Electro-optical Wavelength Selection Enables Confocal Ratio Imaging at Low Light Levels

1995 ◽  
Vol 1 (1) ◽  
pp. 1-11
Author(s):  
Roland Nitschke ◽  
Kenneth R. Spring
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Dyes ◽  
Fluorescence Emission ◽  
Argon Laser ◽  
Tunable Filter ◽  
Transmitted Light ◽  
Light Levels ◽  
Mechanical Filter ◽  
Ratio Imaging ◽  
Illumination Wavelength

A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.

Simultaneous Nomarski and fluorescence imaging during video microscopy of cells

1988 ◽  
Vol 255 (4) ◽  
pp. C566-C571 ◽  
Author(s):  
J. K. Foskett
Keyword(s):  
Fluorescence Imaging ◽  
Fluorescence Emission ◽  
Video Camera ◽  
Light Level ◽  
Optical Path ◽  
Transmitted Light ◽  
Low Light ◽  
Dichroic Mirror ◽  
Wollaston Prism ◽  
Side Port

A video microscope designed to allow low light level fluorescence imaging of cells during simultaneous high-resolution differential interference contrast (DIC) imaging, without the fluorescence light losses of 60-90% normally associated with this contrast-enhancement technique, is described. Transmitted light for DIC imaging, filtered at greater than 620 nm, passes through standard DIC optical components, (1/4 lambda-plate, polarizer, and Wollaston prism) before illuminating the cells. Transmitted light and fluorescence emission pass through a second Wollaston prism but not through the analyzer, which is repositioned more distally in the optical path. Prisms designed to reflect light out a side port of the microscope to a video camera have been replaced with a dichroic mirror. This mirror reflects fluorescence emission out the side port to a low light-sensitive video camera. The spectrally distinct transmitted light continues through the dichroic mirror to an overhead camera through a polarizer (analyzer), which completes the DIC optical path. The fluorescence and DIC images can be viewed simultaneously on side-by-side video monitors, examined sequentially by an image-processing computer, or examined simultaneously using a video splitter/inserter. The ability to image cells with high resolution simultaneously with low light level fluorescence imaging should find wide applicability whenever it is necessary or desirable to correlate fluorescence intensity or distribution with specific cell structure or function.

scholarly journals P039 Tracking intestinal epithelial cells with fluorescent dyes

2019 ◽  
Vol 13 (Supplement_1) ◽  
pp. S105-S106
Author(s):  
J Seidelin ◽  
F H Bergenheim ◽  
O H Nielsen
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Dyes ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial

scholarly journals ANTIGEN-ANTIBODY REACTIONS IN ALLERGIC HUMAN TISSUES

1960 ◽  
Vol 112 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Ben Z. Rappaport
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Dyes ◽  
Fibrous Tissue ◽  
Specific Antigen ◽  
Hair Follicles ◽  
Sweat Glands ◽  
Specific Staining ◽  
Intense Staining ◽  
Egg Albumin

Skin sensitizing human antibodies were conjugated with various fluorescent dyes without significant loss in their ability to combine with specific antigen in vitro. A biopsy of the skin site challenged with egg albumin in a patient sensitive to this antigen could be stained specifically by the fluorescent reagins. The epithelial cells of the epidermis, sweat glands, hair follicles, and sebaceous glands in such a challenged site showed specific staining. In addition to the epithelial cells, the most intense staining was in macrophages and in pericapillary cells. The endothelium of the small blood vessels stained less intensely. Fibrous tissue bundles were specifically stained. The immunologic staining with the conjugated reagins was similar to but more intense than that obtained with conjugated rabbit anti-egg albumin globulins.

scholarly journals Photoinduced and Classical Sol-Gel Synthesis: Spectral and Photophysical Behavior of Silica Matrix Doped by Novel Fluorescent Dye Based on Boron Difluoride Complex

Polymers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 2743
Author(s):  
Jameelah Al-Harby ◽  
Haja Tar ◽  
Sadeq M. Al-Hazmy
Keyword(s):  
Fluorescent Dyes ◽  
Sol Gel ◽  
Stokes Shift ◽  
Fluorescence Emission ◽  
Molar Absorptivity ◽  
Silica Matrix ◽  
Physical Parameters ◽  
Solid Media ◽  
Boron Difluoride ◽  
Glass Matrices

The boron difluoride complex is known as an extraordinary class of fluorescent dyes, which has attracted research interest because of its excellent properties. This article reports the optical properties such as absorption, fluorescence, molar absorptivity, and photo-physical parameters like dipole moment, and oscillator strength of new fluorescent organic dye based on boron difluoride complex 2-(1-(difluoroboraneyl)-1,2-dihydroquinolin-2-yl)-2-(1-methylquinoxalin-2-ylidene) acetonitrile (DBDMA). The spectral characterization of the dye was measured in sol-gel glass, photosol-gel, and organic–inorganic matrices. The absorption and fluorescence properties of DBDMA in sol-gel glass matrices were compared with each other. Compared with the classical sol-gel, it was noticed that the photosol-gel matrix is the best one with immobilized DBDMA. In the latter, a large stokes shift was obtained (97 nm) and a high fluorescence quantum yield of 0.5. Special attention was paid to the addition of gold NPs into the hybrid material. The fluorescence emission intensity of the DBDMA with and without gold nanoparticles in different solid media is described, and that displayed organic–inorganic matrix behavior is the best host.

scholarly journals Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes.

1990 ◽  
Vol 38 (9) ◽  
pp. 1323-1329 ◽  
Author(s):  
J Kapuscinski
Keyword(s):  
Nucleic Acids ◽  
Spectral Properties ◽  
Fluorescent Dyes ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Binding Mode ◽  
Fluorescence Yield ◽  
Cooperative Interaction ◽  
Fluorescence Emission Spectra ◽  
Yield Information

Interaction of cations with nucleic acids (NA) often results in condensation of the product. The driving force of aromatic cation-induced condensation is the cooperative interaction between ligand and single-stranded (ss) NA. This type of reaction is highly specific with regard to the primary and secondary structure of NA, and results in destabilization of the latter. The spectral properties of fluorescent intercalating and non-intercalating ligands [acridine orange, pyronin Y(G), DAPI, Hoechst 33258, and Hoechst 33342]-NA complexes were studied in both the relaxed and condensed form. The changes in absorption, excitation, and fluorescence emission spectra and fluorescence yield that followed the condensation were examined. Although some of these effects can be explained by changes in solvation of the fluorophore and its interaction with NA bases and the solvent, the overall effect of condensation on spectral properties of the complex is unpredictable. In particular, no correlation was found between these effects and the ds DNA binding mode of these ligands. Nevertheless, the spectral data associated with polymer condensation can yield information about the composition and structure of NA and can explain some nonspecific interactions of these probes.

scholarly journals Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

1990 ◽  
Vol 1 (12) ◽  
pp. 951-963 ◽  
Author(s):  
A P Morris ◽  
K L Kirk ◽  
R A Frizzell
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Inhibitory Pathway ◽  
Colonic Epithelial Cells ◽  
Chloride Currents ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Cell Current ◽  
Ht 29 ◽  
Cl Conductance

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.

pH-induced calcium transients in type II alveolar epithelial cells

1993 ◽  
Vol 264 (5) ◽  
pp. L448-L457 ◽  
Author(s):  
G. D. Gerboth ◽  
R. M. Effros ◽  
R. J. Roman ◽  
E. R. Jacobs
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Dyes ◽  
Cultured Cells ◽  
Alveolar Epithelial Cells ◽  
Metabolic Effects ◽  
Type Ii Cells ◽  
Type Ii ◽  
Intracellular Acidification ◽  
Isolated Cells ◽  
Alveolar Epithelial

Although both intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i) are highly regulated and have important metabolic effects in alveolar epithelial cells, little is known about the interrelationship between these two ions in alveolar epithelial cells. The present study examined changes in [pH]i and [Ca2+]i in isolated alveolar epithelial cells using the fluorescent dyes SNARF-1 and fura-2. Basal pHi values in freshly isolated and cultured alveolar epithelial cells were 7.27 and 7.24, respectively. Resting [Ca2+]i values in freshly isolated cells (53 +/- 5 nM) were lower than those in cultured type II cells (107 +/- 21 nM). pHi increased rapidly after addition of 25 mM NH4Cl in both cultured and freshly isolated cells and then decreased back toward baseline over the following 10 min. The rise in pHi was associated with a transient increase in [Ca2+]i. Resuspension of cells in an NH4Cl-free solution resulted in rapid intracellular acidification, which recovered over the subsequent 10 min. Removal of sodium or addition of 1 mM amiloride to the external solution slowed the rate of recovery from intracellular acidification, consistent with the participation of Na(+)-H+ exchanger in this process. In freshly isolated cells, [Ca2+]i increased following acidification and then decreased as the cells recovered from an acid load. In cultured cells, [Ca2+]i also increased following acidification but then remained elevated over the subsequent 10 min. The recovery of [Ca2+]i toward baseline values in fresh cells following acidification was dependent on the presence of external sodium. These data demonstrate that both increases and decreases in pHi of alveolar epithelial cells are associated with increases in [Ca2+]i and suggest that some of the metabolic effects of altering pHi may be secondary to increases in [Ca2+]i. The dependency of [Ca2+]i recovery following acidification on external sodium raises the possibility that freshly isolated type II cells have Na(+)-Ca2+ exchangers that contribute to the regulation of [Ca2+]i.

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