Lung cancer remains the leading cause of cancer death in the United States. It has been estimated that in the year 2001 there will have be more than 157,400 deaths from lung cancer (28% of all cancer deaths) and 169,500 newly diagnosed cases of lung cancer (13.4% of all newly diagnosed cancer) recorded. The significance of this disease requires development of better treatments and of feasible means to prevent the development and progression of lung cancer, especially since the progression of initiation to clinical manifestation may take years. The use of an appropriate animal model is paramount to the study of human disease.

More than 95% of all primary human lung cancer is directly attributed to the use of tobacco products2. The most common forms of human lung cancer include squamous cell carcinoma, adenocarcinoma, and small cell carcinoma .

Immunohistochemistry (IH) has revolutionized tumor diagnosis in recent years. The antigenic/molecular information now available is extraordinarily helpful not only in clinical diagnostic work, but also in improving our understanding of the cell biology of neoplasia. As a result, there is now less need for diagnostic conventional transmission electron microscopic (CTEM) studies on neoplasms. For example, lymphomas and leukemias are now largely defined by IH and related techniques such as flow cytometry, not CTEM. A postive S-100 stain in the right setting usually obviates the need to search for melanosomes.

However, CTEM is still a useful tool for surgical pathologists. One important advantage CTEM has over IH is that - like gross observations and the classic H&E section - CTEM can detect features not suspected in advance. This property of CTEM makes it particularly valuable in studying complex and unusual patients/tumors.

Adipose tumors during the first decade of life are infrequent, composing about 6% of all soft tissue neoplasms in children. Lipoblastomas are rare benign tumors occurring in infancy and early childhood and arising from embryonic white fat. The tumors may infiltrate tissue locally and may be of considerable size, but lack the ability to metastasize. The tumor may be an isolated well-encapsulated mass (lipoblastoma, Figure) or lack encapsulation and be locally infiltrative (lipoblastomatosis). Although a benign tumor, lipoblastomas tend to recur, and may resemble myxoid liposarcoma, a malignant tumor.

We report 26 cases of lipoblastoma/lipoblastomatosis over a 15 year period at Texas Children's Hospital. There was a slight female predilection (1.2 F:1.0 M). The most common symptom was a painless mass of increasing size. The trunk (48%), extremities (33%), head and neck (15%), and lung (4%) were the tumor sites.

Apoptosis peaks within the cardiac outflow tract (OFT) myocardium during chick development (stage 30-31) and coincides with dramatic remodeling of the OFT 4. Apoptosis was assayed by TUNEL, Annexin V, transmission electron microscopy, and Lysotracker Red (Fig. 1-4). These assays reveal that as many as 92% of the apoptotic cells express cardiomyocyte markers. The timing of apoptosis suggests that it may occur to allow proper alignment of the great vessels over their respective ventricles. Our current aims are to determine the consequences of disturbing OFT cardiomyocyte cell death to heart morphogenesis and to elucidate the cellular and molecular components that initiate and regulate this high level of apoptosis.

To enhance apoptosis in the OFT myocardium, recombinant adenoviruses were injected into the pericardial space to deliver the gene for the death ligand Fas-L to the cardiomyocytes of chicken embryos prior to the stages when OFT remodeling occurs.

Apoptotic events can be the result of both cell anchorage-dependent and independent mechanisms. The term “anoikis” has been used to describe apoptotic events resulting from a lack of cell-ECM contact (Frisch and Francis, 1994). The integrin heterodimer α5β1 has been implicated in several systems as providing protection against apoptosis. Previous studies have demonstrated that the α5 integrin subunit is present only during cardiac development and conditions of cardiac hypertrophy and is not found in normal adult rat myocytes (Terracio et al., 1991). Cell anchorage-independent apoptosis can result from a variety of factors including biochemical and mechanical stimulus. Angiotensin II (Ang II) has been implicated as a modulator of myocyte apoptosis acting through the angiotensin II type I receptor (Kajstura et al., 1997). The role of a second Ang II receptor, angiotensin II type 2 receptor, has not been determined. The role of apoptosis in myocyte cell death during heart failure remains unclear.

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the depth of penetration of the laser light into the tissue. We have optimized the confocal microscope performance and developed a sample technique that increases the optical resolution of the system.

CLSM has been used to study cellular death (apoptosis) during GD 8-12 normal rat/mouse embryonic development, neonatal ovarian development (PD10-45) and fetal limb development (GD 11-15). LysoTracker Red (LT) was incorporated into the tissue prior to laser excitation. It is aldehyde fixable stain that concentrates into acidic structures or into cells that have high lysosomal activity.

Many reagents have been developed recently to label living cells with substrates that will become fluorescent if an enzyme is active. The general principle is that the substrate will be taken up by living cells then detected only if the enzyme is active. These substrates work well with isolated individual cells, however, more difficulty can be encountered when studying whole tissues. Problems can range from substrate penetration into whole tissues to being able to detect the label effectively. We have used the chicken corneal epithelia for many studies, but the tissue is to thick to view with a conventional light microscope, therefore we have developed techniques using laser confocal microscopes to view this tissue in with a variety of techniques including in situ hybridization, immunohistochemistry, and vital dyes/stains.

Whole embryonic corneal epithelial sheets can be isolated without the basal lamina.

Mitochondria play an important role in apoptotic and necrotic cell death. Depending on the stimulus, a variety of mitochondrial dysfunctions can trigger onset of cell death. Assessment of mitochondrial dysfunction in living cells in situis important to understand the sequence of events producing cell injury and death. Laser scanning confocal microscopy, a technique that creates submicron thin optical slices through living cells and tissues, allows us to monitor several mitochondrial events simultaneously in intact single cells over time.

During oxidative stress, we monitored mitochondrial redox status, Ca2+ increase, reactive oxygen species (ROS), the mitochondrial permeability transition, and mitochondrial depolarization in cultured hepatocytes. When an oxidant chemical, t-BuOOH, was added to cultured hepatocytes, oxidation of mitochondrial pyridine nucleotides occurred as visualized directly by ultraviolet confocal microscopy of NAD(P)H autofluorescence. This event was followed by an increase of mitochondrial Ca2+ measured by the Ca2+-indicating fluorophore, Rhod-2.

INTRODUCTION: The mitochondrial permeability transition (MPT) is implicated in mediating TNFα-induced apoptosis in cultured hepatocytes. Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the MPT. After the MPT, mitochondria swell, the outer membrane bursts, and pro-apoptotic cytochrome c is released into the cytosol. in isolated mitochondria, ROS formation promotes onset of the MPT. However, how mitochondrial ROS formation regulates the MPT in intact cells in TNFα-induced apoptosis is unknown. AIM: The present study was designed to determine the role of mitochondrial ROS formation in TNFα-induced MPT and apoptosis in cultured rat hepatocytes.

METHODS: Hepatocytes expressing an IkB superrepressor were pretreated with 2 μM t-BuOOH 4 hours before TNFα exposure with and without CsA (2 μM, an inhibitor of the PT pore) and the antioxidants, desferal (0.5 mM) or diphenylphenylendiamine (DPPD, 10 μM). Cell viability was monitored by propidium iodide fluorometry.

In the last few years there has been a mild explosion in both microscopy and cytometry. in part, this has been a more sensitive understanding of new fluorescent chemicals or probes as well as the ability to transfect fluorescent proteins into cells. in part, this has also been a case of substantial improvement in instruments, electronics, computers, lasers and sensors. in this session, we hope to examine correlative and comparative methods that may improve the quality of data.

This is an introduction to session 29, Correlative Fluorescent Microscopy and Flow Cytometry Techniques, and the first time the Microscopy Society of America has sponsored a session that involves flow cytometry. Flow cytometry offers several statistical advantages over standard microscopies in evaluating populations of cells. Flow cytometry also facilitates a statistical precision in evaluating fluorescence and fluorescence changes in both chemical probes and transfected fluorescent proteins.

Introduction This presentation will follow the pathways to convergence of two very complementary technologies - flow cytometry and imaging. Interestingly, each technology has evolved and matured almost exclusively isolated from the other. in the past several years it has become increasingly clear that these technologies are able to deliver complementary solutions and that when combined the end value of each exceeds the sum of the individual components.

History of development Flow cytometry has a rich history over the past 40 years. Ironically, as a technology, flow cytometry was almost a consolation prize to its imaging cousins. in fact, it seems that it might only have been because imaging solutions proved to be technologically difficult that flow cytometry not only thrived, but left the imaging field well behind for nearly 20 years. Initial interest in evaluating differences between normal and cancer cells was almost exclusively attempted by imaging techniques.

Flow cytometry is an analytical technique in which optical measurements are made of cells or other biological particles as the cells or particles flow, ideally in single file, in a fluid stream past one or more optical measurement stations. Modern optical flow cytometers typically measure light scattered at small (1-5°) and large (15°-135°) angles to an illuminating laser beam, and fluorescence emitted in three or more discrete spectral bands; the most complex instruments employ three or four spatially separated illuminating beams at different wavelengths and can measure twelve fluorescence signals from each cell or particle analyzed.

Flow cytometry was developed in the 1960's to speed up and automate microspectrophotometry and microfluorometry, which had previously been microscope-based, in order to facilitate development of observer-independent clinical tests which could replace the Papanicolaou smear and the differential leukocyte count. At that time, high-resolution scanning was an extremely slow process; high-intensity sources were restricted to arc lamps, and memory in which images could be stored was expensive.

As originally developed in the 1960's, flow cytometry was primarily a technique for the analysis of mammalian cells. Analysis of cellular constituents such as DNA or cell surface antigens made fluorescent by a variety of reagents has been the main stay of flow cytometric applications. Over the years, flow cytometric analysis techniques have been developed that range from multicellular spheroids containing a million or more cells down to single molecule detection. An outgrowth of single molecule detection capability is DNA fragment size analysis.

DNA fragment size analysis starts with a sample of naked DNA that can be derived from a variety of sources including PCR products, double stranded viral genomes, BAC/PAC clones, and bacterial genomes. For genomic or cloned DNA, restriction enzyme digests are analyzed to produce a fingerprint pattern. The fingerprint, i. e., the distribution of fragment sizes produced by the restriction enzyme digestion, is characteristic of the source of DNA and forms the basis for identifying the source.

The plasma membrane defines the outer limit of eukaryotic cells and it is here that cells interact with their environment through receptor proteins. Clearly the management of cell surface receptor concentration is critical for normal cellular function, and this is achieved through two key processes, exocytosis and endocytosis. During exocytosis transport vesicles containing both membrane receptors and soluble cargo fuse with the plasma membrane releasing cargo and delivering receptors to the cell surface. During clathrin mediated endocytosis receptors destined for internalisation become concentrated in clathrin coated pits before coat invagination and membrane fission. Since transferrin receptors are recycled and undergo numerous rounds of exocytosis and endocytosis by labeling transferrin receptor with an appropriate fluorophore it is possible to label both exocytic and endocytic organelles in the same cell. We have exploited this phenomenon to visualise the spatial and temporal organisation of constitutive exocytosis and endocytosis at the surface of single cells using dual wavelength evanescent field microscopy.

Intracellular bacterial pathogens such as Salmonella typhimurium secrete proteins into the host cell after infection. These proteins alter the normal structural and metabolic machinery of the host cell and benefit the bacterium by facilitating replication and avoidance of host immune surveillance. Since the host cytoplasmic localization of these proteins infers access to the class-I MHC antigen processing and presentation machinery of the host cell, we collectively refer to these proteins as Class- I Accessible Proteins (CAPs).

The design of vaccines for new and emerging bacterial pathogens is often constrained by the selection of appropriate and specific antigens. While vaccine design is being greatly aided by whole genome analysis of bacterial pathogens, it has been of limited use in the assignment of function and host subcellular localization of a large percentage of bacterial proteins. in addition, analysis of the bacteria/host interaction is further complicated by the complex lifestyle of the pathogen.

Flow cytometry offers several advantages in evaluating cellular fluorescence and separating values from chemical probes or transfected fluorescent proteins from autofluorescence. Autofluorescence can introduce artificial values that cannot be separated by examination of the fluorescence histogram. Often a comparison of fluorescence in two detectors can separate autofluorescence from positive fluorescence. A region can be defined around the cells of interest for further statistical evaluation or for subsequent sorting.

Autofluorescence is also correlated with apoptosis in that apoptotic cells often increase autofluorescence values. A clear separation of living cells from dying cells can be achieved by the introduction of a DNA stain, such as 7AAD, which will penetrate cells with disrupted or damaged cellular membranes. Intact living cells will not show the 7AAD fluorescence.

This separation is important in transfected fluorescent protein studies, as the transfection process is often toxic to cells.

While morphological analysis at both the light and electron microscopical levels has been fully exploited in academic research, its potential in biopharmaceutical industry has only been fully appreciated in recent years. The biotechnology industry has been developing highly specialized therapeutic molecules, particularly proteins and peptides that are directed to specific molecular targets. The pace of the development of new therapeutic molecules is expected to increase dramatically in the close future, as the biopharmaceutical community will capitalize on the new targets identified by the Human Genome Project.

An efficient drug discovery and development effort should not only include the characterization of the tissue and intracellular distribution of potential targets, but also establish unambiguously that the therapeutic entities reach their intended targets. It has become apparent in recent years that a comprehensive view of the pharmacological action of drugs must include not only the pharmacokinetic parameters but also the tissue distribution and the cellular localization.

Digital microscopy, the integration of digital and microscopy technologies, was initiated for quantitative microscopic image analysis, but it is now for almost all microscopy applications. During the past decade, with the advance of digital technologies, digital microscopy imaging is becoming an indispensable technology in drug discovery.

We started establishing state-of-the-art digital microscopy imaging for drug discovery with the investigation of bioimaging applications at our US research site. Our results shown that all the top 5 bioimaging needs require computer-aided microscopy. Based on this investigation and our review of the microscopy imaging applications in the pharmaceutical industry, we determined four directions for microscopy in drug discovery: multidimensional/multimodal microscopy, digitalization, automation, and bioimage informatics.

Multidimensional/multimodal microscopy imaging is required by the nature of biological research, which is fundamental in drug discovery. From genomic imaging to pathology observation, we require biological details and compound activities at the levels from subcellular organelles to organ tissues, from cellular signaling to anatomical locations of compounds.

In spite of improved equipment and software, the routine application of quantitative microscopy to practical problems in drug development has been limited. This is due to a perceived and often real increase in project complexity and time added by quantitation of tissue changes, without an offsetting return of information. A major barrier to quantitative microscopy is the degree of difficulty in collecting data. The more steps in this process, the less attractive the task. Therefore, it follows that the more automated the task, the more likely quantitative methods will be applied. The purpose of this presentation will be to illustrate an approach taken to automate the morphometric quantification of muscle atrophy which follows dennervation.

The traditional morphometric approach to assess myofiber atrophy is based upon laborious planometric measurement of individual muscle fiber cross-sections. Examples of the type of data obtained using the traditional method are shown in text table 1.

Non-invasive imaging technologies such as magnetic resonance imaging are commonly used in clinical practice. in an experimental setting, it is possible to decrease field of view and pixel size, thereby increase image resolution. We tested this magnetic resonance microscopy (MRM) technique in a theopln lline-induced rat model of reproductive toxicity. The goal of this work was to study the sensitivity of MRM to detect or predict changes in tissue proton (i.e. water) characteristics, and confirm those findings with histology.

Male Sprague-Dawley rats were fed either a control or theophylline (1.3-dimethylxanthine. 8000 ppm) diet. Experimental groups of days 8. 16. 24. and 32 animals were imaged with MRM (Figure 1) and tissue morphology confirmed with histology (Figure 2).

Tl-weighted images (with and without gadolinium-DTPA contrast. Magnevist®) and T2-weighted images were acquired at 2 T (spin-echo multislice images. 100 μm2 pixel. 300 μm slice thickness).