Specimen contamination and amorphous irradiation damage severely limit the ability to perform accurate electron microscope analysis of materials, especially as specimen areas of interest decrease in size. To analyze smaller areas of interest, electron probe sizes have decreased, while probe currents have increased. The combination of these two factors results in an increase in the amount of carbonaceous contamination formed on the specimen under the electron beam. Recently, the use of low energy plasmas has been shown to be effective in preventing such contamination from occurring (1-3). For TEM, the simultaneous placement of the specimen holder and specimen into such a plasma for short periods of time (usually less than two minutes) results in the ability to hold a converged probe on the specimen for analysis without contamination becoming an problem. Figure 1 is a TEM micrograph of a 304 stainless steel specimen.

The common theme of most forms of thin specimen preparation for the physical sciences has been the controlled, gradual removal of material, whether by electrolytic dissolution, ion bombardment, careful mechanical dimpling or step-by-step tripod polishing. It is somewhat ironic that a “brute force” method, wherein the material is forced at a high rate against an atomically sharp diamond wedge so that an ultrathin slice, or section, is sheared, or fractured, from a ‘block’ of the material, should be viewed not only as acceptable, but with growing enthusiasm in the last several years. This process, known as ultramicrotomy, originated in life science TEM, but has been modified by materials scientists and applied to an impressive array of materials as illustrated in Table 1, culminating, perhaps, with the recent successful sectioning of diamond film.

Over the past few decades, the demands of modern analytical electron microscopy have increased the need for TEM specimen preparation techniques with a minimum of misleading artifacts in terms of chemical microanalysis. At the same time, the demands of modern industrial materials, be they semiconductor, polymeric or composite in nature, call for speed, flexibility and high spatial resolution as well. The response from the electron microscopy community, especially that portion in the private sector, have been to devise (or advocate) radically different forms of TEM thin specimen preparation from that of classic replication, electropolishing and ion thinning.

This tutorial sets forth the goals of TEM specimen preparation, and the requirements for a "good" TEM specimen. The strategic choices governing which technique to use for preparing a wide variety of specimens will be covered. A TEM Specimen Preparation Flow Chart will be used to plot a course that makes optimum use of the preparation techniques available as a function of the type of specimen to be prepared.

While the techniques offer high spatial resolution in some cases down to the atomic scale and three dimensional mapping of surface topography, there still remain issues related to quantitative interpretation of scanned probe data, particularly in the recently developed phase contrast imaging modes. In this talk, the various modes of force microscopy will be introduced, along with examples. New techniques are starting to emerge which allow us to use the scanned probe microscope to measure properties such as local adhesion and local elastic and plastic deformation of samples.

Each scanned probe technique relies on a very sharp probe positioned within a few nanometers of the surface of interest. Some combination of probe and/or substrate positioning is required to provide sub-nm-resolution, three-dimensional motion of the probe relative to the substrate. When the probe translates laterally (horizontally) relative to the sample, any change in the height of the surface causes the detected probe signal to change.

Many imaging processes, including both conventional wide-field and confocal fluorescence microscopy, can be described to a good approximation as linear and spatially-invariant. Linearity means that the image of an extended specimen is simply the sum of the images of the parts of the specimen - in the limit the specimen can be regarded as a collection of points, and its image is the sum of the images of the points - i.e. weighted instances of the imaging system's point spread function (psf). In the case of spatially invariant imaging the psf is the same over the entire field of imaging. Mathematically, the image is the convolution of the specimen with the system psf. In principle, this convolution can be reversed to remove degradation introduced by the imaging process, a computational procedure often called deconvolution or, more generally, restoration.

This report summarizes our experiences gained with deconvolving 3-d images of various types of biological samples - animal and plant cells; live and fixed. We compared raw, unmodified images with images processed with standard filtering methods and images improved by using deconvolution. The blind decovolution software package from AutoQuant, Inc. was used. All images were acquired using a BioRad MRC 1024 confocal microscope (attached to a Nikon Diaphot microscope), using fluorescence or reflected light. For thin (up to 25 μm) flat specimens a 60x PlanApo NA1.4 lens was used while for thick (25-200 μm) water containing samples a 40x Fluor NA1.15 water immersion lens was used.

In an attempt to test the usefulness and validity of deconvolution for various types of biological samples we adopted the following approaches:

1. collect images of structures that are known to be both fluorescent and reflective - compare the results of deconvolution of fluorescence images with reflected light images that are not deconvolved.

We are using wide-field deconvolution microscopy to study the locomotion of cells within a three-dimensional cell mass. How cells move within such an environment is poorly understood, even though such movement occurs widely, for example, during embryonic development or tumor cell invasion. In these and other cases, cells moving within a cell mass face challenges not encountered by isolated cells crawling on a flat substrate. Cells within a mass must clear a path for themselves, and also gain traction on other cells which themselves may be moving in the same or another direction. To understand the mechanisms of such 3D movement, we observe and analyze it in both normal and mutant cells. The mutant analysis sheds light on which molecules are important for 3D locomotion.

To visualize either normal or mutant cell locomotion within a three-dimensional cell mass, we require time-lapse 3D imaging at light levels low enough to maintain specimen viability.

Fluorescence intensity in a spectrofluorometer is proportional to the concentration of analyte over a wide dynamic range and is, therefore, an excellent quantitative analytical tool. In this paper we use the techniques of deconvolution and convolution to extend the utility of quantitative fluorescence to the measurement of analyte concentrations inside cells and their organelles. This permits us to assess the physiological state of living cells in situ.

To successfully turn a voxel within a 3D image into a microcuvette, 2 conditions must be met. First, to be sure the voxel is associated with only one fluorescent structure, the object that is being measured must be fully resolved from neighboring objects in all three dimensions. For structures separated by distances larger than about 300nm in the xy plane or 600nm in z (where z is the optical axis), this can be achieved with either digital deconvolution of 3D widefield images or by confocal microscopy.

The technologies of nanofabrication as applied to inorganic materials and substrates are advanced and continue to develop. These sophisticated processes enable the formation of complex electronic, optical and mechanical devices with feature sizes down to tens of nanometers. Adaptation of these types of processes to surface chemical patterning and topographical patterning provides a new set of experimental tools for investigating biological systems and realizing sensors and devices that require the interaction of biological systems and fluids with inorganic materials and surfaces.

In this talk we discuss methods of pattering self assembled monolayers, proteins and antibodies on silicon and glass surfaces by lithography and microcontact printing. This is of utility in a variety of experiments in cell-surface interactions and in sensor devices. In certain types of devices the manipulation of fluids and sieving of molecules is a critical function such as in DNA sequencing6 by electrophoretic separation.

Nanobiotechnology is the fusion of biology and nanofabrication (Hoch, et al. 1996). Nanofabricated devices are increasingly being used in biological studies, and surface modification methods are important for interfacing inorganic devices with tissues.

Nanofabricated implants are used to study brain physiology (Najafi and Wise, 1986). These implants can have cross-sectional dimensions as small as 15 μm. However, the implants cause glial scars that inhibit their operation. Figure 1 shows a model implant, with a trapezoidal cross section (base=200 μm, height=130 μm, top=60 μm, length=2 mm), for studying scar formation. Implants were inserted into the brains of rats and tissue was harvested after 1 hr. to 12 wks. Figure 2 shows a thick (100 μm) section of brain prepared 7 days after implantation; the section was immunolabeled for glial fibrillary acidic protein (GFAP) and imaged in a Bio-Rad confocal microscope. The implantation site is surrounded by GFAP positive material indicative of reactive astrocytes.

Novel microfluidic designs are needed for fluid delivery and transport in an integrated micro-genetic analysis system and these designs often lead to complex relationships between the shape and size of the microchannels and the behavior of multi-component fluids. We are studying both pressure-driven and electrokinetic flow in various microfluidic channel designs fabricated from both glass and silicone substrates to understand chemical containment and transport. In conjunction with the experiments, simulations are being performed to predict behavior in microdevices and models are being used to develop a CAD tool for designers.

Fluorescent flow profiles are generated using a charged caged dye which fluoresces upon irradiation with 355 nm light from a Nd:YAG laser. The starting position of the dye is determined by a tightly focused laser spot <20 μm in diameter.

Virtually everyone associated with a science or engineering discipline has some baseline knowledge of optical microscopy; and most attendees at this conference have a reasonable exposure to at least some form of electron microscopy. The many developments in instrumentation and application require the modern microscopist to continuously follow the literature to stay aware of the ongoing improvements and advances in these microscopies. While electron and optical microscopes have been around for many decades, the family of microscopes known as Scanned Probe Microscopy (SPM) are just entering their second decade; and are actually in there first decade of widespread use.

The most commonly used forms of scanned probe microscopy are the force microscopes; commonly referred to as Atomic Force Microscopy (AFM). This includes a wide variety of microscopy modes that are generally made available as modifications of a basic AFM. Before describing the basic versions of AFM, the fundamentals of instrument design must be addressed.

The variety of instrumentation available to the researcher today can be overwhelming and confusing. Scanning Electron Microscopes (“SEM's) are no exception, and choosing one can often serve as an exercise in dealing with complexity. First time purchasers are most at risk, being subject to a barrage of information that attempts to sway the purchaser in one direction or the other. As a result, one can sometimes be drawn to the details of the latest “high end” performance parameter, while overlooking the basics. At its worst, the selection process can degrade to one of vague guesswork with little hard data to serve as a compass.

By applying a methodical approach to define your individual requirements, carefully designed tests of actual instruments, and discussions with your collaborators, potential and experienced users, one can begin to ensure a successful selection process.

The selection of an ultramicrotome should always begin with an evaluation of the immediate as well as the long-term needs of the laboratory. If present projects require ultramicrotomy on a regular basis (e.g., more than 5-10 specimens per week) and if this demand will probably continue for more than one year, the acquisition of an ultramicrotome is justified. An intensive, short-term project or an occasional need with no clear indication of a future requirement for ultramicrotomy could best be accommodated by either leasing an ultramicrotome or contracting the work to an individual or corporation. On the other hand, several laboratories with modest needs may wish to pool resources and purchase a shared instrument capable of fulfilling the requirements of each lab.

Since new ultramicrotomes cost $20k and up, one may be compelled to acquire a used instrument. Depending upon the age and condition, a used ultramicrotome may be purchased at a cost of $2-15k.

The decision to buy a confocal microscope presents the huge task of wading through designs and options from a number of excellent companies. While the principle of confocal imaging - rejection of out-of-focus light - is consistently achieved, each manufacturer does so with a unique design. There is strong competition among manufacturers to have the microscope with the best possible resolution and light transfer efficiency so that maximum specimen detail is detected with the least light damage. There are a number of excellent instruments currently available. In deciding among them consideration should be given to the following: type and number of users, types of imaging required, and your budget. If the microscope is going to be used by a single lab for a single purpose i.e. gfp expression in embryos, you should consider one of the less elaborate “personal confocals” with one laser, one or two detectors and a price tag under $150,000.

Designing a microscopy facility can be completed in a step by step approach, whether it be a completely new facility or an existing one moving into expanded quarters. The procedure is independent of size. There are several major factors that must be taken into consideration. Check lists can be compiled by the microscopist. One should easily be able to start at any point along the way. The time and effort initially put into the design will pay for itself many times over in convenience and efficiency of the resulting laboratory.

Prior to drawing the actual design of the microscopy facility but critical to the design specifics, it is necessary to compile the following information: considerations for determining the size of the facility; general EM lab requirements which affect site determination; activity functional flow diagram; EM room requirements; a list of all the equipment to be placed in the EM facility and exact specifications for each; design features not associated with specific equipment; activities requiring space that are not specific to the equipment; general building requirements not specifically related to microscopy operations; and ergonomic and psychological factors.

Introduction: This is a personal account of the author's attempts to solve some of the problems associated with the fixation and embedding of difficult biological materials such as plant tissues, almost all insect tissues, and large pieces of tissues including whole organs.

Historically, the search began in the 1950's with the study of the maize root tip using the relatively new technique of electron microscopy. This system of cells, separated by thick and partially impermeable walls, did not respond well to the procedures most suitable for animal tissues. New approaches were needed and, in time, some were found that were at least useful if not ideal. A brief account of several of these is given in the following paragraphs.

Fixation of plant cells: Potassium permanganate was chosen very early in the study primarily because it worked better for plant cells than did any of the fixation procedures currently available.

When managing multi-disciplinary materials characterization facilities to help solve scientific materials problems, one needs to confidently operate the instrumentation knowing that the calibrations and operational procedures are as accurate as possible and optimized by periodic evaluation. An overview of the calibrations and some operational pitfalls will be reviewed for the Analytical Electron Microscope. I intend to show examples of various results produced over the years characterizing materials, including some recent data. A short chronological history of my career pertaining to image documentation will show examples from the old days to the present. I pride myself on having a top-notch laboratory so when scientists need relatively fast answers, one knows which direction to proceed and the equipment is capable of achieving the results. When scientific users come to an AEM laboratory, each individual brings a potpourri of materials problems to try and solve.

Casting of hollow spaces with solidifying materials and subsequent removal of surrounding tissues by corrosive alkali and acids and the inspection of the remaining casts by bare eyes or the dissecting microscope is an old anatomical technique.

The introduction of polymerizing resins as casting materials which resulted in durable casts of even the smallest spaces (bile and blood capillaries) and the application of the scanning electron microscope with its high resolution and great depth of focus, enabled the qualitative and the quantitative analysis of the 3D-arrangement of tubular systems by means of their casts.

Presently, scanning electron microscopy of microvascular corrosion casts is used to study growing, stable or regressing blood vessel systems under physiological (e.g. during development, wound healing, metamorphosis) and pathological (e.g. tumor angiogenesis) conditions in qualitative and quantitative terms.

SEM analysis of pulmonary vascular casts shows basket-like alveolar capillaries that are about 6 μm in diameter. Generally pulmonary arteries are adjacent to airways and veins are not. The pleural surface and the airways capillaries have a planar meshwork with few large vessels. Uncommonly are lymphatics cast in normal lungs but they are regularly found in casts of edematous lungs. Casts of pulmonary veins in several species have smooth surfaces with a slight notch occurring at regular intervals; these notches will deepen after physiological interventions and are sharply constricted in spontaneously hypertensive rats (SHR) with pulmonary hypertension and cattle. The circular bands are caused by tufts of smooth muscle cells and have been called pulmonary venous sphincters (Fig. 1). The depth of the constrictions can be measured to show the effect of an experimental intervention on individual small veins, which is important in understanding the pulmonary microcirculation and its control.