James C. Bouwer

Automated microscopy system for mosaic acquisition and processing.

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide‐field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast‐scanning, multiphoton, confocal microscope fitted with a motorized, high‐precision stage and custom‐developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.

Characterization of a direct detection device imaging camera for transmission electron microscopy.

The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics.

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJs could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.

First use of a high-sensitivity active pixel sensor array as a detector for electron microscopy

There is an urgent need to replace film and CCD cameras as recording instruments for transmission electron microscopy (TEM). Film is too cumbersome to process and CCD cameras have low resolution, marginal to poor signal-to-noise ratio for single electron detection and high spatial distortion. To find a replacement device, we have tested a high sensitivity active pixel sensor (APS) array currently being developed for nuclear physics. The tests were done at 120 keV in a JEOL 1200 electron microscope. At this energy, each electron produced on average a signal-tonoise ratio about 20/1. The spatial resolution was also excellent with the full width at half maximum (FWHM) about 20 microns. Since it is very radiation tolerant and has almost no spatial distortion, the above tests showed that a high sensitivity CMOS APS array holds great promise as a direct detection device for electron microscopy.

Future directions for camera systems in electron microscopy.

Publisher Summary Charge-coupled device (CCD) invented in 1970, soon became the sensor of choice in many imaging applications, particularly for video cameras and camcorders. This chapter reviews current efforts to scale up lens-coupled CCD camera and make a system capable of exceeding the spatial resolution of film, while maintaining single-electron sensitivity. This lens-coupled CCD system represents the current state-of-the-art in CCD-based systems, and it also demonstrates the great engineering effort required to achieve these key performance benchmarks when the detector is based on a resolution-limiting scintillation screen. The chapter discusses the development of a parallel effort to produce a radiation-tolerant system that can withstand direct electron bombardment. It also describes efforts required to adapt the pixel array detector (PAD) that is commonly used in X-ray diffraction, and discusses the development of a groundbreaking prototype system based on an active pixel sensor (APS). This early implementation of an APS-based direct detection detector (DDD) has already delivered unprecedented performance in many areas exceeding the fundamental capabilities of CCD-based systems.

Improving Signal to Noise in Labeled Biological Specimens using Energy-Filtered TEM of sections with a Drift Correction Strategy and a Direct Detection Device

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDDs) to increase the signal to noise as compared with CCDs. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.

A new direct detection camera system for electron microscopy

High resolution electron imaging is very important in nanotechnology and biotechnology fields. For example, Cryogenic Electron-Microscopy is a promising method to obtain 3-D structures of large protein complexes and viruses. We report on the design and measurements of a new CMOS direct-detection camera system for electron imaging. The active pixel sensor array that we report on includes 512 by 550 pixels, each 5 by 5 μm in size, with an ~8 μm epitaxial layer to achieve an effective fill factor of 100%. Spatial resolution of 2.3 μm for a single incident e- has been measured. Electron microscope tests have been performed with 200 and 300 keV beams, and the first recorded Electron Microscope image is presented.

The application of energy-filtered electron microscopy to tomography of thick, selectively stained biological samples.

Publisher Summary This chapter describes the use of energy-filtered electron microscopy (EM) to enhance contrast and reduce the chromatic aberration in the imaging of thick, selectively stained specimens. For thick specimens, the resolution of electron microscopy is severely limited by chromatic aberration that results from the inability of current electron lenses to deal uniformly with beam electrons that have a distribution of energies as a result of inelastic scattering in the samples. The chapter describes a technique of automated, most-probable loss (MPL) tomography. It shows that for thick, selectively stained biological specimens, this method produces a dramatic increase in the resolution of the projected images. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. In addition, MPL tomography effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by improving resolution relative to unfiltered transmission electron microscopy (TEM).

The intermediate size direct detection detector for electron microscopy

In a longstanding effort to overcome limits of film and the charge coupled device (CCD) systems in electron microscopy, we have developed a radiation-tolerant system that can withstand direct electron bombardment. A prototype Direct Detection Device (DDD) detector based on an Active Pixel Sensor (APS) has delivered unprecedented performance with an excellent signal-to-noise ratio (approximately 5/1 for a single incident electron in the range of 200-400 keV) and a very high spatial resolution. This intermediate size prototype features a 512×550 pixel format of 5&mgr;m pitch. The detector response to uniform beam illumination and to single electron hits is reported. Radiation tolerance with high-energy electron exposure is also impressive, especially with cooling to -15 °C. Stable performance has been demonstrated, even after a total dose of 3.3×106 electrons/pixel. The characteristics of this new detector have exciting implications for transmission electron microscopy, especially for cryo-EM as applied to biological macromolecules.

Prototype of Kepler Processing Workflows For Microscopy And Neuroinformatics

We report on progress of employing the Kepler workflow engine to prototype “end-to-end” application integration workflows that concern data coming from microscopes deployed at the National Center for Microscopy Imaging Research (NCMIR). This system is built upon the mature code base of the Cell Centered Database (CCDB) and integrated rule-oriented data system (IRODS) for distributed storage. It provides integration with external projects such as the Whole Brain Catalog (WBC) and Neuroscience Information Framework (NIF), which benefit from NCMIR data. We also report on specific workflows which spawn from main workflows and perform data fusion and orchestration of Web services specific for the NIF project. This “Brain data flow” presents a user with categorized information about sources that have information on various brain regions.