Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells

Anyone who has used a light microscope has wished that its resolution could be a little better. Now, after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology

Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines

The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2–3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed

System and method for confining an object to a region of fluid flow having a stagnation point

A device for confining an object to a region proximate to a fluid flow stagnation point includes one or more inlets for carrying the fluid into the region, one or more outlets for carrying the fluid out of the region, and a controller, in fluidic communication with the inlets and outlets, for adjusting the motion of the fluid to produce a stagnation point in the region, thereby confining the object to the region. Applications include, for example, prolonged observation of the object, manipulation of the object, etc. The device optionally may employ a feedback control mechanism, a sensing apparatus (e.g., for imaging), and a storage medium for storing, and a computer for analyzing and manipulating, data acquired from observing the object. The invention further provides methods of using such a device and system in a number of fields, including biology, chemistry, physics, material science, and medical science

Developmental mechanism of the periodic membrane skeleton in axons

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites. DOI: http://dx.doi.org/10.7554/eLife.04581.00

Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities—2D, astigmatic 3D, biplane 3D and double-helix 3D—and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field

3D Multicolor Super-Resolution Imaging Offers Improved Accuracy in Neuron Tracing

The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density

Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS cameras

This work explores the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that industrial grade CMOS cameras approach the performance of scientific grade CMOS cameras at a fraction of the cost. This makes it more economically feasible to construct high-performance imaging systems with multiple cameras that are capable of a diversity of applications. In particular we demonstrate the use of industrial CMOS cameras for biplane, multiplane and spectrally resolved SMLM. We also provide open-source software for simultaneous control of multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images

W.B. Hazen, headquarters, 15th Army Corps, to Captain Heath, commanding Pioneer, 2nd Division

Hazen introduces a man (name illegible) who Hazen wants Heath to employ in the Pioneer Corps.Heath, Alfred H.1860s (1860-1869)Washington (D.C.)600ppiCivil War Military FrontDC046This Civil War Military Front collection was funded by LSTA

W.B. Hazen

Hazen asks for a report regarding the contraband men he sent to Ohio.[Heath, Alfred H.]1860s (1860-1869)Louisville (Ky.)600ppiCivil War Military FrontDC046This Civil War Military Front collection was funded by LSTA