High pressure freezing (HPF) is now widely recognized as an important and even essential method of specimen preparation for many biological samples. As with other rapid freezing methods, HPF immobilizes all molecules in the cytoplasm within a few milliseconds and we can obtain a true snapshot of the cell at that instant in time. HPF differs from other freezing methods in that samples up to several hundred micrometers in size can be well frozen. HPF is especially critical for high resolution studies such as cellular tomography, cryoSEM, EM immunolabeling, and imaging of vitreous cryosections. In this tutorial we will briefly cover the different HPF machines, but we'll mostly focus on how to achieve proper specimen loading because this is the critical step for success with HPF. We'll also explain how to tell the difference between good and bad freezing.

Freeze substitution is the process by which frozen biological samples are dehydrated and fixed at low temperatures, usually in preparation for infiltration and embedding in resin. Freeze substitution should maintain all the structural preservation achieved by ultra rapid freezing, and in the best cases, add con-trast to molecules so they are visible in thin sections. Adopting a freeze substitution protocol typically involves making choices about the following variables: 1) manual vs. automatic processing, 2) organic solvent, 3) additives to the solvent such as fixatives or contrasting agents, 4) starting, intermediate (if any), and ending temperatures, 5) times at each temperature and rate of warm-up in between, and, 6) temperature of resin infiltration and polymerization. Given this many variables, it is not surprising that the literature contains such a diversity of different freeze substitution protocols. In this tutorial, it is our goal to provide some guidance about how to make sense of these many choices.

Currently, methods for freezing samples include the high pressure freezing method, the metal mirror method, and the plunge freezing method. In this tutorial, we will introduce a new freezing method that produces high quality ultrastructure and can be easily conducted in any laboratory.

Electron tomography, a method to reconstruct 3D objects from a series of 2D TEM images, has become firmly established as an important technique in materials science. This tutorial will cover the acquisition of tomographic tilt series, the use of reconstruction techniques and the large choice of image modes available in the microscope for tomography. A number of applications will be shown, ranging from catalysts and biominerals through to semiconductors and metallic alloys, each of which highlights how novel information can be obtained from tomographic reconstructions.

Any introductory course for physical scientists in transmission electron microscopy (TEM) dwells upon the interpretation of contrast arising from coherent diffraction effects. This coherent imaging contrast can be useful when examining a crystalline specimen at the microscope: bend contours indicate the presence of lattice strain; extinction fringes flag the presence of defects. However, these diffraction ef-fects typically dominate the contrast in a TEM image, often obscuring structural features of interest and complicating image interpretation. Coherent imaging contrast can also act as an artifact in quantitative TEM imaging techniques, such as tomography and energy-filtered TEM (EFTEM) elemental mapping. As a consequence, scanning transmission electron microscopy (STEM) - which is less sensitive to coherent diffrac-tion effects - has become the conventional TEM mode for quantitative imaging. This tutorial will outline the theory, practice, and applications of large angular convergence scanned beam illumination (LACSBI), a new incoherent TEM imaging mode that suppresses these coherent diffraction effects. LACSBI is a hybrid imaging mode in which TEM imaging is performed while operating the microscope in STEM mode, effectively providing an incoherent average of image intensity over a range of incident beam orientations. Topics to be discussed include the fundamental basis and a practical step-by-step procedure for performing the technique, as well as the implementation of LACSBI for many common TEM imaging modes, such as strong- and weak-beam bright/dark field, EFTEM, and tomography.

The Atomic Force Microscope (or AFM) is the most common of a family of microscopies known as Scanned Probe Microscopies (SPM). The original SPM was the scanning tunneling microscope invented by Binnig and Rohrer, who shared the 1986 Nobel Prize in Physics with Ruska (the inventor of the TEM). The basic principle of AFM is similar to that of reading Braille, in that a force (or touch) sensor is scanned across a surface and the surface topography is determined via the response of the force sensor. In the most common mode, a micromachined cantilever with a sharp probe is physically scanned across a sample surface, with a simple PID feedback control system used to maintain constant tip-sample interaction. AFM uses no lenses, and therefore does not require focus or stigmation; however, selection of the optimum force sensing probe and gains of the feedback control system are critical. This tutorial will discuss the history and instrumentation aspects of AFM and other SPMs along with a range of illustrative applications.

How many times have you sat down to prepare a talk and can't figure out how to get started? Once you have your thoughts organized, how do you present your data to an audience without losing them halfway through? This tutorial will describe the construction of a high-quality scientific presentation us-ing design elements to add to the impact of your data, illustrating the "what to do" and "what not to do" points, and discussing image resolution and PowerPoint's effect on it. In addition, tips on delivering your talk will be offered.

As technology rapidly advances in capability and cost, and available funds continue to decrease, the game of writing grants to obtain the instruments (toys) we want and need to advance our research takes on added importance. During this tutorial several investigators will briefly present different perspectives on what they have done to successfully obtain extramural funding from a variety of sources. Information will also be presented that represents some "Dos" and "Don't's" that may improve a grant or get it tri-aged, and a mock Study Section will be held to demonstrate what goes on behind closed doors during the review of grants.

The number of x-ray microCT systems has increased enormously over the last decade, as has the number of papers describing the results of this type of 3D, non-destructive imaging modality. The bases of x-ray microCT will be presented. Examples from commercial laboratory microCT systems and from synchrotron microCT facilities will be described to illustrate the capabilities of this imaging approach. This tutorial will also discuss different data analysis strategies for x-ray microCT.

The goal of this "Ask the Experts" session will be to share knowledge and expertise regarding cutting edge optical microscopic methods for live cell imaging. Optical microscopy has dramatically increased in capability over the last few years, and has found utility in increasingly sophisticated cell biological prob-lems that require high spatial and temporal resolution. The advances that permit these applications are principally based on fluorescence microscopy, and depend on microscopic methodologies coupled with fluorescent proteins, new fluorescent dye technologies, highly sensitive detectors, and inexpensive pow-erful computers. A panel of several investigators who are currently exploiting these methods for live cell applications will be available to answer questions and discuss issues currently facing the field. The focus of the information will be determined by the questions asked either in advance or at the session. To pro-pose questions in advance, please email them to Dave.Piston@Vanderbilt.edu.

Recent high-resolution TEMs can provide remarkable insight into the microstructure of materials at the atomic level. However, it is all too easy for a novice operator to record images that are full of artefactual detail, especially for instruments equipped with FEGs. For example, beam misalignment and/or image delocalization can produce apparent lattice fringes in the vacuum beyond the edge of a sample. Discussion will initially center around principles and practice for routine high-resolution imaging (e.g. coma-free alignment, setting optimum focus), with tips on how to avoid common operator errors, but any questions on related topics will be welcomed (e.g. aberration-corrected imaging, sample thickness