Sub-10-nm imaging of nucleic acids using spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON).

Elucidating chromatin structure in vitro requires resolution below 10 nm to visualize the mononucleosome has been an ongoing challenge. In this work, we achieve sub-10-nm imaging of nucleic acids via spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON) without the use of external labels. SICLON leverages two key innovations: using endogenous nucleotides as the emission source and a custom-made imaging system that can simultaneously record the position and optical spectra of emitting molecules. With a novel spectral regression algorithm that identifies the spectroscopic fingerprints of neighboring molecules that were previously indistinguishable, we demonstrate the utility of SICLON by visualizing unlabeled poly-nucleotides and linear single-stranded DNA fibers with a resolution of 6.2 nm.

Stage-Rocked Electron Channeling for Crystal Orientation Mapping.

Microstructural analysis by crystal orientation mapping of bulk functional materials is an essential and routine operation in the engineering of material properties. Far and away the most successfully employed technique, Electron Backscattered Diffraction (EBSD), provides high spatial resolution information at the cost of limited angular resolution and a distorted imaging condition. In this work, we demonstrate a stage-rocked electron channeling approach as a low-cost orientation mapping alternative to EBSD. This is accomplished by automated electron channeling contrast imaging (ECCI) as the microscope stage physically tilts/rotates a sample through a reduced hemisphere of orientations followed by computational reconstruction of electron channeling patterns (ECP). Referred to as Orientation Mapping by Electron Channeling (OMEC), our method offers advantages in terms of local defect analysis, as it combines the advantages of selected area ECP (SACP) and ECCI. We also illustrate dynamic or "adaptive" sampling schemes to increase the throughput of the technique. Finally, we discuss the implications for sample analysis in which large 3D maps of ECCI images can be routinely constructed of challenging crystalline samples. As an electron channeling-based approach to orientation mapping, OMEC may open new routes to characterize crystalline materials with high angular and spatial resolution.

High speed/low dose analytical electron microscopy with dynamic sampling.

Technological advances in electron microscopy, particularly improved detectors and aberration correctors, have led to higher throughput and less invasive imaging of materials and biological structures by enhancing signal-to-noise ratios at lower electron exposures. Analytical methods, such as electron energy loss spectroscopy (EELS) and energy dispersive x-ray spectrometry (EDS), have also benefitted and offer a rich set of local elemental and bonding information with nano-or atomic resolution. However, spatially resolved spectrum imaging with EELS and EDS continue to be difficult to scale due to limited detector collection angles or high signal background, requiring hours or even days for full maps. We present the principle and application of a Multi-Objective Autonomous Dynamic Sampling (MOADS) method which can accelerate spectrum mapping in EELS or EDS by over an order of magnitude. Initial guesses about the true spectrum images are constructed as measurements are collected, which allows the prediction of points which contribute information/contrast. In this fashion, an intelligently selected and reduced set of points which best approximate the true spectrum image are autonomously collected on-the-fly to save considerable time and/or radiative area dose. We implemented MOADS as a software add-on to arbitrary commercial Scanning Transmission Electron Microscopes (STEMs) equipped with a Gatan Digital Micrograph (DM, Gatan ©) interface. We demonstrate the efficacy of our proposed method on several prototypical analytical specimens, as well as dose sensitive materials. It is expected that MOADS and similar supervised dynamic sampling approaches may open the exploration of large area analytical maps or the imaging of beam reactive materials not previously thought feasible.

The effects of chemical fixation on the cellular nanostructure.

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.

Measuring the Autocorrelation Function of Nanoscale Three-Dimensional Density Distribution in Individual Cells Using Scanning Transmission Electron Microscopy, Atomic Force Microscopy, and a New Deconvolution Algorithm.

Essentially all biological processes are highly dependent on the nanoscale architecture of the cellular components where these processes take place. Statistical measures, such as the autocorrelation function (ACF) of the three-dimensional (3D) mass-density distribution, are widely used to characterize cellular nanostructure. However, conventional methods of reconstruction of the deterministic 3D mass-density distribution, from which these statistical measures can be calculated, have been inadequate for thick biological structures, such as whole cells, due to the conflict between the need for nanoscale resolution and its inverse relationship with thickness after conventional tomographic reconstruction. To tackle the problem, we have developed a robust method to calculate the ACF of the 3D mass-density distribution without tomography. Assuming the biological mass distribution is isotropic, our method allows for accurate statistical characterization of the 3D mass-density distribution by ACF with two data sets: a single projection image by scanning transmission electron microscopy and a thickness map by atomic force microscopy. Here we present validation of the ACF reconstruction algorithm, as well as its application to calculate the statistics of the 3D distribution of mass-density in a region containing the nucleus of an entire mammalian cell. This method may provide important insights into architectural changes that accompany cellular processes.

Flexible ferroelectric organic crystals.

Flexible organic materials possessing useful electrical properties, such as ferroelectricity, are of crucial importance in the engineering of electronic devices. Up until now, however, only ferroelectric polymers have intrinsically met this flexibility requirement, leaving small-molecule organic ferroelectrics with room for improvement. Since both flexibility and ferroelectricity are rare properties on their own, combining them in one crystalline organic material is challenging. Herein, we report that trisubstituted haloimidazoles not only display ferroelectricity and piezoelectricity-the properties that originate from their non-centrosymmetric crystal lattice-but also lend their crystalline mechanical properties to fine-tuning in a controllable manner by disrupting the weak halogen bonds between the molecules. This element of control makes it possible to deliver another unique and highly desirable property, namely crystal flexibility. Moreover, the electrical properties are maintained in the flexible crystals.