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PubMed1983

Enumeration of human lymphocyte subpopulations by immunofluorescence: a comparative study using automated flow microfluorometry and fluorescence microscopy.

These studies reveal that the enumeration of peripheral blood mononuclear cells by fluorescence microscopy and automated flow microfluorometry show a high degree of correlation whether the cells came from normals, individuals with common variable immunodeficiency, chronic lymphocytic leukemia of B cell origin or chronic lymphocytic leukemia of T cell origin. There was excellent agreement between these two methods when counting positive cells stained by the pan-T monoclonal antibodies OKT3 and Leu-1, the helper T reagents OKT4 and Leu-3a, and the suppressor T antibodies OKT8 and Leu-2a. The values obtained for B cells using a pan-B (HB-2) cell antibody analyzed by fluorescence microscopy and automated flow microfluorometry gave a correlation coefficient of 0.86. The percentage of cells identified by antibodies with reactivities toward peripheral blood monocytes (MMA or Leu-M1), the HLA-DR determinant, and HNK-1 (Leu-7) positive cells gave correlation coefficients of 0.90, 0.90, and 0.80 respectively when compared by the 2 methods mentioned above. These data suggest that comparable values for lymphocyte subpopulations in human blood samples can be obtained using the most convenient and available technology.

Antibodies, Monoclonal↗

PubMed1983

Differences in daunomycin retention in sensitive and resistant P388 leukemic cells as determined by digitized video fluorescence microscopy.

Cellular uptake and binding of daunomycin were studied using the digitized video fluorescence microscopy technique, in a sensitive and a resistant subline of P388 leukemic ascites tumor cells. When a 60-min time course of uptake was monitored, the sensitive cells had a 4-fold greater uptake than did the resistant cells. When the cells were perfused with drug-free medium, identical exchangeable levels of the drug were lost from both sublines. The difference in drug uptake could be accounted for entirely on the basis of differences in a slowly exchanging drug fraction which probably represents bound intracellular drug. In glucose-free medium, uptake of daunomycin was accelerated by metabolic inhibition to a greater extent in resistant than in sensitive cells. Furthermore, there was minimal decrease in the fluorescence when both sublines were perfused with drug-free medium. The addition of glucose to this medium induced a significant decrease in fluorescence in resistant but not in sensitive cells. These data raise the possibility that decreased drug uptake in resistant cells associated with decreased slowly exchanging drug fraction may be associated with an inherent defect in drug binding which is reversed by inhibition of energy metabolism. Parallel in vitro and in vivo studies revealed the presence of uptake heterogeneity; both sensitive and resistant cells contained subpopulations (20 to 30%) that have less or more fluorescence than the predominant pattern. This observation demonstrates the possible use of the digitized video fluorescence microscopy for recognizing subsets of cells with different drug susceptibility and to monitor the emergence of anthracycline-resistant cell populations.

Animals↗

PubMed1998

Effect of 2'-OH acetylation on the bioactivity and conformation of 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol. A NMR-fluorescence microscopy study.

The relationship between conformation, 2'-OH acetylation, and bioactivity of two fluorescent taxoids has been investigated by a combination of NMR and fluorescence microscopy techniques. These taxoids present the structure of taxol with the 7-OH group esterified with the N-(4'-fluoresceincarbonyl)-L-alanine group and with the 2'-OH group free (taxoid 2) or acetylated (taxoid 3). The larger water solubility of 2 and 3 compared with taxol allowed a detailed NMR study in DMSO-d6/D2O (3/7), showing that both taxoids adopt a similar collapsed conformation in which the hydrophobic groups 2-O-benzoyl, 3'-phenyl and 4-O-acetyl are in close proximity, with the fluorescein group displaying unrestricted motion. On the other hand, while taxoid 2 retains essentially the ability of taxol to induce in vitro microtubule assembly and to bind to cell microtubules, the 2'-acetylated derivative 3 does not show immediate activity. However, when taxoid 3 is left in the cell culture, the slow hydrolysis of the 2'-acetate group in the medium liberates the cytotoxic, microtubule-specific taxoid 2. The intense emission of this active derivative (2) allows the accurate recording of the drug-cell interaction from the very initial steps using fluorescence microscopy. These experiments show conclusively, for the first time in cell cultures, that a free 2'-OH group in taxol is essential for the recognition of the drug by the binding site of cellular microtubules.

Acetylation↗

PubMed1983

Comparison of analysis of bovine surface immunoglobulin bearing and peanut agglutinin binding lymphocytes by flow cytometry and fluorescence microscopy.

Bovine peripheral blood lymphocytes were examined for their binding to anti-immunoglobulin serum, peanut agglutinin, and mu, alpha, and epsilon heavy chain specific antisera by immunofluorescence. The percentage of total lymphocytes with positive staining was determined independently by flow cytometry and fluorescence microscopy. The correlation of data from both methods was best for analysis of total surface immunoglobulin and IgM bearing cells. The percentage of lymphocytes bearing surface immunoglobulin (B cells) was determined using both whole antiserum and a F(ab')2 reagent. Quantitation by flow cytometry did not show a significant difference when the two reagents were used, whereas fluorescence microscopy revealed a significant difference (p less than .05). The mean percent of total surface immunoglobulin bearing cells was 30 +/- 3% by either method. Flow cytometry gave significantly larger values than fluorescence microscopy for samples stained with fluorescein conjugated peanut agglutinin. Peanut agglutinin binding cells comprised 70 +/- 3% by flow cytometry and 51 +/- 3% by fluorescence microscopy. Similarly, there was a significant difference between both methods when IgA bearing lymphocytes were examined. Percentages of immunoglobulin E, A, and M bearing lymphocytes as well as total B and T cells in spleen and bronchial lymph node were determined by immunofluorescence using the cytofluorograph. Peanut agglutinin binding cells were less numerous in spleen and lymph node than in peripheral blood. Immunoglobulin E bearing lymphocytes increased from 0.07% in peripheral blood to 4% in spleen and 1.9% in lymph node. In this paper we demonstrate how flow cytometry can be used to examine a large number of samples in a rapid and reproducible manner. This is the first report in which bovine lymphocytes bearing surface IgE are quantitated.

Animals↗

PubMed2006

Characterization of mixed alcohol monolayers adsorbed onto a Au(111) electrode using electro-fluorescence microscopy.

A single-crystal Au(111) electrode modified with an adsorbed layer of 1-octadecanol (C18OH) or oleyl alcohol (OLA) in pure or mixed composition was characterized using electrochemical and in situ fluorescence microscopy. Cyclic voltammetry and differential capacitance measurements revealed a repeatable, potential-induced adsorption/desorption process of the surfactant to/from the electrode surface while charge density and film pressure measurements indicated quasi-ideal mixing of the two adsorbed alcohols. A layer less defective than pure C18OH was created with incorporated OLA. Optical characterization was accomplished using epi-fluorescence microscopy combined with electrochemistry (electro-fluorescence microscopy) through the incorporation of two fluorescent probes into the adsorbed surfactant layer. Since molecular luminescence is quenched by a nearby metal, fluorescence was only observed when the fluorescent dye/alcohol layers were desorbed and therefore separated from the metal surface. When desorbed, the structure of the alcohol layers were similar in character, revealing aggregated features which did not change in morphology over numerous desorption/re-adsorption cycles. We have also used the electro-fluorescence technique to estimate the distance separating the metal and desorbed surfactant and believe that the molecules are displaced from the electrode surface by a distance not more than 40 nm.

Adsorption↗

PubMed2004

Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.

This study investigated the capability of fibered confocal fluorescence microscopy (FCFM) to provide in vivo microvascular observations. FCFM is specifically designed for in vivo in situ observation thanks to a probe composed of a fiber bundle and micro-optics having a diameter as small as 650 microm. In the first part of the study, we compared the main characteristics of FCFM with those of intravital fluorescence microscopy (IFM). A mouse cremaster preparation was used as a common basis to allow for imaging with both modalities. We discussed the feasibility of obtaining quantitative measurements usually provided by IFM in the context of FCFM: morphometry, capillary permeability, functional capillary density, vasoconstriction and dilation effects. In addition, the possibility to visualize fluorescent red blood cells or leukocytes was also evaluated. Phototoxicity issues and limitations of FCFM were also discussed. We showed that FCFM allows observations and measurements usually provided by IFM and that the real-time capability of the system, as well as the flexibility and small diameter of the optical probe enable micro-invasiveness and can extend imaging capabilities for in vivo in situ observations when compared to IFM.

Animals↗

PubMed2003

Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy.

In a novel application of dual-channel high-speed two-photon fluorescence microscopy, the skin autofluores-cence and the transdermal fluorescent model drug spatial distributions were imaged simultaneously over precisely the same spatial coordinates. The dual channels enable the detection of the fluorescence emission wavelengths characteristic of the endogenous (intrinsic) skin fluorophores, as well as of the rhodamine-based model drug intensity emission at a different wavelength range of the fluorescence emission spectrum. These fluorescent model drugs delineate the oleic acid induced changes in permeant diffusion with respect to the skin structural features over the 0.3 mm by 0.3 mm skin area imaged per skin sample. The dual-channel high-speed two-photon fluorescence microscopy studies presented here provide evidence for the existence of intracorneocyte diffusion in addition to the commonly cited lipid multilamellar transdermal pathway. The image quantification analysis methodology introduced in this paper reveals that intracorneocyte diffusion exists for the hydrophobic (rhodamine B hexyl ester) and for the hydrophilic (sulforhodamine B) model drugs, in the absence of oleic acid chemical enhancer action. The mechanism of oleic acid chemical enhancer action, however, depends on the model drug physicochemical properties, where the oleic acid induces hydrophobic model drug localization to the lipid multilamellar region, while increasing the hydrophilic model drug lipid to corneocyte partitioning.

Biological Transport↗

PubMed1952