed to increased dry mass, rather than the activities preparing for cell division. This interpretation is consistent with recent observations that the energy required for the preparation of cell division is much smaller than that for maintaining house-keeping proteins.

Snapshot projection optical tomography.

We present a new plenoptic microscopy configuration for 3D snapshot imaging, which is dual telecentric and can directly record true projection images corresponding with different viewing angles. It also allows blocking high-angle stray rays without sacrificing the light collection efficiency. This configuration named as snapshot projection optical tomography (SPOT) arranges an objective lens and a microlens array (MLA) in a 4-f telecentric configuration and places an aperture stop at the back focal plane of a relay lens. We develop a forward imaging model for SPOT, which can also be applied to existing light field microscopy techniques using an MLA as tube lens. Using the developed system, we demonstrate snapshot 3D imaging of various fluorescent beads and a biological cell, which confirms the capability of SPOT for imaging specimens with an extended fluorophore distribution as well as isolated fluorochromes. The transverse and vertical resolutions are measured to be 0.8 µm and 1.6 µm, respectively.

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.

Phase-contrast imaging with a compact x-ray light source: system design.

X-ray phase-contrast imaging (XPCI) overcomes the problem of low contrast between different soft tissues achieved in conventional x-ray imaging by introducing x-ray phase as an additional contrast mechanism. This work describes a compact x-ray light source (CXLS) and compares, via simulations, the high quality XPCI results that can be produced from this source to those produced using a microfocus x-ray source. The simulation framework is first validated using an image acquired with a microfocus-source, propagation-based XPCI (PB-XPCI) system. The phase contrast for a water sphere simulating a simple cyst submersed in muscle is evaluated and the evolution of PB-XPCI signal as the object to detector distance is increased is demonstrated. The proposed design of a PB-XPCI system using the CXLS is described and simulated images of a coronary artery compared between CXLS and microfocus source PB-XPCI systems. To generate images with similar noise levels, a microfocus source would require a 3000 times longer exposure than would the CXLS. We conclude that CXLS technology has the potential to provide high-quality XPCI in a medical environment using extremely short exposure times relative to microfocus source approaches.

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.