MICROSCOPY RESEARCH AND TECHNIQUE 47:291–301 (1999) 5TH California Microscopy Colloquium The California State University & Northern California Society for Microscopy Saturday, October 2, 1999 ALEXANDER, C.G. Department of Biology, San Francisco State University, San Francisco CA 94132. Parasites as Art I began photographing parasites when I was working on shark tapeworms for my doctorate thesis at UCLA in 1953. After I began teaching Parasitology in 1955 at San Francisco State I developed a system of preparing color-slides using a photomicrographic outfit obtained with a grant from the National Institutes of Health. I now have several thousand slides of prepared and stained specimens of virtually every species of parasite from the lab studies of my class and from my own research. I put together a show using these slides initially to introduce the students on the first day of class to a consideration of parasites not as something to be viewed with distaste and horror but as one integral part of the whole of “creation,” each with its own beauty and fascination of complexity. One cannot help but wonder - why is such intricate design of structure developed simply to grasp and hang on to the villus of a shark’s intestine? ANTIPA G.A. Department of Biology, San Francisco State University, San Francisco CA 94132. Hi Res/Lo Tech computer imaging for the classroom I have found that scanning micrographs, either prints or 35mm slides, at reasonably high resolution (i.e., 2–5 Mb/image) provide image files that can be “zoomed-in” during a class session. This allows the opportunity to select parts of an image and magnify just that section for the class. The continuity between ALL low magnification and high magnification images adds significantly to clarity for all students, a clarity not generally available in textbooks or standard slide presentations. That’s the good news. Unfortunately, most existing slides or prints were not produced with © 1999 WILEY-LISS, INC. that objective in mind. As a consequence, they are either not in proper focus or with sufficient resolution to be used in the zoomed-in mode. Of approximately 1000 slides of micrographs of three-dimensional objects that I use routinely in classroom presentations, perhaps as few as five (5) met the criteria for zoom presentation; new images must be produced. One of the most effective presentations of these images makes use of a digital to analog converter, a standard classroom TV (available routinely in many classrooms), and any version of Photoshop™. Photoshop™ automatically centers the feature selected to magnify or demagnify. The TV presentation is very effective for images but ineffective for text. TV presentation is especially functional as an enhancement for class sizes below thirty. A computer projector while also effective (especially for larger classes) is seldom as convenient or available. BANISH, N., WEISS BIZZOCO, R. L. W., LU, M., AND SAAVEDRA, S. Department of Biology, San Diego State University, San Diego CA 92182-0057. DAPI based discovery of a new group of extremophiles: hyperthermal acidophilic rods We report here on the discovery of a new group of rod shaped hyperthermal acidophiles living in nature at temperatures of 70–80°C at pH 2–3. At pH 2 the maximum temperature at which these organisms were observed was 88°C. That these rods are organisms is supported by DNA specific DAPI staining and combined phase contrast fluorescence microscopy. In most acid hot springs examined, these rods were the predominant organisms above 70°C. They were found from 65–88°C, pH 2–3. Two types of rod shaped cells exist, one with a cell wall structure that resembles the Sulfolobus wall and others that have a multilayered Gram negative type cell wall. 24 hour slide immersion studies at 65–75°C showed rods were the only attached entities, other than sulfur. 292 5TH CALIFORNIA MICROSCOPY COLLOQUIUM BARANSKI, M.1, BERDOUGO, E.1,3, SANDLER, J.S.1,3, DARNELL, D. K.2, AND BURRUS, L.W.1,4 Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco CA 94132 Department of Biology, Lake Forest College, 555 N. Sheridan Road, Lake Forest, IL 60045 3 These two authors contributed equally to the work. 4 Corresponding author: LBurrus@sfsu.edu 1 2 The dynamic expression pattern of frzb-1 suggests multiple roles in chick development The Wnt family of secreted proteins has been shown to have multiple roles in embryonic development. Frzb proteins have been shown to inhibit Wnt signaling. In order to gain a better understanding of the potential roles of Frzb-1 in chick development, we utilized the polymerase chain reaction (PCR) to isolate a partial cDNA of the chick orthologue of frzb-1 and compared its expression pattern to that of Wnt-1 and Wnt-5a, by whole mount in situ hybridization. The earliest expression of cfrzb-1 is in cells fated to become neural ectoderm in streak stage embryos. After primitive streak stages, cfrzb-1 expression is gradually attenuated in the closing neural tube of the trunk and is concomitantly up-regulated in neural crest cells. Finally, cfrzb1 appears in the condensing mesenchyme of the bones in both the limb and the trunk. Expression patterns for Wnt-1 and Wnt-5a suggest possible antagonistic relationships between cFrzb-1 and these Wnt proteins. BARLOW, S.B. Electron Microscope Facility, San Diego State University, San Diego CA 92182-4614. Education outreach: resources for microscopy in education Microscopy is a powerful tool for studying and comprehending structure. It is now possible to operate several different kinds of microscopes with a desktop computer and the appropriate software, regardless of whether the microscope is adjacent to that computer or located at a distance. With high-speed Internet access and the increasing sophistication of both computer hardware and software, students and researchers can remotely access instruments not owned by their own laboratory. However, simply making the transmission electron microscope, the scanning electron microscope, the atomic force microscope, and the confocal light microscope remotely accessible does not insure the incorporation of these tools into the precollege and college academic curriculum. Several steps can be taken toward this end. First, instructors need to write laboratory exercises that can be incorporated into student laboratories or daily lesson plans. These exercises would emphasize student involvement in planning and preparing samples to be viewed either in local or remotely accessible instruments. Once written, these exercises would be collated and disseminated. Second, students can use computer simulations that mimic digital microscope operation. Third, microscopists can make available collections of microscope images that can be used as part of the lesson plan. These three activities are being carried out by the Microscopy Society of America sub-committee on Education Outreach. Further descriptions of microscopy outreach resources are available at http://www.sci.sdsu.edu/emfacility/outreach.html BART, K.M.1, AND BAILEY, D.G.2 Department of Biology, Hamilton College, Clinton NY 13323 Department of Geology, Hamilton College, Clinton NY 13323. 1 2 Enhancing undergraduate education through remote operation of an SEM/ EDS system At Hamilton College we have tried to broaden and diversify the pool of students that are able to use the EM facilities. We have done this by providing remote access to the SEM/EDS system in large undergraduate courses. Recent advances in digital technologies have provided new opportunities for dramatically increasing the number of students exposed to SEM and X-ray microanalytical systems. Recently, students in a 200-level Geology course (Mineralogy) have used the system by remote operation from their normal classroom. During class, approximately 25 students are able to examine and investigate geological samples in real time. It is one of the best ways to teach students about the chemistry and structure of complex, polyphase inorganic materials. The hardware and software needed to control the system from a remote computer terminal are relatively inexpensive and simple to configure. The main requirements are: 1) a 10 BaseT Ethernet network, 2) computer with remote access software (e.g. LapLink™); and 3) a data projection system. Use of the EM facilities has expanded enormously. Most significantly, many more students are now using the instrument for senior research projects. We believe that remote operation of microscopy facilities will become one of the most efficient and effective ways to teach important scientific techniques and concepts to large numbers of students. BIZZOCO, R.L.W., AND DIAZ, R.G. Molecular Biology Institute, San Diego State University, San Diego CA 92182. Effect of carbohydrate on the function of the contractile vacuole in Chlamydomonas reinhardtii When Chlamydomonas reinhardtii 406 (mt-), a wall-less, flagella-less mutant is incubated in low concentrations of sorbitol (10 to 70 mM), the time of the contractile vacuole cycle (filling to emptying) is increased. We have examined this response using both phase-contrast and electron microscopy. In normal control cells filling starts when small vesicles fuse into a large vacuole. Water expulsion begins with a rapid vesiculation of the contractile vacuole. This causes a reduction in vacuole volume and an abrupt collapse. During volume reduction water is somehow forced out of the cell at the vacuole-plasma membrane juncture. After sorbitol treatment the contractile vacuole cycle time increases and pulsation of the two vacuoles 5TH CALIFORNIA MICROSCOPY COLLOQUIUM becomes asynchronous, i.e. they fill at the same time. The contractile vacuole in normal control cells has a vectoral tendency towards a permanent attachment site on the inner plasma membrane surface. We observed this vectoral membrane flow during vacuole collapse both by video microscopy and gluteraldehydeosmium tetroxide fixed cells. We present micrographs that depict these chemically fixed cells and cells prepared by rapid freezing in liquid propane, followed by freeze infiltration in osmium tetroxide. Cells exposed to hypertonic medium, such as sorbitol, would ordinarily be expected to lose water and crenate as a consequence of high osmotic strength in the external medium. These cells do not crenate. Instead, the contractile vacuole behavior becomes abnormal. Although it is unclear why the contractile vacuole changes, we offer a contractile vacuole pumping model. BLAKE, D. NASA Ames Research Center, Moffett Field CA 94035-1000. Life on Mars, and life in the Mars meteorite. Has the latter taken on a life of its own? In 1996, McKay and co-authors announced the discovery of evidence of ancient life in a meteorite from Mars. The data consist of five lines of evidence which, while individually weak, according to the authors, together provided compelling evidence of ancient bacterial life on Mars. Since the original proposal was made, three of the lines of evidence have been shown to be either inconclusive, or to have no bearing on life. The two remaining lines of evidence are difficult to either prove or disprove, but are, in the opinion of many researchers, not persuasive. Regardless of the resolution of the Mars meteorite controversy, many believe that Mars could have harbored early life. Indeed, the search for evidence of life is one of the central themes of NASA’s Mars exploration program. BLINK, A. 1,2, KWONG, A.1,2, ROZENFELD, S.1, LARGMAN, C.1, AND KOMUVES, L.G.2 Molecular Hematopoiesis Laboratory Microscopy and Molecular Histology Laboratory, V. A. Medical Center, UCSF, San Francisco CA 94121. 1 2 The localization of PRX2, a homeobox gene in developing human skin PRX2 is a homeobox protein thought to regulate nuclear transport of interacting homeobox proteins. Although earlier we established the spatial and temporal changes in the expression of a set of homeobox genes during fetal skin development, the role of PRX2 in skin is not known. The aim of this investigation was to determine PRX2 localization in developing human skin. At 10 weeks both the fetal epidermis and periderm stained strongly for PRX2. However, at 17 weeks the basal cells stained only weakly whereas the suprabasal cells continued to display a strong staining for PRX2. Moreover, whereas PRX2 clearly localized to the cytoplasm of basal cells, both cytoplasmic and nuclear localization was seen in suprabasal cells. The anlangens of the forming hair follicles and cells in the 293 dermal papillae did not stain for PRX2. At 21 weeks epidermal staining was similar to that observed at 17 weeks. In the fully formed hair follicles strong staining was observed in the outer root sheath and the bulge. No staining was seen in the dermal papillae and fibroblasts. In adult skin a decreased cytoplasmic PRX2 staining was seen in the spinous and granular layers. However, intense staining persisted in the hair follicles and sweat glands. BUCHANAN, J., AND SMITH, S.J. Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford CA 94305. Observations on the fine structure of cell cultures prepared by microwave processing Microwave processing is a rapid and reliable method used to prepare monolayer cell cultures for E. M. studies. We have studied hippocampal neurons and MDCK cells by combining confocal and correlative electron microscopy. Glass coverslips can be processed in less than two hours using conventional fixatives and resin mixtures. The ultrastructure of these cultures is similar to bench processed tissue. Observations on early contacts between developing neurons reveal the presence of synaptic vesicles and dendritic filopodia. CHANG, E. AND ANTIPA, G.A. Department of Biology, San Francisco State University, San Francisco CA 94132. Morphogenesis in the ciliate Conchophthirus: structural detail of thigmotactic field formation The ciliature and infraciliature of the thigmotactic field of C. curtus (Scuticociliatida) is distinctive. Morphogenesis was studied by Antipa and Hatzidimitriou (J. Protozool. 28:206-214). Here we report further study by TEM and provide additional developmental detail. Within the nine stages of morphogenesis previously described by Antipa and Hatzidimitriou, we have distinguished two major phases of thigmotactic field development. Phase 1 includes stages 1 through 5 during which the replication zone is formed by duplication of basal bodies within all kinetal rows. Monokinetids initially replicate to produce dikinetids, and then trikinetids, and finally quadkinetids. This results in a band dense with basal bodies that encircles the cell and demarcates the presumptive proter from the opisthe. All of these new basal bodies become ciliated. Phase 2 begins at stage 6, in which the linear arrangement of the replication zone basal bodies becomes remodeled. On the left side of the organism, in the area of the opisthe’s developing thigmotactic field, there is one more round of basal body proliferation. This produces one additional basal body for each basal body already present and establishes a zig-zag arrangement of basal bodies characteristic of the thigmotactic field. Each new basal body becomes ciliated at the end of stage 6, and the thigmotactic field of the opisthe is complete. The proter inherits the parental thigmotactic field. 294 5TH CALIFORNIA MICROSCOPY COLLOQUIUM CISNEROS-MORENO, Y., AND BIZZOCO, R.L.W. Department of Biology, San Diego State University, San Diego CA 92182-0057. Contractile vacuole function in Chlamydomonas This study defines the membrane events in the contractile vacuole cycle of Chlamydomonas reinhardtii, a single cell green alga. It provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis. The contractile vacuole (CV) is an organelle that serves primarily as a pump to remove excess water entering the cell by osmosis from dilute solutions. It has been reported that fluid discharge from contractile vacuoles is caused by the systolic pressure and that the rate of discharge is higher when the vacuole size at the start of discharge (systole) is greater. Our study stands in contrast to previously published work in Chlamydomonas which offers a fusion model (CV-plasma membrane) for CV discharge. A loss of both water and CV membrane is postulated to occur as a result of CV-plasma membrane fusion. Our data show no such loss and that CV membrane persists in the cytoplasm throughout the pumping cycle. This provides evidence against a fusion mechanism. We also show continuous attachment of the main CV membrane to the plasma membrane. No previous study has shown this. This study defines the different stages of the contractile vacuole cycle by correlating observations made by video- and electron microscopy. The results we present indicate that the complete cycle consists of slow filling and rapid emptying and that the CV may act as a pump, releasing water through a pore at a plasma membrane attached site. CONTI, N., AND ANTIPA, G.A. Department of Biology, San Francisco State University, San Francisco CA 94132. Buccal ultrastructure of Conchophthirus curtus: Implications in the arrangement of vestibular basal body fibrillar structures The buccal ultrastructure and infraciliature of C. curtus has been examined by transmission electron microscopy. Dikinetal vestibular rows have zigzag ciliated basal bodies where both have kinetodesmal fiber, transverse fiber, transverse and postciliary microtubular ribbons. Monokinetal vestibular rows have ciliated basal bodies each with kinetodesmal fiber, transverse fibers, transverse and postciliary microtubular ribbons. Three polykineties contain ciliated basal bodies in three main kinetal rows with a fourth offset. These basal bodies have kinetodesmal and transverse fibers and postciliary microtubular ribbons. Haplokinety has zigzag ciliated basal bodies with transverse fibers and postciliary microtubular ribbons. DKU has monokinetal unciliated basal bodies with postciliary microtubular ribbons. Oral rib ridge has three cytoplasmic layers and membrane junctions with four and two microtubules apiece. Cytopharynx has numerous stacked microtubular ribbons along one side. Previous studies show that basal bodies of the locomotor region are monokinetal and each has a kinetodesmal fiber, transverse fiber, transverse and postciliary microtubular ribbons; those of the thigmotactic region are dikinetal, zigzag and have posterior basal body only with kinetodesmal fiber, transverse fiber and postciliary microtubular ribbons. Comparisons of dikinetal rows with these latter regions suggest that dikinetal vestibular rows (which are somatic in origin) probably receive some cellular signal that makes the dikinetal rows have a characteristic paired zigzag arrangement and imposes locomotor infraciliature over both basal bodies. CORTES, P., AND YOUNGBLOM, J. Department of Biology, California State University, Stanislaus, Turlock CA 95382. The effect of cigarette tar extract on sister chromatid exchanges in Leber’s Hereditary Optic Neuropathy Leber’s Hereditary Optic Neuropathy (LHON) is a maternally inherited disorder that involves various genes associated with the electron transport system in the mitochondria. The primary clinical manifestation associated with this condition is loss of vision, usually bilaterally. Several environmental factors have been implicated in exacerbating the onset of the symptoms. Two frequently cited factors are excessive cigarette smoking and alcohol consumption. Despite these speculations, there have been no reported systematic studies to examine the role of these factors in the LHON disorder. In this study, we investigated the effect of varying doses of aqueous cigarette tar (ACT) extract on the level of sister chromatid exchanges in one LHON patient and one control subject. The sister chromatid exchange (SCE) assay is a highly sensitive method for detecting DNA damage to cells upon exposure to various agents. The more damaging the agent, the higher the level of sister chromatid exchanges. Lymphocyte cultures were prepared from whole blood samples obtained from the LHON and control individuals. The cells were exposed to varying doses of ACT added at the initiation of the cell cultures. To visualize the SCEs, the chromosomes were stained with propidium iodide and analyzed with a confocal microscope. Using the Mann-Whitney test, the results showed a significantly higher level of SCEs in the LHON patient following exposure to ACT as compared with the control individual. This study provides the first direct evidence that specific components of cigarette smoke have a more deleterious effect on LHON cells than normal cells. DEMAREE, R.S., AND FOX, N.E. Department of Biological Sciences, California State University, Chico, Chico CA 95929. One hour microwave sample preparation for SEM. The use of microwave-assisted sample preparation for TEM is slowly gaining acceptance as a routine method of tissue processing (Giberson, Demaree and Nordhousen, 1997. J. Vet. Diagn. Invest. 9:61-67). We 5TH CALIFORNIA MICROSCOPY COLLOQUIUM have recently reported a one hour microwave enhanced procedure for preparing bacteria for SEM (Fox and Demaree, 1999. Microsc. Res. Tech. 46:338–339, 1999). We have extended these studies and now report that we can successfully prepare eukaryotic cells for SEM in under one hour with microwave enhancement. Fixatives successfully employed include Parducz’ fixative, glutaraldehyde and osmium tetroxide. Either ethanol or acetone may be used to dehydrate cells. Drying was most rapid using hexamethyldisilazane in a convection oven, but critical point drying can also be used. We have prepared HeLa cells, human lymphocytes, Peranema sp., Spirostomum sp., as well as various plant and animal cells. Examination by field emission SEM at magnifications up to 100,000× revealed no preparation artifacts. EVANS, K. L. Department of Biological Science, California State University, Hayward CA 94542. Using scanning electron microscopy to characterize previously unreported freshwater sponges from the California Delta The California Delta forms an inland coastal environment and is one of the most extensive bodies of water in California. Sponges previously unreported were found there year round, mainly growing on the pondweed Egeria but also on the side of certain covered marina floats. Scanning electron microscope (S.E.M.) techniques were used to identify the most common small gray sponges based on structure and asexual reproductive stages. In freshwater sponges one method of asexual reproduction is by gemmules, which have a resistant coat containing unique gemmoscleres made of silica. Such silica spicules interfered with additional transmission electron microscopy. Gemmosclere rotules (“teeth”) were detected, which numbered fewer than 12 and were 20mm in length, suggesting the species Ephydatia muelleri (Lieberkuhn). One problem with this identification is the presence of a microsclere as part of the gemmule coat; these are not reported to be present in this species. A similar sponge is Spongilla, also reported in much of North America. Invertebrates growing over the sponge surface also were examined, indicating the bryozoan Plumatella, growing within and upon some sponges. The ecological role of such animals is hypothesized as patchy but active filters of very small particles, occupying a niche very different from other animals. Keywords: California Delta, freshwater sponges, Ephydatia muelleri, gemmules, gemmoscleres, rotules, bryozoan. GAERTIG, J. Department of Cellular Biology, University of Georgia, Athens GA 30602-2607. Molecular and microscopic analysis of organelle assembly in Tetrahymena Microtubules form structural frameworks of cells and organelles which are essential for maintaining 295 shape and play major roles in cell movement, intracellular transport and cell division. Different microtubule systems (e.g. cytoplasmic network, axonemes, centrioles, and mitotic spindle) contain MTs that have distinct lengths, spatial arrangement, and dynamics. Our goal is to uncover the molecular mechanisms which regulate the assembly and maintenance of distinct microtubular organelles. The ciliated protozoan Tetrahymena thermophila maintains at least 17 distinct microtubule structures in a single cytoplasm. Our studies implicate two mechanisms in the assembly and maintenance of distinct microtubular systems 1) targeting of specific motor proteins to specific microtubules and 2) post-translational modifications of a subset of microtubules. We found that kinesin-II, a microtubular motor protein, is preferentially targeted to cilia and plays an essential role in ciliary assembly and maintenance. Although disruption of kinesin-II genes gave a complex phenotype including paralysis, cell division arrest and lethality, all these changes appear to be induced by the loss of cilia. Distinct microtubules are subjected to unique combinations of several types of postranslational modifications. We show that polyglycylation, a postranslational modification specific to cells with axonemes, is essential for survival and required for normal functions of multiple types of microtubules. GRIFFITH, E. Boston Technology Center, FEI Company, 66A Concord Street, Wilmington MA 01887-2185. New types of imaging and analytical data available from an ESEM™ In an SEM, the ability to image materials without a conductive coating is the starting point for enhancing the level of imaging information available from the material. Additionally, the ability to image materials in an SEM in different states and the ability to image materials as they change state, provides opportunities for collecting new sample information. The Environmental Scanning Electron Microscope (ESEM™) has a specimen chamber operating mode that allows for a low pressure range of up to 20 torr of operator specified gases. These gases can be inert, reducing, or oxidizing. Water vapor is also a gas that can be used in ESEM™;. The use of sample stages with temperature control, in combination with an operator specified gas, provides for dynamic, in-situ, sample experimentation. Examples of imaging materials wet and hot will be explored in this session. GRIZZI, F.1, INTERNATI, C.M.2, AND DIOGUARDI, N. 3 1 Researcher of Scientific Direction, Istituto Clinico Humanitas, Rozzano, Milano, Italy, 2 Myeloma and Transplantation Research Center, Arkansas Cancer Research Center, UAMS, Little Rock, Arkansas, 3 Scientific Director of Istituto Clinico Humanitas, Rozzano, Milan, Italy; President Centro Medicina Teoretica, University of Study, Milan, Italy Functional and morphological quantitative evaluation of human myeloma dendritic cells 296 5TH CALIFORNIA MICROSCOPY COLLOQUIUM Dendritic cells are considered the most important professional antigen presenting cells, distinguished by their potency and ability to initiate primary T cell responses in vitro and in vivo. The most common characteristic of these cells is the irregular morphology which is strictly linked to their immunological function. Despite the qualitative visual perception of this morphological complexity, is not available a quantitative method for measuring the irregular shape of these cells for comparing this quality with the specific immunological function of these cells. In fact, the dendritiform morphology cannot be correctly measured by conventional Euclidean geometry which is limited to regular structures, practically unknown in nature. Instead, natural forms can be measured by the fractal dimension, which represents the space-filling property of an irregular object. In this paper, we describe a fractal system, for the quantitative analysis of structural changes during dendritic cell differentiation from blood monocytes, obtained by human myeloma blood. Fractal dimension was compared with the expression of MHC surface antigens and adhesion/costimulation molecules. Moreover, our study was undertaken to clarify the morphometric estimations by an image analysis system, of various cytological features that characterize these cells. The fractal and morphometric analyses provided data (p < 0.0005) which suggest that the early development of irregular dendritiform morphology reflects high modifications in immunological properties of the cell. Furthermore, the study of the fractal properties of dendritic cells is likely to reveal more about its morphology as well as the kinetics of expression of several immunologically important surfaces antigens during the monocyte differentiation pathway to dendritic cells. INGMIRE, P.D., LALLY, D., AND HE, Z.H. Department of Biology, San Francisco State University, San Francisco CA 94132. Tissue and cellular characterization of cell Wall-Associated receptor protein Kinases (WAK) in transgenic plants The plant cell wall plays a dynamic role and functions not only as a mechanical structure but as an important component in signaling pathways as well. The members of a family of transmembrane proteins, called cell Wall-Associated receptor protein Kinases (WAK), are tightly associated with the cell wall and play a critical role in mediating signals between the extracellular environment and the cytoplasm. The five members of the WAK family exist as a highly conserved gene family clustered together as a 30 kb region on chromosome one in Arabidopsis thaliana. They are characterized as having a cytoplasmic serine/threonine kinase which shares high homology among the members, a single transmembrane spanning region, and extracellular domains unique to each of the members that could serve to bind ligands specific for each receptor. Genetic manipulation of members of the WAK fam- ily results in severe morphological alterations in the plant. Transgenic plants show stunted growth, advanced senescence, and necrosis. Microscopic analysis of leaves stained with lactophenol trypan blue and examined using a dissecting microscope confirms cell death in manipulated transgenic plants. Using scanning electron microscopy (SEM), it was also shown that manipulated dwarf plants do not show markedly fewer cell numbers but rather reduced cell size, suggesting that disruption of members of the WAK family affects normal cell expansion. SEM analysis also shows developmental disruption of trichome formation on young leaves, and reduced root hair and lateral root formation on affected plants. This work was supported by NIHMBRS grant 2 S06 GM52588-04 and NIH-RIMI grant (Z.-H. H). ISAACSON, M. Cornell University, Ithaca NY 14853. Nanoscopy: A new paradigm for microscopy in the next century The last decade has seen a veritable explosion of new microscopies aimed at the exploration of inner space. In the past however, the tools used for this exploration were large scale complex instruments, scanning transmission and scanning electron microscopes for example. In fact, the smaller the object to be explored, the larger and more complex was the instrument. This has changed with advent of scanned tip instruments; not necessarily the complexity, but the size scale of the “interrogator.” As we look at the next century there will be this paradigm shift in how we go about the exploration of inner space. No longer will the observing tools be orders of magnitude larger than the object to be viewed, but rather on the same order of magnitude in size. This shift has occurred largely thru the ability to use large scale tools to create small scale devices. In this talk, we will explore this paradigm shift, and review the micro observational tools we have at hand and predict what we might have as we begin the new millennium. KOMUVES, L. G. Microscopy and Molecular Histology Laboratory, University of California, San Francisco, V. A. Medical Center (190), San Francisco CA 94121. Activation of peroxisome proliferator-activated receptor-α regulates keratinocyte differentiation Ligands of peroxisome proliferator-activated receptor-α (PPARα) stimulate keratinocyte differentiation and accelerate epidermal development in fetal rat skin explants (Komuves et al., J. Invest. Dermatol. 1998, 111: 429-433). The aim of the present study was to determine if topically applied PPARα ligands (clofibrate and Wy-14,643) could regulate keratinocyte differentiation in adult epidermis. Topical treatment with PPARα activators resulted in a decreased epidermal thickness. The expression of 5TH CALIFORNIA MICROSCOPY COLLOQUIUM structural proteins of spinous and granular layers (involucrin, filaggrin, loricrin) increased following topical treatment with PPARα activators. This treatment also decreased cell proliferation and increased apoptosis in the epidermis. Furthermore, topical treatment with PPARα activators resulted in rapid normalization of both subacute (induced by repeated barrier abrogation) and chronic (caused by essential fatty acid deficiency) hyperproliferative skin conditions. Topical treatment with PPARα activators resulted in a decrease in epidermal hyperplasia in both models of hyperproliferation. Following topical clofibrate treatment, PCNA-expressing cells were restricted to the basal layer, similar to normal epidermis. In hyperproliferative epidermis the expression of involucrin, filaggrin, and loricrin was decreased. Following topical treatment with PPARα activators staining for these mRNAs and proteins increased towards normal levels. Finally, topically applied clofibrate also increased apoptosis. The present study demonstrates that topical PPARα activators have profound effects on gene expression, cell proliferation and apoptosis both in normal and hyperproliferative epidermis. This study identifies PPARα activators as novel potential skin therapeutic agents. KWONG, A.1,2, ROZENFELD, S.1, LARGMAN, C.1, AND KOMUVES, L. G.2 Molecular Hematopoiesis Laboratory Microscopy and Molecular Histology Laboratory, V. A. Medical Center, UCSF, San Francisco CA 94121. 1 2 Immunohistochemical localization of HoxB13 during fetal mouse development Homeobox genes regulate embryonic development, including organogenesis. To understand the role of HoxB13 in mouse development (E13-E17) we analyzed HoxB13 localization by immunohistochemistry. Strong HoxB13 staining was seen in fetal epidermis but not in hair follicles and dermis. Interestingly, the stratified keratinizing epithelium of the tongue did not stain for HoxB13 whereas strong staining was seen in the oral mucosa. No HoxB13 staining was found in developing teeth. Striated muscle cells, including skeletal muscles, the diaphragm, the musculature of the tongue, heart muscles stained strongly for HoxB13 whereas no staining was seen in smooth muscle. The liver parenchyma did not stain for HoxB13 during E1315, but was weakly positive later. The thymus and haemopoietic tissues (liver and bone marrow) did not stain for HoxB13. Similarly, no staining was seen in the pancreas and in the salivary glands. In the lung HoxB13 staining was restricted to the airways, no staining was seen in the respiratory epithelium. Weak staining was found in the collecting ducts in kidney. No HoxB13 staining was observed in the intestinal tract (stomach, small intestine, large intestine). Whereas the ependymal cells in the 297 choroid plexus displayed intense HoxB13 staining, no staining was seen in the central nervous system (brain and spinal cord). Therefore, HoxB13 has complex expression pattern during organogenesis in fetal mouse. It is mainly expressed in organs of ectodermal and mesodermal origin (such as epidemis, striated muscles), whereas it is not present in organs originating from the endoderm (such as the intestinal tract and digestive glands). LAU, B.1,2,3, KWONG, A.1,2, LARGMAN, C.1, AND KOMUVES, L.G.2 1 Molecular Hematopoiesis Laboratory, V. A. Medical Center, UCSF, San Francisco CA 94121 2 Microscopy and Molecular Histology Laboratory, V. A. Medical Center, UCSF, San Francisco CA 94121. 3 B. Lau (A. Lincoln High School) was supported by the UCSF Science and Health Education Partnership Summer Internship Program. Double immunofluorescent staining with tyramide-mediated signal amplification using mouse monoclonal antibodies Fluorescence-based immunohistochemical detection methods are essential in double-labeling studies where information about the spatial relationship between different proteins is sought. The feasibility of double-labeling experiments, however, is often compromised by the limited availability of primary antibodies raised in different species. Our goal was to determine whether mouse monoclonal antibodies could be used for double immunofluorescence with tyramide-mediated signal amplification (TSA). To establish doublelabeling TSA on paraffin-embedded sections, we used monoclonal antibodies against human keratin 14 and filaggrin. These proteins were specifically detected in single-labeling TSA, using an HRP-conjugated antimouse IgG as a bridge reagent. In the double-labeling experiments the antigens were detected sequentially, with FITC- or Cy3-labeled tyramide reagents. A set of controls established that TSA during the localization of the first antigen did not interfere with TSA detection of the second antigen. Moreover, TSA prevented the non-specific deposition of the bridge antibody to the sections during the detection of the second antigen. Using this technique we were able to identify a novel cell in dysplastic epidermis of basal cell carcinomas. These solitary cells are wedged between basal cells and possess an unusual phenotype, expressing both early (keratin 14) and late markers (filaggrin) of keratinocyte differentiation. LAUZON, C.R., AND POTTER, S. . Department of Biological Sciences, California State University, Hayward, Hayward CA 94546. Nitrogen utilization by bacteria in the gut of two tephritid species. Tephritid flies consume a variety of microorganisms while feeding on natural food sources such as bird feces, plant structures, and aphid honeydew. Despite the consumption of an array of microbial species, only two bacterial species are consistently recovered from 298 5TH CALIFORNIA MICROSCOPY COLLOQUIUM their intestines. This selection suggests that these bacteria contribute to the welfare of the insects. Although the exact mechanism(s) for selection of these bacterial species remains to be defined, we present evidence that the bacteria participate jointly in nitrogen cycling for the fly using biochemical assays alone and coupled with electron microscopy. LIM, J.J., AND KOCH, R.A. Department of Biological Science, California State University, Fullerton, Fullerton CA 92834. Integrins and cadherins as cell adhesion molecules in ascidian sperm mitochondrial translocation. Mitochondrial translocation in the sea squirt, Ascidia ceratodes, is the movement of the sperm mitochondrion from the head to tail during fertilization and is an actin:myosin- dependent process that defines sperm activation and provides the force for penetration (Lambert & Lambert, Dev. Biol. 106:307, 1984). Activation of myosin filament formation has been previously supported by the work of Koch et al. (Dev. Biol., submitted). Actin polymerization, a process not well studied in ascidian sperm, requires the appropriate actin binding proteins and multi-protein complexes that anchor species-selective cell adhesion molecules to the sperm cell plasma membrane for stability and initiation of signal transduction. These cell adhesion molecules mediate the association of actin with egg surface anchor proteins to form multi-protein complexes that recruit signaling molecules and activate actin-myosin dependent motility (Porter & Hogg, Trends in Cell Biol., 8:10, 1998). I hypothesize that integrins, a family of transmembrane glycoproteins that consist of noncovalent heterodimers, are present in A. ceratodes sperm and are responsible for anchoring and initiating mitochondrial translocation during fertilization. However, integrins have not yet been identified in the ascidian sperm. To test this hypothesis, I plan to use fluorescently tagged and colloidal gold tagged antibodies to detect the presence of integrin in A. ceratodes sperm using light and electron microscopy. Preliminary evidence at the light microscope level using anti-integrin monoclonal antibodies (Transduction Labs, #141720) supports the hypothesis. The alternative hypothesis that adhesion is via cadherin molecules is currently being tested. Electron microscopy will follow. (Supported by CSUF Biology; NIH R15HD36500.) MATEY, V.E., DEVAUX, C.A., AND WALSH, T.A. da is similar in both sexes and formed by two large lips. Each lip is armed with tripartite tooth, external tooth and numerous lateral teeth. Two knob-like papillae, anterior sensory amphid, and three sensitive porous structures are placed on the lip surface. Dilated cuticle surrounding cephalic end forms the collarette with two papillae. Posterior end of male and female worms is different. Male: caudal alae is well developed and supported by four pairs of pedunculated papillae. Cloaca is located in the upper part of tail, number of spicules varies. Two spicules were found in four males, one spicule in three males and none in two males. Seven button-like papillae are associated with cloaca, another three pairs are situated below it. Posterior receptors, phasmids, are placed before the second pair of papillae. Ventral surface of the male tail shows three different patterns of ornamentation specific for this parasite. Female: vulva is located in the anterior part of body. Conical posterior end bears crescent anal opening and few phasmids. MILLER, J.J., AND ANTIPA, G.A. San Francisco State University, San Francisco CA 94132. Ultrastructure of Fernandez-Galiano stained Conchophthirus curtus The Fernandez-Galiano ammoniacal silver carbonate method is frequently used for viewing the macronucleus and the kinetal rows of ciliates by means of light microscopy. Light micrographs of Conchophthirus curtus indicate that ciliary tips, basal bodies, kinetodesmal fibers, and the macronucleus are stained. The DKU (a putative group of submerged basal bodies) does not appear to stain. This could be due to differences in the fiber system of the DKU when compared to other kinetal rows. Transmission electron micrographs confirm that ciliary tips, basal bodies (particularly the transition zone and the cartwheel region), kinetodesmal fibers, and the macronucleus are stained. In addition, the transverse fiber appears to pick up the stain. Staining of the transverse fiber and the ciliary tips is a novel finding. It is possible that some feature of the cilium confers thigmotactic ability to C. curtus which is also responsible for the argentophilicity of the tips. However, cilia in all areas of the cell stained in the same way. Could it be that all of the cilia of C. curtus are potentially thigmotactic and the density of cilia in the thigmotactic region as compared to the other regions makes this region sticky? MORGAN, C.L.1, LAUZON, C.R.2, AND SMITH, N. R.2 Department of Biology, San Diego State University, San Diego CA 92182. Department of Mathematics and Computer Science; Department of Biological Sciences, California State University, Hayward CA 94542. Surface ultrastructure of parasitic nematoda Physaloptera turgida For the first time, SEM was used to study key taxonomic characters of Physaloptera turgida Rud., 1819 (Nematoda: Spiruroidaea) from the opossums Didelphis virginiana. 10 male and 10 female worms from stomach of 5 infected animals trapped in San Diego County were examined. Cephalic end of P. turgi- Development of a standard model for remote control of scientific instrumentation Faculty and staff of the Microscope and Graphic Imaging Center (MAGIC) at California State University, Hayward (CSUH) have been involved for several years in developing software for a standardized architecture for remote control of scientific instrumentation. Commercial software and freeware are 1 2 5TH CALIFORNIA MICROSCOPY COLLOQUIUM available for connecting computers to control and communicate with scientific instruments, but they do not afford the flexibility, the security, and the scalability that custom software can offer. When we acquired our Philips (FEI) XL-40 scanning electron microscope, the challenge to develop a software system to gain remote users a standardized Internet-accessible system was undertaken. Using the remote control software developed by faculty and students in the Department of Mathematics and Computer Science, the SEM has been controlled locally, nationally and internationally. The SEM has been used by sister CSU campuses, local community colleges and high schools, and through inhome use by staff and faculty. An adjunct project involving HPLC remote instrumentation control incorporates the same basic computer-system architectural design. This model system can be used to control any digital visual-output instrumentation. MORIMUNE, M.1, 2, 3, KWONG, A.2, ROZENFELD, S.1, LARGMAN, C.1, AND KOMUVES, L.G.2 Molecular Hematopoiesis Laboratory Microscopy and Molecular Histology Laboratory, V. A. Medical Center, UCSF, San Francisco CA 94121. 3 M. Morimune (A. Lincoln High School) was supported by UCSF Science and Health Education Partnership Summer Internship Program. 1 2 Immunohistochemical localization of HOXB4 during fetal human skin development Our previous studies have established the spatial and temporal changes in the expression of several homeobox genes during fetal skin development (Differentiation 62:33-41, 1997; J. Invest. Dermatol. 110:110-115, 1998). To expand these studies, we report here the developmental changes in HOXB4 expression in fetal human skin. Whereas at 10 weeks only weak staining was seen in the epidermis, at 17 weeks the basal cells strongly stained for HOXB4. Moreover, several cells stained in the core of the forming hair follicles. No staining was seen in the dermal papillae. At 21 weeks, in the stratified epidermis the basal cells (which are proliferating cells) stained strongly for HOXB4. Moderate staining was also observed in the suprabasal cells. In the fully formed hair follicles the cells in the outer root sheath, in the bulge (where the epidermal stem cells are located), and in sebaceous glands stained for HOXB4. However, no staining was seen in the dermal papillae and fibroblasts. This expression pattern (presence of HOXB4 in proliferating cells and in stem cells) suggests that HOXB4 plays a role in the regulation of cell proliferation and/or stem cell renewal in developing fetal human epidermis. MURPHY, J. A. San Joaquin Delta College, Microscopy Technology Center, 5151 Pacific Ave., Stockton CA 95207. firstname.lastname@example.org. The Challenges of Teaching Research Microscopy at a Community College San Joaquin Delta College has a unique 2 yr Microscopy Certificate Program which successfully places 95% of its students either in the job market 299 directly or as transfers to other 4 yr institutions. Microscopy, in general, is research oriented in that it provides tools to solve problems. The program has advanced project courses which train the student to do a research project from the very beginning stages of experimental design through data collection and analysis. There are however, many challenges for teaching research microscopy at the community college level, e.g., library resources, how class logistics affect experiments, equipment necessary, cost, staff, and space required. Although research is not the mission of most Community Colleges, in order to provide high technology skills required in today’s job market, they need to address the elements of scientific method, e.g., critical thinking, problem solving, organization, and team work. Instrument based programs need more financial and space support than traditional programs and absolutely need District and Administrative support at both the local and state level minimally. In addition, funding agencies need to re-think how they distribute funds, i.e., funding of research training activities such as for supplies, instrumentation, and buildings, regardless if it is a 2 yr, 4 yr, or graduate institution. We must meet these challenges in innovative ways as in the end, it all reflects on our society’s commitment to our young people, to make them the best that they can be. They are, after all, our future. PARVIN, B.1, TAYLOR, J.1, AND O’KEEFE, M.A.2 ICSD, Lawrence Berkeley Laboratory, Berkeley CA 94720 MSD, Lawrence Berkeley Laboratory, Berkeley CA 94720. 1 2 Deepview: A channel for distributed microscopy. The Materials Microcharacterization Collaboratory (MMC), a pilot project of DOE2000, unites DOE user facilities at LBNL, ANL, ORNL, the University of Illinois and NIST. The MMC offers materials scientists access to an on-line virtual laboratory that collectively houses the nation’s most advanced electron microscopes, expert microscopists, and other microcharacterization tools. As an integral part of the MMC, we have developed a generalized “Microscopy Channel” over the wide area network. A microscopy channel advertises a listing of available online microscopes, where users can seamlessly participate in an experiment, acquire expert opinions, collect and process data, and store this information in their electronic notebooks. This channel is a collaborative problem-solving environment (CPSE) that allows for both synchronous and asynchronous collaboration. Deepview, provides a layer of abstraction for controlling any type of microscope, a common set of utilities for information management and transaction, and the analytical capabilities needed for online microscopy. With Deepview, we have provided remote users with collaborative access to electron microscopes in Berkeley (LBNL) and Tennessee (ORNL) from several materials science laboratories sited at locations distributed across the country. 300 5TH CALIFORNIA MICROSCOPY COLLOQUIUM POTTER, S.E., AND LAUZON, C.R. Department of Biological Sciences, California State University, Hayward, Hayward CA 94542. Bacterial assemblages in the gut of two tephritid species. The true fruit flies are destructive insect pests of economically-important agricultural crops such as coffee, citrus, and nuts. Current control methods for these insects include the use of pesticides that affect nontarget species, pollute the environment, and are expensive. These are incentives for researchers to find new control methods that are species specific. In order to achieve this, the biology of the insect must be more completely understood. We have looked closely at the bacteria that reside in the intestines of two tephritid pests. It has been hypothesized that bacteria are important in the life cycle of these insects. We have found a close association exists between the insect gut tissue and bacteria. Bacteria attach to the peritrophic matrix and form a biofilm. The biofilm includes packets of bacteria, an architecture conducive to the flow of nutrients in and the excretion of waste products out of the gut. These data provide evidence that physical factors may be involved in selection of bacteria within the fly gut. SADREDDIN, A., LUONG, J., LEE, R., CARMEN, G., AND BRECKLER, J. Department of Biology, San Francisco State University, San Francisco CA 94132. Immunolocalization of cytoskeletal elements in retinal pigment epithelium (RPE) during phagocytosis RPE are phagocytic cells in the vertebrate eye that engulf rod outer segments (ROS) that are shed with light on-set. Phagocytosis is an actin and microtubuledependent process. Therefore we are studying actin and microtubule cytoskeleton during RPE phagocytosis. We are particularly interested in ADF (actin depolymerizing factor). ADF depolymerizes actin filaments in the cytoskeleton and allows them to perform and rearrange for phagocytosis. Immunocytochemistry is used to study the distribution of ADF using antibodies and secondary fluorescent detection methods. In addition, we are using Rhodamine-Phalloidin staining method to localize the F-actin in the cell and compare this localization pattern to the ADF pattern. We hope to answer the following questions: Where is ADF more concentrated, and does it localize in areas where phagocytosis is taking place? Does ADF co-localize with F-actin? In order to follow microtubules during phagocytosis, we are using anti-tubulin. We also plan to use anti-myosin VI to see if this particular type of myosin is involved. Two sets of experiments are in progress. One is on Sunfish and the other is on albino (unpigmented) rat eyes. In order to look at the presence of ADF in cells that are actively phagocytizing, we examined different time points. We performed rat eye dissection one hour after light on-set (8 am), at which there is a burst of ROS shedding and most of phagocytosis is taking place. Another experiment is the late afternoon dissection (approximately 8 hrs. after light on-set). In our study, we are comparing and contrasting these time points in order to determine any shift in distribution of ADF, actin, microtubules and myosin VI during several stages of engulfment in vitro. SMITH, N.R. Microscope and Graphic Imaging Center (MAGIC), California State University, Hayward CA 94542. Teaching Microscopy at CSUH: Then and Now Electron microscopy classes were first taught at CSUH in 1971. The classes were small in size with a maximum of six students for the transmission electron microscopy course and as many as 14 for the scanning microscopy course. Then, as now, students were required to produce a finished project at the end of each term. However, now rather than spending many hours in the darkroom, students are producing images on their computers. The development of remote microscopy has changed our perspective on how microscopy courses are taught and has reduced the limitations on class size. The one instructor, one student approach to teaching microscopy is elevated to another level and it can now be done in a lighted room, thanks to digital microscopy. Using telemicroscopy methods we can bring images into classrooms on our campus and outreach to local community colleges and high schools through the Internet thus serving many more students than in the past. More students can be exposed to the direct experience of all types of digital microscopy. Digital output on TEMs and SEMs has saved students a tremendous amount of time in producing micrographs and has reduced costs to the facilities. Students are able to save images on disks and output them from their homes or schools and incorporate them into imaging enhancing and analytical software. THOMPSON, J.M. Biology Department, California State University, San Bernardino CA 92407. Remote Microscopy - current status and future promise Remote microscopy can take several forms. On a simple level, it is nothing more than sending a micrograph to a colleague. This is a technology which has existed for years, but which limits the immediate interaction and value to both participants. New technologies now allow for real-time interaction between microscopists and colleagues who are at remote, often distant, sites. This interaction can take the form of person to person communication using videoconferencing hardware and software through internet connections, and multiple remote observers can also participate with the appropriate software. If the microscope can be controlled by a computer, the interaction may also occur by remote control of the microscope by the colleague at the remote site. Limitations in existing hardware and software include slow rates of internet transmission, low resolution images, small screen size of images, and often a lack of audio capabilities. In this symposium, we will discuss the hardware and software currently available, as well 5TH CALIFORNIA MICROSCOPY COLLOQUIUM as future developments to enable these remote interactions. Demonstrations of current remote microscopy from several sites will also be presented. TIFFANY, M.A. Department of Biology, San Diego State University. San Diego CA 92128. Studies on the skeletal development of Hermesinum adriaticum Zacharias, a flagellate from the Salton Sea, California. Hermesinum adriaticum is a rarely reported unicellular biflagellated organism of uncertain classification. Unlike silicoflagellates it has a solid internal siliceous skeleton. It was found in cyanobacterial mats and in the sediments of the Salton Sea, a salt lake in California, USA. Stages of the developing skeleton were studied with SEM and the progression from small tetraxial daughter skeletons to complete asymmetrical adult skeletons is presented. VANDIVER, A. AND MENON, J. Department of Biology, William Paterson University of NJ, Wayne NJ 07470. Retinoic acid and apoptosis The thyroid hormone-induced tissue remodeling that occurs during metamorphosis is generally accomplished by the death of larval cells (via apoptosis) and the growth and differentiation of stem cells into an adult organ. Retinoids (include retinol, retinal, trans and cis form of retinoic acid) have been linked to the induction of apoptosis in several in vivo and in vitro models. Thyroid hormone receptors and retinoic acid receptors belong to the same family of nuclear receptors. In Xenopus laevis it is shown that thyroid hormone receptors (TRs) and retinoic acid X receptors (RXRs) function together in vivo to regulate T3 response genes that are normally expressed during metamorphosis. Whole tails of Xenopus laevis were treated in vivo to regulate T3 response genes that are normally expressed during metamorphosis. Whole tails of Xenopus laevis were treated in vitro with retinol palmitate (2IU/ml). The tail epidermis was processed for scanning and transmission electron microscopy. Under SEM, retinol treated tail epidermis showed a loss of intercellular connections as well as microvilli at their apical surface. Also there was pitting on epidermal surface of retinol treated tail. In contrast, microvilli were clearly visible in epidermal cells was observed following retinol treatment. Muscle fibers were also affected: muscle bundles were less numerous and more degenerated in retinol treated tissue than in the control. Role of retinoids in inducing apoptosis will be discussed. WRIGHTING, D., AGNEW, B. AND BRECKLER, J. Department of Biology San Francisco State University, San Francisco CA 94132. Involvement of the actin cytoskeleton in the phagocytosis of rod outer segment (ROS) disks by retinal pigment epithelial (RPE) cells The Retinal Pigment Epithelium (RPE) consists of supportive cells beneath the vertebrate retina. One of 301 the primary functions of the RPE is to phagocytose rod outer segment (ROS) disks shed daily from the distal tips of the photoreceptors. The function of RPE phagocytosis is critical to retinal integrity, adhesion and survival. Striking evidence has been collected for the role of actin cytoskeletal elements during the process of phagocytosis and specifically during phagocytic cup formation. The initial purpose of our study is to directly visualize the dynamics of rapid actin filament turnover during RPE phagocytosis. Once G-actin addition involved in cup formation in normal cells has been characterized, we will study the specific roles of actin depolymerizing factor (ADF) in this process by transfecting cells with adenoviral vectors containing mutant ADF isoforms. In our studies, the use of the RPE-J cells, an immortalized RPE rat cell line, has proved invaluable. The RPE-J cells possess the important aspects of RPE morphology and function, including the ability to recognize and phagocytose ROS in vitro. We have developed an assay that incorporates exogenous fluorescent and biotinylated G-actin into RPE-J cells to visualize actin rearrangements during phagocytosis by both light and electron microscopy. Support for this research provided by NIH-RIMI #RR11805 to JB and NIH-BRIDGES to DW. YOUNGBLOM, J.H., WILKINSON, J., AND YOUNGBLOM, J.J./ Department of Biology, California State University, Stanislaus, Turlock CA 95382. Remote access confocal microscopy. In recent years, there has been a growing interest in the development of remote access capabilities to a variety of microscopy systems. While certain types of microscopes, such as electron microscopes and scanning probe microscopes have been well established for telepresence microscopy, there have been no reports of similar developments for the confocal microscope. At California State University-Stanislaus, home of the CSUPERB (California State University Program for Education and Research in Biotechnology) Confocal Microscope Core Facility, we have established a remote access confocal laser scanning microscope facility that allows users with virtually any type of computer platform to connect to our system. Our Leica TCS NT confocal system, with an interchangeable upright and inverted microscope set up, is accessible to any authorized user via the Internet by using a free software program called VNC (Virtual Network Computing). Once connectivity is established, users are able to control virtually all the functions to conduct real time image analysis and quantitative assessments of their specimen. The ease of connectivity to the microscope system and the relatively rapid speed of image transfer allow accessibility of this highly sophisticated instrument to the larger global community. A shared resource facility of this type, without geographic constraints, helps to catalyze the interaction of individuals with diverse interests and expertise, and promotes a center for academic collaboration and innovation.