Fluorometric Quantification of Specific Chemical Species in Single Cells

Dolbeare, Frank

doi:10.1007/978-1-4684-1092-1_6

Cited by 7 publications

(3 citation statements)

References 128 publications

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“…Fluorescence techniques have become important tools in the study of molecular dynamics in cells. Fluorescent probes have been developed that are specific for certain macromolecules (13), organelles (14,15), compartments (16), and physiologically important parameters, such as membrane potential (17)(18)(19), pH (20), pCa (21), and specific enzymes (22). The development of fluorescent analogue cytochemistry has made possible the study of specific molecules and specific molecular interactions in living cells (23)(24)(25).…”

Section: Tin-containing Stress Fibers Terminate At Focal Contacts (4-10)mentioning

confidence: 99%

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

Lanni¹,

Waggoner²,

Taylor³

1985

The Journal of Cell Biology

134

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We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the watersoluble cationic dye, diI-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with diI-Ct~-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.Mammalian fibroblasts that are grown on a planar substrate exhibit a laminar morphology that is a result of spatial constraints and the forces of adhesion and motility. Cell-substrate contacts are classified by degree of apposition and by size. Reflection-interference microscopy (1-3) and electron microscopy (4) have been used to demonstrate the existence of close contacts (extended domains in which the distance between cell membrane and substrate is ~50 nm) and focal contacts (compact domains in which the separation may be as little as 10 nm).The distribution and interaction of the cytoskeletal and contractile proteins responsible for cell-substrate attachments and, by implication, the sites of application of forces to the substrate are now partly under...

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Section: Tin-containing Stress Fibers Terminate At Focal Contacts (4-10)mentioning

confidence: 99%

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

Lanni¹,

Waggoner²,

Taylor³

1985

The Journal of Cell Biology

134

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“…Also, microfluorometry is more convenient in measuring the DNA content of each cell for a chain-forming species. However, compared to flow cytometry, microfluorometry is slow, and its variation seems to be larger (Dolbeare 1981). The microfluorometry system used in this study is equipped with a low Light television camera and a video digitizer to detect the fluorescence intensity.…”

mentioning

confidence: 99%

Species-specific phytoplankton growth rates via diel DNA synthesis cycles. II DNA quantification and model verification in the dino-flagellate Heterocapsa triquetra

Chang¹,

Carpenter²

1988

Mar. Ecol. Prog. Ser.

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The DNA content of individual cells, after being stained by a DNA-specific fluorescent dye, was quantified by a TV camera-based microfluorometry system. Under proper dye concentrations, fluorescence intensity of a stained nucleus was in proportion to the amount of nuclear DNA. Durations of die1 cell cycle phases were estimated with DNA histograms from 2 partially synchronized cultures of the dinoflagellate Heterocapsa tn'quetra. Estimates of growth rate were calculated from phase durations and compared to the growth rates estimated by cell counts. Growth rates calculated by various mathemahcal procedures and sampling intervals were simllar, and the calculated growth rate via DNA synthesis gave a reasonably good estimate of the rate calculated by increase in cell density. This technique is promising in its application to species-specific growth rates in the field.

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“…Most of them have been constructed from chromophores that have either an amino or a hydroxyl group that participates in the electronic conjugation. Naphthylamine, amino coumarin, amino quinolines and rhodamine have served as chromophore bases for fluorogenic peptidase substrates [52,110]. Hydroxycoumarin and fluorescein have most often been used for construction of glycosidase, sulfatase, and phosphatase substrates [198].…”

Section: Fluorophores Used In Flow Cytoenzymologymentioning

confidence: 99%

Assessment of fluorochromes for cellular structure and function studies by flow cytometry

Petit¹,

Denis-Gay²,

Ratinaud³

1993

Biology of the Cell

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Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.

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Fluorometric Quantification of Specific Chemical Species in Single Cells

Cited by 7 publications

References 128 publications

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

Species-specific phytoplankton growth rates via diel DNA synthesis cycles. II DNA quantification and model verification in the dino-flagellate Heterocapsa triquetra

Assessment of fluorochromes for cellular structure and function studies by flow cytometry

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