This study involved the application of the Scanning Electron Microscope (SEM) for the characterization of a GaN-based Light Emitting Diode (LED) by the Electron Beam Induced Current (EBIC) method. The diffusion length of the minority carriers in both the p and n-type regions of a GaN-based LED structure was determined with the EBIC technique in the line scan configuration. The diffusion length is a critical physical parameter that directly affects the electrical and optical properties of these devices. This value is generally understood to be a material property of the layers within a device. However, the measured values of the diffusion length are sensitive to the sample design, experimental conditions, measurement technique, and data analysis model and method utilized.

The EBIC measurements were performed at room temperature in a Hitachi S-3200N SEM using a GW Electronics Type 31 Specimen Current Amplifier.

Many different techniques have been used in attempts to minimize defects in single crystal semiconductors. This study examines semiconductors grown in the presence of a rotating magnetic field (RMF). The RMF method is commonly used in metallurgy to stir an electrically conducting liquid during the casting process which can reduce the effects of buoyancy driven convection and enhance the mass transfer process. The variation of heat and mass transfer processes by RMF can be controlled by selecting a specific frequency and strength of the magnetic field. Both numerical modeling and space-based crystal growth experiments using RMF indicate that the application of RMF to solidification of semiconductors will dramatically minimize defects and inclusions.

A ground based program in the Microgravity Research Division at NASA's Marshall Space Flight Center has been studying the effects of RMF on various semiconductor compounds grown by the traveling heater method (THM).

The ternary II-VT alloy Hg1-xCdxTe has become the material of choice for many infrared detector applications. Current state of the art Hg1-xCdxTe infrared focal plane arrays (IRFPAs) are constructed as hybrid structures consisting of an epitaxial sensing layer of Hg1-xCdxTe on either a CdTe or Cd1-xZnxTe substrate, hybridized to a silicon readout circuit chip. For backside illuminated structures, like the typical infrared Hg1-xCdxTe detector array, multilayer antireflective coatings (AR) are required on the backside of the detector chip. The next generation of higher performance IRFPAs will be based on high densities of smaller detector pixels fabricated on large area monolithic heteroepitaxial substrate materials. Since the ultimate performance of photovoltaic diodes of this type is determined by the signal to noise ratio of the device, reducing the size of the pixels while lowering the undesirable noise currents in the devices also reduces the amount of signal generated by the diode.

Two-dimensional dopant profiling continues to be a topic of great interest and importance to the semiconductor industry. Off-axis electron holography provides access to the phase of the electron wavefront that has traversed a sample, and it thus enables voltage contrast to be visualized. The technique was used by McCartney et al. (1994) to observe the potential distribution across Si/Si p-n junctions [1], and it was later also used by Rau et al. (1999) to map the two-dimensional electrostatic potential in deep-submicron transistor structures [2]. in this paper, we have used electron holography and a known test structure to demonstrate that accurate voltage profiles can be extracted under carefully controlled sample preparation conditions. Carrier information can be obtained using a simulation program. Experimental factors that affect quantitative measurement are also discussed.

The sample studied was a test structure fabricated at IBM for comparison of various profiling methods [3]. Both p-n junctions and p-p+ regions were available for study.

Even though all real objects are three dimensional, imaging and metrology performed by using electron-beam tools such as scanning electron microscopes is inherently two dimensional. Any information about the third dimension must therefore be obtained by inference, or by time consuming special methods such as stereo-photogrammetry. If, however, the structures of interest are thin enough to be electron transparent then quantitative three dimensional metrology can be performed directly by using off-axis transmission electron holography. Here we demonstrate the application to a SCALPEL lithography mask which consists of chromium lines on a silicon support film. The off-axis holography was performed in a field emission transmission electron microscope, a Hitachi HF2000 operated at 200keV. The sample is positioned so that half the beam passes through the specimen while the rest travels only through the vacuum. An electrostatic biprism then recombines these two components to form the hologram which is recorded onto a CCD camera.

The mechanism of action of the tumor promoter, phorbol 12-myristate 13-acetate (PMA), depends on its ability to substitute for an endogenous second messenger, diacylglycerol, and thereby activate certain members of an enzyme family known as protein kinase C. Previous work from this laboratory showed that the quantitative shape phenotype of cells treated with PMA resembled the phenotype of bona fidecancer cells. The effect of PMA on this phenotype was transient, and was restricted to a period of two- to five-hours after exposure to PMA. When the shape phenotype was dissected into components by relating different variable's values to shape features, several of the altered values appeared to rely upon a declining number of sharp features, such as filopodia and microspikes, at the cell edge.

Filopodia and microspikes are in turn regulated by a GTPase of the Rho family, Cdc42, which modulates actin architecture.

The extracellular matrix (ECM) plays a critical role in directing cell behaviour and morphogenesis by regulating gene expression and nuclear organization. Using non-malignant (S1) human mammary epithelial cells (HMECs), it was previously shown that ECM-induced morphogenesis is accompanied by the redistribution of nuclear mitotic apparatus (NuMA) protein from a diffuse pattern in proliferating cells, to a multi-focal pattern as HMECs growth arrested and completed morphogenesis . A process taking 10 to 14 days.

To further investigate the link between NuMA distribution and the growth stage of HMECs, we have investigated the distribution of NuMA in non-malignant S1 cells and their malignant, T4, counter-part using a novel model-based image analysis technique. This technique, based on a multi-scale Gaussian blur analysis (Figure 1), quantifies the size of punctate features in an image. Cells were cultured in the presence and absence of a reconstituted basement membrane (rBM) and imaged in 3D using confocal microscopy, for fluorescently labeled monoclonal antibodies to NuMA (fαNuMA) and fluorescently labeled total DNA.

Using human peritoneal explants, we have previously demonstrated that endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs) attach to intact mesothelium. Attachment occurs within one hour and mesothelial invasion occurs within 18 hours (Figure 1). We have also demonstrated that, in vivo, the mesothelium overlies a continuous layer of collagen IV (Col IV).

More recently we have used CLSM, to study the mechanism and time course of ESC and EEC attachment and invasion through mesothelial monolayers. in these studies, CellTracker® dyes were used to label cells. Mesothelial cells were labeled with chloromethylbenzoylaminotetramethylrhodamine (CellTracker Orange). Mesothelial cells were then plated on human collagen IV coated, laser etched coverslips. Mesothelial cells were cultured to subconfluence. ESCs and EECs, labeled with chloromethylfluorscein diacetate (CellTracker Green) were plated on the mesothelial monolayers. Cultures were examined at 1, 6, 12 and 24 hours with simultaneous differential interference contrast and CLSM.

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

Specific lipid self-associations in the fluid bilayer, particularly involving sphingolipids and cholesterol, are believed to result in the formation of membrane microdomains or “rafts” that mediate recycling of proteins between the cell surface and intracellular compartments or function as platforms for signal transduction events. Such membrane microdomains may be recovered in a low-density fraction that is insoluble in Triton X-100 at 4°C and referred to as detergent-insoluble, glycolipid-enriched complexes (DIGs). Membrane microdomains that constitute cell surface invaginations called caveolae can also be recovered in the form of DIGs. Caveolae are typically associated with a protein called caveolin, which coats their cytoplasmic surface.

Various glycosyl-phosphatidylinositol (GPI)-anchored proteins such as the folate receptor (FR) are believed to be associated with membrane rafts and to constitutively internalize and recycle to the cell surface although FR is the only known GPI-anchored protein with a transport function, i.e.,

Co-administration of Vitamin C (VC) and Vitamin K3 (VK3) combination (in a VC/VK3 ratio of 100:1) to several human carcinoma cell lines resulted in equivalent antineoplastic activity at concentrations that were 10-50 times lower than when either vitamin was administered alone. Peroxides generated by VC resulted in membrane lipid alteration. VC and VK3 combination resulted in cytoskeletal alterations, DNAses and RNase activation. This report documents LM and SEM morphological changes of ovarian carcinoma cells which accompany vitamin treatment and lead to autoschizic cell death.

MDAH cells were cultivated in titer dishes and on coverslips coated with polylysine. After exposure to either VC, VK3, or the VC/VK3 combination for 1 hour, cells on coverslips were fixed and processed for SEM. Cells in dishes (105 cells) were harvested, centrifuged (<500 r/min), fixed and embedded in PolyBed. Sections, oneurn thick, were stained by Toluidine blue prior to LM and TEM studies.

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.