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PubMed1984

In situ evaluation of the functional state of chromatin by means of Quinacrine Mustard staining and time-resolved fluorescence microscopy.

A pulsed laser microfluorometer with high spatial and temporal resolution was employed to study the functional state of chromatin, using Quinacrine Mustard as the fluorescent probe. Corresponding segments of polytene chromosomes of embryo suspensor cells of Phaseolus coccineus and parenchymal cells of Helianthus tuberosus, in phases not involving DNA synthesis, were selected as models. The fluorescence decay time turned out to be a discriminating parameter for the chromatin fractions of differing functional engagement. The results were interpreted on the basis of a different accessibility of the DNA to the intercalating agent, as a result of the different structural situation of the chromatin. This fact determines modifications of the energy transfer rates between dye molecules in different quantum efficiency conditions, which results in variations of fluorescence decay times.

Chromatin↗
PubMed1995

Analysis of antifading reagents for fluorescence microscopy.

The utility of p-phenylenediamine, 1,4-di-azobicyclo-(2.2.2.)-octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half-life times of 96, 330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH 8.5), these half-life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes.

Coumarins↗
PubMedDate not supplied

Electrophoretic charge density and persistence length of DNA as measured by fluorescence microscopy.

Individual ethidium-stained DNA molecules, embedded in an agarose gel made with electrophoresis buffer (0.05 molar salt), are observed using a fluorescence microscope. In the first experiment, open circular 66 kilobase pair (kbp) plasmids, immobilized by agarose fibers threaded through their centers, display entropic "rubber" elasticity. The charged molecules extend in an electric field of several volts per centimeter and contract to a compact random coil when the field is removed. The extension of the plasmids as a function of field strength is consistent with the freely jointed chain model when the effective electrophoretic charge density is set at 15 e-per persistence length. In a second experiment, stained linear 48.5 kbp DNA molecules are observed as random coils immobilized in agarose. A measure of their size, here named the "maximal-X-extent," is taken for 100 molecules and found to average 1.47 mu. A Monte Carlo computer simulation of random coils (freely jointed chain model) gives the same maximal-X-extent value when the persistence length is set at 0.08 mu.

Chemical Phenomena↗
PubMed2005

In vivo analysis of angiogenesis in endometriosis-like lesions by intravital fluorescence microscopy.

OBJECTIVE: To establish a novel endometriosis model that allows for repetitive in vivo analyses of angiogenesis in ectopic endometrial tissue. DESIGN: Intravital fluorescence microscopic study. SETTING: Institute for Clinical and Experimental Surgery, University of Saarland. ANIMAL(S): Female Syrian golden hamsters equipped with skinfold chambers. INTERVENTION(S): Large (0.5 mm2) and small (0.1 mm2) endometrial fragments were mechanically isolated and transplanted autologously into skinfold chambers of untreated hormonally synchronized or bilaterally ovariectomized hamsters. MAIN OUTCOME MEASURE(S): Angiogenesis, vascularization, and microhemodynamics were analyzed over a 14-day period. RESULT(S): In untreated controls, endometrial fragments developed complete microvascular networks during the experimental observation period. Interestingly, microvascular blood flow was higher in large than in small fragments. Histologic examinations revealed proliferating endometriosis-like lesions with dilated endometrial glands surrounded by a richly vascularized stroma. Vascularization of endometrial fragments in synchronized animals did not differ from that of untreated controls. In contrast, endometrial fragments in ovariectomized animals showed a delay in angiogenesis and a significantly decreased blood perfusion, indicating the essential role of ovarian estrogens for ectopic vascularization and perfusion of endometrial tissue. CONCLUSION(S): This novel model of endometrial tissue transplantation is a useful experimental approach, not only to focus on the in vivo pathogenesis of endometriosis but also to develop antiangiogenic strategies for the treatment of this disease.

Animals↗
PubMed1999

Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles.

Using the sectioning effect of the two-photon fluorescence microscope, we studied the behavior of phospholipid giant unilamellar vesicles (GUVs) composed of pure diacylphosphatidylcholine phospholipids during the gel-to-liquid crystalline phase transition. We used the well-characterized excitation generalized polarization function (GP(ex)) of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN), which is sensitive to the changes in water content in the lipid vesicles, to monitor the phase transition in the GUVs. Even though the vesicles do not show temperature hysteresis at the main phase transition, we observed different behaviors of the vesicle shape, depending on how the GUV sample reaches the main phase transition. During the cooling cycles, we observed an increase in the vesicle diameter at the phase transition ( approximately 0.5-1%), followed by a decrease in the diameter when the vesicle reached the gel phase. During the heating cycles and close to the phase transition temperature, a surprising behavior is observed, showing a sequence of different vesicle shapes as follows: spherical-polygonal-ellipsoidal. We attribute these changes to the effect of lipid domain coexistence on the macroscopic structure of the GUVs. The "shape hysteresis" in the GUVs is reversible and largely independent of the temperature scan rate. In the presence of 30 mol% of cholesterol the events observed at the phase transition in the GUVs formed by pure phospholipids were absent.

2-Naphthylamine↗
PubMed1997

High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.

The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.

Animals↗
PubMed2001

[The electric pulse caused pore in the skin observed by fluorescence microscopy].

The objective of this study was to enunciate the difference in skin morphological changes between iontophroresis and electroporation for transdermal drug delivery (TDD). Fluorescence microscope was employed to observe the skin structure of human and snake in passive diffusion (control); iontophoresis was performed using direct current(0.5 mA/cm2) and electroporation was performed using intensive 380 V, pulse rate 4 ppm, pulse duration 5.5 ms, pulse number 100 and electric capacity 22 microF. Fluorescein was FITC. The results showed that for human skin in the passive, these were a few highlight fluorescent blots with scale of 15-30 microns and rough edge, but the edge of the blots became smooth and the scale did not change in the iontophoresis; when the scale of the blots was extended to 80-90 microns, the edge was smooth. For the snakes shin, the highlight blots were not sighted in the passive and iontophoresis, but they were found to be 25-30 microns in the electroporation. These data demonstrate that electric pulse can cause pore in the skin, thus creating a new route for drug permeation through skin.

Administration, Cutaneous↗
PubMed2003

Direct visualization of minimal cerebral capillary flow during retrograde cerebral perfusion: an intravital fluorescence microscopy study in pigs.

BACKGROUND: Retrograde cerebral perfusion (RCP) is used in some centers during aortic arch surgery for brain protection during hypothermic circulatory arrest. It is still unclear however whether RCP provides adequate microcirculatory blood flow at a capillary level. We used intravital microscopy to directly visualize the cerebral capillary blood flow in a piglet model of RCP. METHODS: Twelve pigs (weight 9.7 +/- 0.9 kg) were divided into two groups (n = 6 each): deep hypothermic circulatory arrest (DHCA) and RCP. After the creation of a window over the parietal cerebral cortex, pigs underwent 10 minutes of normothermic bypass and 40 minutes of cooling to 15 degrees C on cardiopulmonary bypass ([CPB] pH-stat, hemocrit 30%, pump flow 100 mL x kg(-1) x min(-1)). This was followed by 45 minutes of DHCA and rewarming on CPB to 37 degrees C. In the RCP group the brain was retrogradely perfused (pump flow 30 mL x kg(-1) x min(-1)) during DHCA through the superior vena cava after inferior vena cava occlusion. Plasma was labeled with fluorescein-isothiocyanate-dextran for assessing microvascular diameter and functional capillary density (FCD), defined as total length of erythrocyte-perfused capillaries per observation area. Cerebral tissue oxygenation was determined by nicotinamide adenine dinucleotide hydrogen (NADH) autofluorescence, which increases during tissue ischemia. RESULTS: During normothermic and hypothermic antegrade cerebral perfusion the FCD did not significantly change from base line (97% +/- 14% and 96% +/- 12%, respectively). During retrograde cerebral perfusion the FCD decreased highly significantly to 2% +/- 2% of base line values (p < 0.001). Thus there was no evidence of significant capillary blood flow during retrograde cerebral perfusion. The microvascular diameter of cerebral arterioles that were slowly perfused significantly decreased to 27% +/- 6% of base line levels during RCP. NADH fluorescence progressively and significantly increased during RCP, indicating poorer tissue oxygenation. At the end of retrograde cerebral perfusion there was macroscopic evidence of significant brain edema. CONCLUSIONS: RCP does not provide adequate cerebral capillary blood flow and does not prevent cerebral ischemia. Prolonged RCP induces brain edema. However, there might be a role for a short period of RCP to remove air and debris from the cerebral circulation after DHCA because retrograde flow could be detected in cerebral arterioles.

Animals↗
PubMed2001

Frequency-domain fluorescence microscopy with the LED as a light source.

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz--250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10--15 microm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C(12),with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.

3T3 Cells↗
PubMed1990

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles.

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36-48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell-cell contacts involved in a) the formation of the thyroid follicular lumen and b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC-Dextran and Cascade Blue conjugated to bovine serum albumin: CB-BSA). LY microinjected into IL of 2-9-day-old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 microns. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC-Dextran or CB-BSA microinjected into IL behaved as LY; the probes were restrained into the lumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15-30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2(+)-dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FITC-Dextran or CB-BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell-to-cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell-to-cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.

Acridine Orange↗
PubMed1991

Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells.

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells.

Animals↗