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PubMed2006

Spatial refractive index measurement of porcine artery using differential phase optical coherence microscopy.

BACKGROUND AND OBJECTIVES: We describe a methodology to record spatial variation of refractive index of porcine renal artery using differential phase optical coherence microscopy (DP-OCM). STUDY DESIGN/MATERIALS AND METHODS: The DP-OCM provides quantitative measurement of thin specimen phase retardation and refractive index by measuring optical path-length changes on the order of a few nanometers and with a lateral resolution of 3 microm. The DP-OCM instrumentation is an all-fiber, dual-channel Michelson interferometer constructed using a polarization maintaining (PM) fiber. RESULTS: Two-dimensional en face dual-channel phase images are taken over a 150 x 200 microm region on a microscopic slide, and the images are reconstructed by plotting a two-dimensional refractive index map as the OCM beam is moved across the sample. CONCLUSIONS: Because the DP-OCM can record transient changes in the optical path-length, the system may be used to record quantitative optical path-length alterations of tissue in response to various stimuli. A fiber-based DP-OCM may have the potential to substantially improve in vivo imaging of individual cells for a variety of clinical diagnostics, and monitoring applications.

Animals↗
PubMed1959

A cellular reaction to antibody in tissue culture studied with electron microscopy.

The reaction of embryonic chick heart cells grown in tissue culture to specific guinea pig antiserum has been studied with electron microscopy. Heart fragments from chick embryos were cultured with a plasma clot. After being tested with antiserum or normal serum, they were fixed with buffered osmium tetroxide and embedded in butyl methacrylate before removal from the glass culture chamber. Thin cells found by phase microscopy to have reacted were sectioned in a plane parallel to the glass surface on which they had grown. The results confirm and extend observations made previously while the reactions were occurring. The plasma membrane, like that of the red cell, becomes disrupted or less resistant to trauma following the action of antiserum. The membranes of mitochondria and endoplasmic reticulum vesiculate and swell. Before nuclear shrinkage becomes prominent, the outer nuclear membrane separates over a large portion of the nuclear envelope and forms one or more large swollen blebs. Thus, the outer nuclear membrane shows a reactivity similar to endoplasmic reticulum. It is suggested that the various physical and chemical changes observed to follow the action of antibody and complement on fibroblasts may be explained by osmotic pressure differences between various cell components. Some basic similarities to the action of hemolytic agents on red cells are noted.

Animals↗
PubMed1984

A method for dissociation of viable human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labeling.

Collagenase dissociation, performed on 40 human breast cancers, yielded between 1 million and 50 million cells from less than 1 g of tissue from each tumor. Approximately 60% of cells (mean) was considered viable as judged by trypan blue exclusion and phase microscopy. On subsequent flow cytometric analysis, 20 cancers (50%) were considered diploid, three were tetraploid, and the remainder, hyperdiploid. Thymidine labeling (TLI) and flow cytometry following mechanical dissociation also were performed on 23 of these 40 tumors. Among this group of 23 cases, the median percentage of S-phase cells obtained by collagenase dissociation was 5.4, by TLI was 5.7, and by mechanical dissociation was 9.7. There was excellent correlation between the percentage of S-phase cells obtained by collagenase and TLI (r = 0.847, p = 0.0001) but only fair correlation between the percentage of S-phase cells obtained by mechanical dissociation and TLI (r = 0.597, p = 0.0027). The percentage of S-phase cells obtained by either collagenase or mechanical dissociation predicted whether a tumor was above or below median TLI in 19 of 23 cases (p = 0.0018). Estrogen receptor positivity or negativity did not predict whether a tumor was above or below median TLI (r = 0.283, p = 0.130) or above or below median S-phase fraction following collagenase dissociation (r = 0.218, p = 0.182), nor did quantitative estrogen receptor correlate significantly with TLI (r = 0.283, p = 0.13) or S-phase fraction (r = 0.218, p = 0.18).

Breast Neoplasms↗
PubMed2004

Effects of phase change on reflection in phase-measuring interference microscopy.

We show by analytical and numerical calculations that the phase change on reflection that occurs in interference microscopy is almost independent of the numerical aperture of the objective. The shift of the microscope interferogram response due to the phase change on reflection, however, increases with the numerical aperture. Measurements of the interferogram shift are made with a Linnik interference microscope equipped with various numerical-aperture objectives and are reported and compared with theory.

Journal Article↗
PubMed1971

Platelet storage at 22 degrees C; metabolic, morphologic, and functional studies.

Platelets stored at 22 degrees C for transfusion purpose have been examined with metabolic, morphologic, and functional studies. Evaluations were made of platelet-rich plasma (PRP) stored for 3-4 days and platelet concentrates (PC) stored for 24 hr. During these periods, lactate accumulated continuously without significant change in platelet count, pH, or plasma glucose. Platelet glycogen fell dramatically both chemically and by electron microscopy, but adenosine triphosphate (ATP), adenosine diphosphate (ADP), and intracellular potassium did not change. After storage, the cell's capacity for glucose utilization through glycolysis, the hexose monophosphate shunt, and the tricarboxylic acid cycle appeared to be intact. Although platelet volume during storage did not change, disc to sphere transformation was observed by phase microscopy. Platelet aggregration with ADP was reduced even after 1 day of storage. After transfusion of stored platelets to thrombocytopenic recipients, recovery of platelet glycogen and capacity for aggregation occurred within 24 hr. In summary, the platelet remains surprisingly intact during the intervals studied; those defects which do develop are reversible in the circulation of a thrombocytopenic recipient if viability has been maintained. A "storage lesion" responsible for loss of viability has not been defined.

Adenine Nucleotides↗
PubMed2005

On the physical transformations of processed pharmaceutical solids.

Atomic force microscopy Phase Imaging, an adaptation of Tapping Mode AFM was used to visualise physico-mechanical variations on the surface of crystalline materials after being subjected to mechanically induced lattice damage. Large crystals (100-500 microm) of lactose were nucleated on AFM sample stubs, imaged and subjected to a milling process. The milled samples were then imaged at specific humidity using Phase Imaging. Phase and Amplitude images of the re-crystallised lactose suggested an ordered crystalline state with multiple platelets present across the surface. In comparison, the morphology and surface properties after a 1-min mill time suggested milling had a dramatic effect on the surface characteristics of the re-crystallised lactose. Phase and Topographical imaging during exposure to elevated humidities (70% RH) indicated both morphological and physico-mechanical changes that may be linked to surface amorphous re-crystallisation.

Crystallization↗
PubMed1984

Contraction bands: differences between physiologically vs. maximally activated single heart muscle cells.

High resolution interference and phase microscopy were used to inspect the striations' appearance in shortening rat heart cells. Isolated cells were treated with detergent so that shortening could be graded by addition of calcium. Upon activation sarcomeres shortened to form (a) contraction densities in the middle of the A band at 1.7 micrometer (b) disappearance of the I bands and (c) phase brightening of the A bands at 1.6 micrometer and (d) dense Cz contraction bands at shorter lengths. These changes are totally consistent with the uniform sliding of myofilaments of previously accepted fixed dimensions. However, the striated patterns differed significantly in intact cells which were electrically stimulated to shorten. Here individual A bands remained distinct, without phase brightening or contraction band formation despite sarcomere shortening to less than the length of the A band as measured in the unstimulated cell. Maximal activation of intact cells by barium contracture elicited the full sequence of striation changes (a-d) seen in the chemically skinned cells. Light diffraction analysis gave comparable interpretation, i.e., the protein within the shortened sarcomere in the physiologically activated cardiac cell is more narrowly distributed than expected for thick filaments of fixed dimensions. These optical differences may reflect the restricted presence of the globular myosin heads at the ends of the cardiac sarcomere. This situation would explain the narrow range of the cardiac length-tension relation.

Animals↗
PubMed1974

Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells.

Myosin in human, rat, mouse, and chicken fibroblasts was localized by indirect immunofluorescence microscopy using antibodies prepared in rabbits against highly purified chicken gizzard myosin. Filaments containing myosin span the interior of the cells and are often parallel to each other. The majority of the fibers are concentrated toward the adhesive side of the cell. Most of the myosin-containing filaments show "interruptions" or "striations." From a comparison of these fibers in fluorescence and phase microscopy and from previous results on actin-containing fibers, we conclude that at least some of the cytoplasmic myosin can be found in the actin-containing fibers, which themselves have been shown to be very similar or identical to the microfilament bundles. The occurrence of both myosin and actin in the microfilament bundles provides a basis for the motility and contractility of the cell.

Animals↗
PubMed1967

Large bodies of Mycoplasma and L-form organisms.

The large bodies of various Mycoplasma and L-form organisms were studied by ultraviolet fluorescence microscopy of preparations stained with various fluorochromes. Primuline and Thioflavine S specifically stained the outer portion or rim of the large bodies, and the fluorescence characteristics of the stained bodies differed from those for other microorganisms and for spheroplasts and protoplasts. Small granular structures similar in size and morphology to minimal reproductive units were observed within some of the large bodies by phase microscopy and by fluorescence microscopy with acridine orange or Coriphosphine O. Micromanipulation probing of the large bodies revealed their elastic nature; many of the large bodies could be subdivided into two or more smaller circular bodies, each retaining the fluorescence staining properties of the parent body. Under these conditions, however, a few of the large bodies were ruptured, leaving the stainable outer boundary area as a stable residual structure. The large bodies were somewhat resistant to various rigorous treatments normally employed to eliminate viability of Mycoplasma and L-form cultures. Structures similr to large bodies were observed in various natural tissues, and structures resembling large bodies in size, morphology, fluorescence staining characteristics, and reaction to micromanipulation probing were reconstructed from an acetone extract of egg yolk. Overall, the large bodies of Mycoplasma and L-form organisms appeared to be structures resulting from accumulations of metabolic by-products and medium components within or on which minimal reproductive units had become entrapped, although it could not be ruled out that they might be defined structures specifically formed during culture as protective lipoidal sacs for the minimal reproductive units.

Bacteria↗
PubMed1987

Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy.

Examination of Giardia muris cysts stained with the fluorogenic dyes, fluorescein diacetate (FDA) or propidium iodide (PI), by either Nomarski differential interference contrast (DIC), phase, or brightfield (BF) microscopy revealed a direct correlation between morphologic appearance and uptake of FDA or PI. Cysts incorporating FDA were all morphologically identical and exhibited (1) a clearly delineated cyst wall, (2) the presence of a distinct space between cyst wall and cytoplasm, and (3) flagella recognizable at one pole of the cyst. FDA-positive cysts also had a hyaline appearance of the cytoplasm (examined at multiple focal planes with DIC) that made it very difficult to detect the presence of nuclei, intracellular axonemes of flagella, or curved elements of the adhesive disc. However, PI-stained cysts possessed a distinct morphology that was clearly different from that of FDA-stained cysts. Examination of PI-stained cysts demonstrated the presence of well-defined nuclei, intracellular axonemes, and curved elements of the adhesive disc. The cytoplasm of PI-stained cysts contained a fine granular texture as opposed to the hyaline appearance of FDA-stained cysts, and no space was observed separating the cyst wall from the underlying cytoplasm in the PI cyst. This light microscopic comparison of viable FDA- and nonviable PI-stained cysts of G. muris demonstrates that 2 types of cysts can be distinguished and implies that structural differences can be used to identify these subpopulations of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Inhibitory effects of Celecoxib and Sc-58125 on proliferation of human carcinoma of larynx Hep-2 in vitro.

The inhibitory effects of two kinds of selective cyclooxygenase-2 inhibitors on the proliferation of human carcinoma of larynx Hep-2 in vitro and their corresponding mechanisms were investigated. Hep-2 cells were cultured with two kinds of selective cyclooxygenase-2 inhibitors (Sc-58125 and Celecoxib) at various concentrations for 24 h. Morphological changes were observed under the phase microscopy and the growth suppression was detected by using MTT colorimetric assay. Apoptotic DNA fragments were observed by agarose gel electrophoresis, and the cell cycle and apoptotic rate were detected by flow cytometry (FCM) respectively. Hep-2 cells became rounded and detached from the culture dish after being treated with Celecoxib for 24 h, however, they remained morphologically unchanged with Sc-58125. Sc-58125 could increase G2 phase cells, whereas, Celecoxib rose G1 phase cells. Both of the two effects were dose-dependent. Moreover, the Hep-2 cells cultured with 50 micromol/L and 100 micromol/L Celecoxib showed obvious apoptosis, with the nuclear DNA of cells exhibiting characteristic DNA ladder. So Sc-58125 could inhibit the proliferation of Hep-2 cells by altering the G2 phase cells. However, Celecoxib had the same effect by changing the G1 phase cells and inducing apoptosis at higher concentration.

Antineoplastic Agents↗
PubMed1983

An historical review of quality control in hematology.

As the use of laboratory techniques in diagnostic medicine increase, it became obvious that standardized reference methods and stable controls were essential in order for results obtained in different laboratories to be comparable. Certain procedures and standards have been adopted to provide QC in hematology. Currently procedures include the following: cyanmethemoglobin for HGB determinations; particle counter for RBC and WBC counts; phase microscopy for PLT counts; and packed cell volume for HCT determinations. These serve as acceptable reference methods for multichannel hematology instruments. Instruments that are used must be carefully calibrated. Calibration of the spectrophotometer should be performed using an acceptable standard for hemoglobin. Calibration of the particle counter to determine RBC and WBC counts should be performed using a fresh sample of anticoagulated (EDTA) blood. PLT counting by phase hemacytometry should be used to obtain reference values for automated PLT counters. Centrifuges that are used to obtain microhematocrit values should be calibrated for maximum packing times and times checked against an electric clock. After the instruments have been calibrated, fresh whole blood is measured to determine a target value for each determination. Primary calibration of multichannel instruments is performed by calibrating each parameter to the target value. This is performed initially and then once each week. Two techniques that are commonly used to monitor instrument performance are: 1) the use of at least two levels of commercially prepared control samples to prepare either Levey-Jennings charts or the simple Cusum charts, which detect presymptomatic instrument problems; and/or 2) the use of 500-1,000 patient indices and mean WBC counts analyzed by the XB statistic to establish limits for monitoring instrument performance.

Hematology↗
PubMed1979

Phagocytosis of chrysotile fibers by pleural mesothelial cells in culture.

Pleural mesothelial cells (PMC) from the parietal pleura of rats were incubated in culture with UICC A chrysotile fibers. The sequence of events in phagocytosis was studied by electron microscopy: phases of attachment sequestration, and degranulation of lysosomal content into the phagocytic vacuole were observed. This demonstrates that PMC can engage in phagocytosis of chrysotile fibers.

Animals↗
PubMed2005

Biostability and macrophage-mediated foreign body reaction of silicone-modified polyurethanes.

In this study, the effect of soft segment chemistry on the phase morphology and in vivo response of commercial-grade poly(ether urethane) (PEU), silicone-modified PEU (PEU-S), poly(carbonate urethane) (PCU), and silicone-modified PCU (PCU-S) elastomers were examined. Silicone-modified polyurethanes were developed to combine the biostability of silicone with the mechanical properties of PEUs. Results from the infrared spectroscopy confirmed the presence of silicone at the surface of the PEU-S and PCU-S films. Atomic force microscopy phase imaging indicated that the overall two-phase morphology of PEUs, necessary for its thermoplastic elastomeric properties, was not disrupted by the silicone modification. After material characterization, the in vivo foreign body response and biostability of the polyurethanes were studied using a subcutaneous cage implant protocol. The results from the cage implant study indicated that monocytes adhere, differentiate to macrophages which fuse to form foreign body giant cells on all of the polyurethanes. However, the silicone-modified surfaces promoted apoptosis of adherent macrophages at 4 days and high levels of macrophage fusion after 21 days. These results confirm that the surface of a biomaterial may influence the induction of apoptosis of adherent macrophages in vivo and are consistent with previous cell culture studies of these materials. This study validates the use of our standard cell culture protocol to predict in vivo behavior and further supports the hypothesis that interleukin-4 is the primary mediator of macrophage fusion and foreign body giant cell formation in vivo. The impact of these findings on the biostability of polyurethanes is the subject of current investigations. Attenuated total reflectance-Fourier transform infrared analysis of explanted specimens provided evidence of chain scission and crosslinking at the surface of all of the polyurethanes. The silicone modification did not fully inhibit the oxidative biodegradation of the polyether or polycarbonate soft segments; however, the rate of chain scission of PEU-S and PCU-S seemed to be slower than the control polyurethanes. To verify this finding and to quantify the rate of chain scission in order to predict long-term biostability, an in vitro environment that simulated the microenvironment at the adherent cell-material interface was used to accelerate the biodegradation of the polyurethanes. Polyurethane films were treated in vitro for up to 36 days in 20% hydrogen peroxide/0.1M cobalt chloride solution at 37 degrees Celsius. Characterization with attenuated total reflectance-Fourier transform infrared and scanning electron microscopy showed soft segment and hard segment degradation consistent with the chemical changes observed after long-term in vivo treatment. The biostability ranking of these four materials based on rate of chain scission and surface pitting was as follows: PEU < PEU-S PCU < PCU-S. The silicone modification increased the biostability of the PEU and PCU elastomers while maintaining the thermoplastic elastomeric properties.

Biocompatible Materials↗
PubMed2004

Effects of glycolic acid on the induction of apoptosis via caspase-3 activation in human leukemia cell line (HL-60).

Apoptosis is a particular process that leads to the programmed cell death, and it has been a potentially therapeutic target of cancer. In this study, we evaluated the possible apoptotic effects of glycolic acid on human leukemia cell line (HL-60) in vitro. The morphological changes, cell viability, apoptosis induction, and caspase-3 activity were measured by phase microscopy, flow cytometry, and Western blot analysis. Morphological changes including shrinkage of cells were clearly demonstrated in HL-60 cells treated with increasing concentrations of glycolic acid. Cell viability was significantly affected by glycolic acid treatment in a dose- and time-dependent manner. In comparison to the control group, glycolic acid treatment had a profound effect in the induction of apoptosis by flow cytometric assays. In the cell cycle analysis, glycolic acid caused the increased percentage of cells in G2/M phase and the decreased expression of the cyclin A and cyclin B1, suggesting the induction of G2/M arrest of cell cycle by glycolic acid. Moreover, glycolic acid treatment promoted caspase-9 and -3 activity in a dose-dependent manner, but caspse-8 activity was not affected during the same process. Glycolic acid co-administrated with broad-spectrum caspase inhibitor, z-VAD-fmk, caspase-3 activity was blunted and apoptosis was also markedly blocked in HL-60 cells. In conclusion, glycolic acid-induced apoptosis in HL-60 cells may be through the activation of caspase-3. Future studies focusing on cell signaling and biological significance of glycolic acid-induced apoptosis would lead to exploring the mechanisms of chemotherapeutic potency of glycolic acid in human cancers.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Solid-phase immune electron microscopy with human immunoglobulin M for serotyping of Norwalk-like viruses.

A solid-phase immune electron microscopy method that uses protein A, goat anti-human immunoglobulin M (IgM), and human serum is described. Evaluation of the method with different immunoglobulin fractions showed that human IgM constituted the major virus capture antibody. The method appeared to distinguish between two Norwalk-like virus serotypes and demonstrated specific IgM responses to these serotypes in infected individuals. Further work is being carried out to define the relationship of these two serotypes to the previously described Norwalk agent (A. Z. Kapikian, R. G. Wyatt, R. Dolin, T. S. Thornhill, A. R. Kalica, and R. M. Chanock, J. Virol. 10:1075-1081, 1972), and four subsequent hospital outbreaks are being studied.

Humans↗
PubMed1962

Cellular injury in vitro: phase contrast studies on injured cytoplasm.

Unfixed, compressed acinar cells of rat pancreas, isolated by mechanical and enzymatic means, were examined by phase microscopy and photomicrographed using 35 mm film and electronic flash illumination. Similarly, observations were made on Walker carcinoma cells; in addition, these cells were treated with solutions containing either phosphatidase A or enzyme inhibitors. Acinar cells contained, besides nuclei, perinuclear droplets and secretion granules, various membranous and vacuolar structures. The basal cytoplasm showed parallel dark lines interpreted as endoplasmic reticulum. In some cells, fragmentation of the reticulum was followed by the direct incorporation of fragments into simple myelin figures. In other cells it appeared that phase-lucent linear structures and vacuoles were derived by dilatation of cisternae of the endoplasmic reticulum. Perinuclear fluid collections arose either by dilation of the perinuclear cisternae of the endoplasmic reticulum or by fluid dilatation of the nuclear envelope. Phosphatidase A disrupted early vacuoles of Walker carcinoma cells. From this and the direct involvement of elements of the endoplasmic reticulum in myelin figures, it was concluded that the membranes limiting the endoplasmic reticulum incorporate phosphatides in continuous layers. While many severely injured cells formed large vacuoles, others developed concentrically laminated myelin figures; it was concluded that both types of structure derived from phosphatides liberated intracellularly, the vacuoles by vesicular myelin figure formation.

Animals↗