James B. Pawley

Fundamental Limits in Confocal Microscopy

The previous chapter described how the confocal approach developed from conventional light microscopy and outlined the basic advantages gained by the use of confocal sampling techniques, primarily that the exclusion of light by the confocal pinhole makes it possible to record data from optical sections. This chapter will discuss the fundamental considerations that limit the performance of all confocal microscopes. Though at present no commercially available equipment approaches these limits, some simple tests will be described to help the user assess how well a given instrument performs. Additional information to help the user to operate the confocal microscope in an optimal manner can be found in Chapter 35, “ATutorial on Confocal Microscopy,” and Chapter 36, “Practical Confocal Microscopy.” These also include methods for measuring resolution and other useful parameters.

Low voltage scanning electron microscopy

The scanning electron microscope (SEM) is usually operated with a beam voltage, V0, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V0=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V0 to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de‐localized nature of the inelastic scattering event and radiation damage are also discussed.

High-resolution scanning electron microscopy

The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal. Any practical consideration of the high-resolution performance of the SEM must therefore also involve a knowledge of the contrast available from the signal producing the image and the radiation sensitivity of the specimen. With state-of-the-art electron optics, resolutions of the order of 1 nm are now possible. The optimum conditions for achieving such performance with the minimum radiation damage to the specimen correspond to beam energies in the range 1-3 keV. Progress beyond this level may be restricted by the delocalization of SE production and ultimate limits to electron-optical performance.

Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen

The other chapters in this book give the reader an in-depth description of every important aspect of biological confocal microscopy that we could think of. This chapter is to provide the novice user of this instrument with a basic understanding of the practical information needed to use it effectively. Because the computer interfaces of the various commercial instruments vary greatly, this chapter will stress the important features of microscopical optics and the basics of sampling that are common to all instruments.

Points, Pixels, and Gray Levels: Digitizing Image Data

Microscopical images are now almost always recorded digitally. To accomplish this, the flux of photons that forms the final image must be divided into small geometrical subunits called pixels. The light intensity in each pixel will be stored as a single number. Changing the objective magnification, the zoom magnification on your confocal control panel, or choosing another coupling tube magnification for your charge-coupled device (CCD) camera changes the size of the area on the object that is represented by one pixel. If you can arrange matters so that the smallest feature recorded in your image data is at least 4 to 5 pixels wide in each direction, then all is well.

Cryo-crinkling : what happens to carbon films on copper grids at low temperature

A study of the surface flatness of carbon films on copper grids used for cryo-electron microscopy has been carried out using a Hitachi S-900 low-voltage SEM. Dramatic changes in flatness were observed after cooling from room temperature to -170 degrees C. The changes were similar both for carbon films that had been floated from a mica surface and for those initially deposited on the surface of plastic films. Results demonstrate that films prepared on copper grids that appear flat at room temperature become extensively, but reversibly, puckered at -170 degrees C. The linear thermal expansion coefficient (alpha) for copper is 16.2 x 10(-6)/degrees C and the puckering can be explained by assuming that the coefficient for amorphous carbon is substantially less. Measurements on grids made of titanium, molybdenum and tungsten (coefficients 8.5, 5 and 4.5 x 10(-6)/degrees C, respectively) showed significantly less puckering.

Disk-Scanning Confocal Microscopy

Rapid biological imaging of faint fluorophores in living cells — especially in four dimensions [three dimensions + time] — imposes different instrumentation challenges from slowly acquiring a single high-resolution confocal snapshot of fixed tissue. High acquisition speeds with acceptable contrast and minimal photobleaching suddenly become essential, all without losing the instantaneous optical sectioning that a confocal microscope affords. Of particular interest here, disk-scanning confocal microscopes are proving to be a powerful tool in rapid imaging of live cells in space and time. While the principle is relatively old, new instrument developments, both in optics and in novel ultra-sensitive chargecoupled device (CCD) cameras are greatly expanding the versatility and scope of this approach. However, knowing which system is best for a given question and understanding the inherent strengths and weaknesses is not easy, especially as many of the choices involve complex trade-offs between resolution, speed, sensitivity, 1 and signal-to-noise ratio (S/N). The goal of this chapter is to provide both a theoretical and a practical guide, with more emphasis on the latter especially when theoretical considerations are covered elsewhere (Chapters 22, 23, and 34, this volume). Here the relative merits, strengths, and weaknesses of using a disk-scanning confocal microscope for biological imaging will be examined and sample applications shown.

LVSEM for High Resolution Topographic and Density Contrast Imaging

Publisher Summary The chapter discusses some of the contrast mechanisms that contribute to the image in these instruments. The most recent generation of low-voltage scanning electron microscopes yields resolutions well below one nanometer thanks to the use of field-emission guns. This chapter also considers aspects of scanning electron microscopes (SEM) design and operation that affect the contrast and resolution of the secondary emission (SE) and the backscattering (BSE) image. The SEMs developed and used world-wide since that time owe most of their important features to early developments by this group. An SE image of a rough microscopic specimen can be easily and accurately interpreted in terms of its topographic shape. The total BSE signal is a strong function of the density of the specimen under the beam, and BSE images are therefore primarily two dimensional maps of material density versus position. In addition, as the image information is carried as a time-varying electronic signal, a variety of analog and digital signal processing procedures can easily be applied to this signal to emphasize the particular aspects of it that are of interest to the viewer.

High-resolution scanning electron microscopy of frozen-hydrated cells

Cryo‐fixed yeast Paramecia and sea urchin embryos were investigated with an in‐lens type field‐emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low‐temperature scanning electron microscope (LTSEM).

Freeze‐fracture of 3T3 cells for high‐resolution scanning electron microscopy

Triton‐extracted, freeze‐fractured 3T3 cells have been examined in the Hitachi S‐900 field‐emission SEM, after light platinum coating, at low beam voltage to evaluate the performance of the microscope under these conditions. For unstained material fixed in glutaraldehyde alone, high‐resolution images can be obtained, at accelerating voltages of 1.5‐5kV, after rotary deposition of platinum to an average thickness of 1.5‐3nm. Comparisons are made between these results and those of studies by TEM of deep‐etch replicas of similar material previously published.