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.

The primary use of vascular corrosion casting has been and likely will continue to be for describing the 3-dimensional anatomy of the microvasculature of organs and tissues. In these studies the assumption is made that the corrosion cast faithfully replicates the anatomy, dimensions, and distribution of the vasculature of these tissues and organs. If this assumption is accurate, and it appears to be nearly so in tissues where measurements have been made, it follows that the casts should lend themselves to quantitative measurements. I summarize here some simple quantitative measurements we have made from corrosion casts. For these studies, cannulas were inserted into arteries leading to the organ of interest in anesthetized animals, and the blood was removed by flushing with Ringer solution or buffered saline, at physiological temperatures and pressures. Resin consisting of Mercox (or Mercox diluted with methyl methacrylate monomer) and catalyst was infused via the same cannula until the onset of polymerization.

The interaction of cells with biomaterials is influenced by a number of factors including the chemical composition, surface charge, hydrophobicity, porosity and texture of the biomaterial. Changes in texture alone have been shown to result in reduced platelet adhesion on ventricular assist devices, reduced scar tissue formation around implanted prostheses, and enhanced biocompatability of vascular stents. In vitro growth of certain cells requires extracellular matrix components to provide the right physical and chemical stimuli for adherence, growth and differentiation. What role the texture of these materials plays, and the mechanisms responsible for the effects of substrate texture on the behavior of cells are not well understood. Previously we have used microcorrosion casting technology to replicate lymph node and other vasculature. More recently, we have used corrosion casting to replicate of the subendothelial matrix exposed when cells are damaged due to the passage of air emboli through a vessel.

Several authors have been studying the microvascularization pattern of the ovary in different animals. To determine the microvascularization pattern using the corrosive-resin casting method, casts of the vascular network were prepared and examined by scanning electron microscopy. However, the same procedure has rarely been used to study the microvascularization pattern of the uterine tube itself. The microvascularization of the uterine tube has always been associated microvascularization studies of the ovary. This study analyses the microvascularization of the uterine tube, pointing out differences between the region of the tube itself and it's infundibulum. In this study we used uterine tubes from adult female rabbits. Anesthesia was carried out with Ketalar (5mg/kg body weight); after that saline solution and 2,5% glutaraldehyde were injected through the left ventricle. This procedure was followed by mercox 2B injection. After polymerization the samples were collected and immersed in warm water (60°C). Then samples underwent tissue digestion.

The purpose of this study has been to undertake a three-dimensional analysis of the vascular network of the lamina propria in the lingual mucosa of the rat. Using the corrosive-resin casting method, casts of the vascular network were prepared and examined by scanning electron microscopy. We used 21th day normal wistar rats. They were anesthetized and perfused with saline solution and after with mercox (25ml of resin, 5ml of methyl methacrylate monomer). Then, the tongues were resected and placed in warm water bath at 60° C for 4 h. The tongue epithelium were digested with 20% NaOH for 4 days. The vascular resin casts washed in distilled water, several times. The dried vascular casts were mounted on the stubs with conductive paste, coated with gold and examined with a JEOL JSM- 6100 scanning electron microscope. In the SEM images, the lingual mucosa revealed arteriolae which divide into smaller branches entering into connective tissue papillae.

The application of two or more imaging-based technologies to portions of a sample prepared in parallel or preferably to the exact same sample in series is the basis for correlative microscopy. Simultaneous visualization of structure in conjunction with other cellular parameters such as cell motility, intracellular ion concentration, receptor expression, and protein distribution/movement can be followed in living cells by various forms of photon, acoustic, and force based microscopy. The same cells can subsequently be prepared by various physical or chemical based procedures and reexamined by other microscopic methodologies to gain additional qualitative and quantitative information often at significantly increased levels of resolution. Recent advances key to correlative microscopy have been the development of high sensitivity, high resolution video cameras, and computer image processing, image enhancement, image analysis, and reconstruction capabilities. New instrumentation such as video enhanced, rectified interference-based light microscopy,LM; confocal LM, and two and three photon LM also have been critical.

Atomic force microscopy (AFM) provides unique opportunities to study cell-surface and molecular scale interactions in three dimensions under aqueous conditions. Two plasma proteins, von Willebrand Factor (vWf) and fibrinogen, play central roles in the regulation of hemostasis and thrombosis by participating in coagulation and facilitating adhesion and aggregation of activated platelets. vWf and fibrinogen are believed to facilitate platelet adhesion under regions of relatively high and low vascular wall shear stress, respectively. Consequently, elucidating vWf and fibrinogen structure-function relations under shear is of considerable importance in developing a comprehensive understanding of the pathophysiology of thrombogenesis. Previously, we reported on molecular level AFM images of human vWf during shear-induced structural transition and human fibrinogen under aqueous conditions when both proteins were adsorbed on a hydrophobic surface. This presentation will report on the shear dependent interactions of dimeric and multimeric vWf with a hydrophobic surface and the substrate-dependent interactions of fibrinogen.

Although significant advances have been made in the development of biocompatible materials, currently available biomaterials still present a number of problems for in vivo applications. One of the attempts to improve the biocompatibility, especially blood-compatibility, of biomaterials has been surface modification. Typically, poly(ethylene glycol) (PEG), albumin, heparin, and phospholipid molecules are grafted to the surface to prevent protein adsorption and cell adhesion. We have been modifying biomaterial surfaces by covalent grafting of PEG and albumin. The control and modified surfaces were examined using an atomic force microscope (AFM). In this study, we examined the surface topography changes by surface modification using PEO grafting to glass as a model system.

Glass surfaces were modified with PEO using (N-triethoxysilylpropyl)-Omonomethoxy PEG urethane (PEG-Si), a PEG derivative containing a hydrophobic carbon chain and triethoxysilyl group at one end of the PEG chain. The presence of the hydrophobic carbon chain allowed self-assembly on the surface and triethoxysilyl resulted in covalent bonding to glass surfaces