Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM).

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Joint Estimation of Metal Density and Attenuation Maps with Pencil Beam XFET.

X-ray fluorescence emission tomography (XFET) is an emerging imaging modality that images the spatial distribution of metal without requiring biochemical modification or radioactivity. This work investigates the joint estimation of metal and attenuation maps with a pencil-beam XFET system that allows for direct metal measurement in the absence of attenuation. Using singular value decomposition on a simplified imaging model, we show that reconstructing metal and attenuation voxels far from the detector is an ill-conditioned problem. Using simulated data, we develop and compare two image reconstruction methods for joint estimation. The first method alternates between updating the attenuation map with a separable paraboloidal surrogates algorithm and updating the metal map with a closed-form solution. The second method performs simultaneous joint estimation with conjugate gradients based on a linearized imaging model. The alternating approach outperforms the linearized approach for iron and gold numerical phantom reconstructions. Reconstructing an (8 cm)3 object containing gold concentrations of 5 mg/cm3 and an unknown beam attenuation map using the alternating approach yields an accurate gold map (NRMSE = 0.19) and attenuation map (NRMSE = 0.14). This simulation demonstrates an accurate joint reconstruction of metal and attenuation maps, from emission data, without previous knowledge of any attenuation map.

Mantis: high-throughput 4D imaging and analysis of the molecular and physical architecture of cells.

High-throughput dynamic imaging of cells and organelles is important for parsing complex cellular responses. We report a high-throughput 4D microscope, named Mantis, that combines two complementary, gentle, live-imaging technologies: remote-refocus label-free microscopy and oblique light-sheet fluorescence microscopy. We also report open-source software for automated acquisition, registration, and reconstruction, and virtual staining software for single-cell segmentation and phenotyping. Mantis enabled high-content correlative imaging of molecular components and the physical architecture of 20 cell lines every 15 minutes over 7.5 hours, and also detailed measurements of the impacts of viral infection on the architecture of host cells and host proteins. The Mantis platform can enable high-throughput profiling of intracellular dynamics, long-term imaging and analysis of cellular responses to stress, and live cell optical screens to dissect gene regulatory networks.

Spatio-angular fluorescence microscopy III. Constrained angular diffusion, polarized excitation, and high-NA imaging.

We investigate rotational diffusion of fluorescent molecules in angular potential wells, the excitation and subsequent emissions from these diffusing molecules, and the imaging of these emissions with high-NA aplanatic optical microscopes. Although dipole emissions only transmit six low-frequency angular components, we show that angular structured illumination can alias higher-frequency angular components into the passband of the imaging system. We show that the number of measurable angular components is limited by the relationships between three time scales: the rotational diffusion time, the fluorescence decay time, and the acquisition time. We demonstrate our model by simulating a numerical phantom in the limits of fast angular diffusion, slow angular diffusion, and weak potentials.

Spatio-angular fluorescence microscopy I. Basic theory.

We introduce the basic elements of a spatio-angular theory of fluorescence microscopy, providing a unified framework for analyzing systems that image single fluorescent dipoles and ensembles of overlapping dipoles that label biological molecules. We model an aplanatic microscope imaging an ensemble of fluorescent dipoles as a linear Hilbert-space operator, and we show that the operator takes a particularly convenient form when expressed in a basis of complex exponentials and spherical harmonics-a form we call the dipole spatio-angular transfer function. We discuss the implications of our analysis for all quantitative fluorescence microscopy studies and lay out a path toward a complete theory.

Spatio-angular fluorescence microscopy II. Paraxial 4f imaging.

We investigate the properties of a single-view fluorescence microscope in a 4f geometry when imaging fluorescent dipoles without using the monopole or scalar approximations. We show that this imaging system has a spatio-angular band limit, and we exploit the band limit to perform efficient simulations. Notably, we show that information about the out-of-plane orientation of ensembles of in-focus fluorophores is recorded by paraxial fluorescence microscopes. Additionally, we show that the monopole approximation may cause biased estimates of fluorophore concentrations, but these biases are small when the sample contains either many randomly oriented fluorophores in each resolvable volume or unconstrained rotating fluorophores.

Single-fluorophore orientation determination with multiview polarized illumination: modeling and microscope design.

We investigate the use of polarized illumination in multiview microscopes for determining the orientation of single-molecule fluorescence transition dipoles. First, we relate the orientation of single dipoles to measurable intensities in multiview microscopes and develop an information-theoretic metric-the solid-angle uncertainty-to compare the ability of multiview microscopes to estimate the orientation of single dipoles. Next, we compare a broad class of microscopes using this metric-single- and dual-view microscopes with varying illumination polarization, illumination numerical aperture (NA), detection NA, obliquity, asymmetry, and exposure. We find that multi-view microscopes can measure all dipole orientations, while the orientations measurable with single-view microscopes is halved because of symmetries in the detection process. We also find that choosing a small illumination NA and a large detection NA are good design choices, that multiview microscopes can benefit from oblique illumination and detection, and that asymmetric NA microscopes can benefit from exposure asymmetry.

Improved deconvolution of very weak confocal signals.

Deconvolution is typically used to sharpen fluorescence images, but when the signal-to-noise ratio is low, the primary benefit is reduced noise and a smoother appearance of the fluorescent structures. 3D time-lapse (4D) confocal image sets can be improved by deconvolution. However, when the confocal signals are very weak, the popular Huygens deconvolution software erases fluorescent structures that are clearly visible in the raw data. We find that this problem can be avoided by prefiltering the optical sections with a Gaussian blur. Analysis of real and simulated data indicates that the Gaussian blur prefilter preserves meaningful signals while enabling removal of background noise. This approach is very simple, and it allows Huygens to be used with 4D imaging conditions that minimize photodamage.

Cost-effectiveness Analysis of Vascular Access Referral Policies in CKD.

BACKGROUND: The optimal timing of vascular access referral for patients with chronic kidney disease who may need hemodialysis (HD) is a pressing question in nephrology. Current referral policies have not been rigorously compared with respect to costs and benefits and do not consider patient-specific factors such as age. STUDY DESIGN: Monte Carlo simulation model. SETTING & POPULATION: Patients with chronic kidney disease, referred to a multidisciplinary kidney clinic in a universal health care system. MODEL, PERSPECTIVE, & TIMEFRAME: Cost-effectiveness analysis, payer perspective, lifetime horizon. INTERVENTION: The following vascular access referral policies are considered: central venous catheter (CVC) only, arteriovenous fistula (AVF) or graft (AVG) referral upon HD initiation, AVF (or AVG) referral when HD is forecast to begin within 12 (or 3 for AVG) months, AVF (or AVG) referral when estimated glomerular filtration rate is <15 (or <10 for AVG) mL/min/1.73m2. OUTCOMES: Incremental cost-effectiveness ratios (ICERs, in 2014 US dollars per quality-adjusted life-year [QALY] gained). RESULTS: The ICER of AVF (AVG) referral within 12 (3) months of forecasted HD initiation, compared to using only a CVC, is ∼$105k/QALY ($101k/QALY) at a population level (HD costs included). Pre-HD AVF or AVG referral dominates delaying referral until HD initiation. The ICER of pre-HD referral increases with patient age. Results are most sensitive to erythropoietin costs, ongoing HD costs, and patients' utilities for HD. When ongoing HD costs are excluded from the analysis, pre-HD AVF dominates both pre-HD AVG and CVC-only policies. LIMITATIONS: Literature-based estimates for HD, AVF, and AVG utilities are limited. CONCLUSIONS: The cost-effectiveness of vascular access referral is largely driven by the annual costs of HD, erythropoietin costs, and access-specific utilities. Further research is needed in the field of dialysis-related quality of life to inform decision making regarding vascular access referral.