G.Y. Fan

Digital imaging in transmission electron microscopy

The digital revolution currently under way, as evidenced by the rapid development of the Internet and the world‐wide‐web technologies, is undoubtedly impacting the field of transmission electron microscopy (TEM). Digital imaging systems based on charge‐coupled device (CCD) technologies, with pixel array size up to 2 k × 2 k at the present and increasing, are available for TEM applications and offer many attractions. Is it time to phase out film cameras on TEMs and close the darkrooms for good? This paper reviews digital imaging technologies for TEM at different voltages, and contrasts the performance of digital imaging systems with that of TEM film. The performance characteristics of CCD‐based digital imaging systems, as well as methods for assessing them, are discussed. Other approaches to digital imaging are also briefly reviewed.

Implementing a Collaboratory for Microscopic Digital Anatomy

The Collaboratory for Microscopic Digital Anatomy (CMDA) will allow biologists at remote locations to acquire over the network three-dimensional data on biological structure using a sophisticated high-voltage electron mi croscope (HVEM) located in San Diego. The CMDA will transparently distribute to high-performance machines on the network computationally intensive tasks for the deriva tion of three-dimensional datasets from HVEM images using tomography and for volume visualization. Implemen tation of the CMDA includes development of automated microscope functions for specimen positioning, image fo cus, registration, and acquisition; an asynchronous com munications system for transporting messages and large data sets between processes; a resource manager for access control and routing of tasks to computing or acqui sition resources; and systems for tomographic reconstruc tion and three-dimensional volume visualization. The CMDA is designed to be extensible to other unique cen tralized instruments. It will increase access to and use of these instruments and high-performance computers by biologists and facilitate remote collaboration.

High-sensitivity lens-coupled slow-scan CCD camera for transmission electron microscopy

A lens-coupled slow-scan CCD camera has been built for transmission electron microscopy (TEM) applications. In this design, a leaded glass window, which is coated with a 20 microns layer of red P20 phosphor, is mounted on the bottom of the microscope. A lens assembly and mirror prism, located outside the microscope vacuum below the leaded glass, relays the image onto a back-thinned 1kx1k charge-coupled device (CCD) detector. This CCD is electronically cooled to below -30 degrees C during operation. It is found that X-ray irradiation, generally found to be annoying in fiber-optically coupled CCD cameras, is completely eliminated by this configuration. The collection efficiency of this system, although not as high as some of the fiber-optically coupled CCD cameras, is high enough to achieve single-electron sensitivity under a high-gain mode.

ASIC-based event-driven 2D digital electron counter for TEM imaging

A two-dimensional application specific integrated circuit (ASIC) based detector, designed for X-ray protein crystallography, has been tested to determine its suitability as a direct electron detector for TEM imaging in the voltage range of 20-400 keV. Several markedly different properties of this device distinguish it from the charge coupled device (CCD) detectors: (1) the ASIC detector can be used directly under electron bombardment in the voltage range stated above, therefore requiring no scintillator screen; (2) each active pixel of the device is an electron counter and generates digital output independently; (3) the readout of the device is frameless and event driven; (4) the device can be operated at the room temperature and is nearly noise free; and (5) the counting dynamic range of the device is virtually unlimited. It appears that an imaging system based on this type of device would be ideal for low-dose TEM imaging and online diffraction observation and recording, as well as more conventional imaging, providing the many advantages of direct digital readout for almost all applications.

Multiport-readout frame-transfer 5 megapixel CCD imaging system for TEM applications

A multiport-readout, frame-transfer charge-coupled device (CCD) digital imaging system has been successfully developed and tested for intermediate-high-voltage electron microscopy (IVEM) applications up to 400 keV. The system employs a back-thinned CCD with 2560 x 1960 pixels and a pixel size of 24 microm x 24 microm. In the current implementation, four of the eight on-chip readout ports are used in parallel each operating at a pixel rate of 1- or 2-MHz so that the entire CCD array can be read out in as short as 0.6 s. The frame-transfer readout functions as an electronic shutter which permits the rapid transfer of charges in the active pixels to four masked buffers where the charges are readout and digitized while the active area of the CCD is integrating the next frame. With a thin film-based phosphor screen and a high-performance lens relay, the system has a conversion factor of 2.1 digital units per incident electron at 400 keV, and a modulation transfer function value of 14% at the Nyquist frequency.

Optimization of thin-foil based phosphor screens for CCD imaging in TEM in the voltage range of 80–400 kV

The voltage dependence characteristics of thin-foil based phosphor screens in the thickness range of approximately 10-60 microns are examined for CCD imaging in transmission electron microscopy (TEM) in the voltage range of 80-400 kV. The brightest screen is obtained with a P20 layer of about 12 microns at 80 kV, and a thicker screen lowers both the screen brightness and resolution. The thickness of the brightest screen is higher at higher voltage, but other considerations for a practical CCD imaging system suggest that the P20 layer should not be greater than approximately 18 microns for the voltage range stated above.

In vivo calcineurin crystals formed using the baculovirus expression system

Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen‐activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses. Although these in vivo crystals are low in population, ranging only 0–3 per cell, they are extremely large, over 10 μm in some cases. Biochemical assays confirm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoylated forms are expressed. The lattice structure of the in vivo crystals, with a spacing of 5.5 nm, is preserved with the regular electron microscopic (EM) specimen preparation procedure.

Performance of thin foil scintillating screen for transmission electron microscopy.

Scintillating screens made by coating a thin metal foil with a layer of phosphor appear to be attractive for transmission electron microscopy applications. We report here the brightness and resolution in the voltage range of 100-400 kV of such a screen made of a 10 microns layer of P20 phosphor on a 2 microns Al foil. Both brightness and resolution are superior to that of a screen made by coating a glass plate with a similar layer of phosphor. An exciting property of such a device is that its resolution improves slightly at higher voltages. This, combined with its excellent resistivity to radiation damage and stability under the electron beam, makes it a good candidate for high-voltage applications.

Stereoscopy by tilted illumination in transmission electron microscopy

We describe a new technique for stereoscopic observation in transmission electron microscopy, employing tilted illumination. A triple-hole objective aperture is used so that the bright beam can pass through with or without tilt. Stereo views can be acquired by tilting the illumination such that the bright beam passes through a pair of symmetrically arranged apertures alternately. The advantages of this technique as compared to the commonly used method of single-axis tilt are: (i) greater speed, potentially at or close to video rate so that live 3D observation is possible; (ii) elimination of specimen movement associated with stage tilt; and (iii) perspective views corresponding to multiple tilting axes which can be realized by installing more aperture-holes and choosing their positions properly. The main limitation is that the angle of tilt is limited by the extent to which the astigmatism, introduced by beam tilt, can be compensated by the objective lens stigmators.