Multimodal nonlinear optical microscopic imaging provides new insights into acetowhitening mechanisms in live mammalian cells without labeling

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

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Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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Roughness measurements of nonlinear optical polymer films by scanning tunneling microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152082 ◽

1989 ◽

Vol 47 ◽

pp. 22-23

Author(s):

I. H. Musselman ◽

R.-T. Chen ◽

P. E. Russell

Keyword(s):

Surface Roughness ◽

Scanning Tunneling Microscopy ◽

Nonlinear Optical ◽

Polymer Surface ◽

Light Extinction ◽

Scanning Tunneling ◽

Silicon Substrates ◽

Tunneling Microscopy ◽

Optical Polymer ◽

Total Light

Scanning tunneling microscopy (STM) has been used to characterize the surface roughness of nonlinear optical (NLO) polymers. A review of STM of polymer surfaces is included in this volume. The NLO polymers are instrumental in the development of electrooptical waveguide devices, the most fundamental of which is the modulator. The most common modulator design is the Mach Zehnder interferometer, in which the input light is split into two legs and then recombined into a common output within the two dimensional waveguide. A π phase retardation, resulting in total light extinction at the output of the interferometer, can be achieved by changing the refractive index of one leg with respect to the other using the electrooptic effect. For best device performance, it is essential that the NLO polymer exhibit minimal surface roughness in order to reduce light scattering. Scanning tunneling microscopy, with its high lateral and vertical resolution, is capable of quantifying the NLO polymer surface roughness induced by processing. Results are presented below in which STM was used to measure the surface roughness of films produced by spin-coating NLO-active polymers onto silicon substrates.

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Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158388 ◽

1990 ◽

Vol 48 (3) ◽

pp. 168-169

Author(s):

M. H. Chestnut ◽

C. E. Catrenich

Keyword(s):

Acridine Orange ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015900x ◽

1990 ◽

Vol 48 (3) ◽

pp. 290-291

Author(s):

Gustav Ofosu

Keyword(s):

Mammalian Cells ◽

Antitumor Agent ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Limiting Factor ◽

Sarcoma 180 ◽

Electron Microscopic ◽

Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

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