Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorescence Microscopy”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1989

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.

DNA↗
PubMed1998

Examination of blood-brain barrier transferrin receptor by confocal fluorescent microscopy of unfixed isolated rat brain capillaries.

Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti-transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer-aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.

Animals↗
PubMed2004

Two-color two-photon 4Pi fluorescence microscopy.

In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength.

Equipment Design↗
PubMed2005

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone.

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses. METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography. RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure. CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.

Animals↗
PubMed2004

Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents.

Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels. Pancreatic K(ATP) channels are composed of two subunits, a pore-forming inwardly rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing SUR1 in intact pancreatic beta-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact beta-cells.

Animals↗
PubMed1981

Evaluation of vasogenic edema in experimental brain tumors by cathodoluminescence and fluorescence microscopy.

Cathodoluminescence and fluorescence microscopy have been used to study vasogenic edema in experimentally induced brain tumors in rats. Both methods are suited for the demonstration of FITC- or TRITC-coupled antiserum, and thus allow the evaluation of serum protein extravasation. Cathodoluminescence is more time consuming and laborious than fluorescence microscopy, but it has distinct advantages: Contrast enhancement improves the differentiation between certain cell types, and the higher resolution of the scanning electron microscope allows the identification of subcellular regions which cannot be recognized by conventional fluorescence microscopy.

Animals↗
PubMed1983

[Bone structure in aseptic necrosis of the femoral head by light microscopy, contact microradiography, and fluorescent microscopy --comparing with osteoarthritis and rapidly destructive coxarthrosis].

In order to obtain further information of the pathological changes and bone structure of aseptic necrosis (AN), the present author carried out histological investigation on 20 femoral heads removed by prosthesis or total hip replacement from 18 patients. Both decalcified and undecalcified sections were investigated by microscopy, contact microradiography (CMR) and fluorescent microscopy. Schemata of individual cases were drawn to know the relationship between femoral head deformity and bone reactivity. Moreover, 19 femoral heads with secondary osteoarthritis (OA) removed from 18 patients and 7 with rapidly destructive coxarthrosis (RDC) from 6 patients were similarly investigated to compare with AN. The following results were obtained; Histologically, there are four different areas in AN. From the top of the femoral head, the lesion consisted of (1) subchondral necrosis, (2) granulation tissue, (3) an area of variable bone formation, and (4) normal bone tissue. As the advancement of the head deformity, fibrous component in the granulation increases and seems to divide the femoral head into necrotic and living areas, which by fibrous as well as enchondral ossifications become more prominent in the edge of the demarcation. As for the bone formation, four different types of bone tissues are discernible by CMR; (1) low calcified appositional bone and its absorption, (2) low calcified, appositional bone on wide trabeculae, (3) low calcified appositional bone on narrow trabeculae, and (4) irregularly calcified woven bone. Although during the comparatively early stage, the bone formation is seen in almost all of the living area, as the advancement of the head deformity, its activity decreases and results in overt head deformity to resemble that of OA which would be caused by mechanical factor. Increase of fibrous component in the granulation along with decrease of the bone formation would reduce the repairing activity of the femoral head. In the femoral heads with OA, a wedge-shaped area of bone formation is formed of which peak is located on the medial thickened cortex. In the femoral heads with RDC, a large amount of woven bone with irregular calcification is found in the bone formation area, in the upper part of a femoral head.

Adult↗
PubMed2006

Direct observation of amyloid growth monitored by total internal reflection fluorescence microscopy.

Most morphological investigations of amyloid fibrils have been performed with various microscopic methods. Among them, direct observation of fibril growth is possible using atomic force microscopy and fluorescence microscopy. Direct observation provides information about the rate and direction of growth at the single fibril level, which cannot be obtained from averaged ensemble measurements. In this chapter, we describe a new technique for the direct observation of amyloid fibril growth using total internal reflection fluorescence microscopy (TIRFM) combined with amyloid-specific thioflavin T (ThT) fluorescence. TIRFM has been developed to monitor single molecules by effectively reducing the background fluorescence in an evanescent field. One of the advantages of TIRFM is that one can selectively monitor fibrils lying along a glass slide, so that one can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent fibril growth of amyloid beta (1-40). The fibril growth was a highly cooperative process, with the fibril ends extending at a constant rate. Because ThT binding is common to all amyloid fibrils, the present method will have general applicability to the real-time analysis of amyloid fibrils.

Amyloid↗
PubMed2005

Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study.

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Microradiography and fluorescence microscopy of bone remodeling on the basal crypt of permanent mandibular premolars in dogs during eruption.

Alveolar bone of erupting teeth was studied in order to define the types of calcified tissues deposited as well as the rate of tooth growth. The third (P3) and fourth (P4) mandibular premolars of 30 dogs aged 12-24 weeks were analyzed by microradiography and microscopy in fluorescent and ordinary light. The bone plate separating P3 and P4 from the mandibular canal presented a complex arrangement of lamellar and woven bone, and even of chondroid tissue. During the pre-eruptive phase, this plate shifted towards the base of the mandible by means of selective resorption and apposition activities. As soon as the furcation was formed, bone apposition appeared on the alveolar side and became the main activity under P3 at the outset of eruption. Under the roots of P4 it occurred 4 weeks later. Dynamic morphometry in fluorescence microscopy showed that eruption progressed faster than the radicular growth. The formation of interradicular bone underwent the same acceleration as the eruption. However, though the tissues were formed at a high rate, it cannot be inferred therefrom that they are responsible for tooth shifting. They might just fill the space left by the erupting tooth.

Alveolar Process↗
PubMed2003

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over-expression of enhanced green fluorescent protein-tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge-coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.

Animals↗
PubMed1981

The effect of concentration on the binding of compound 48/80 to rat mast cells: a fluorescence microscopy study.

A fluorescent analog of the chemical histamine liberator, compound 48/80, has been synthesized by the covalent attachment of rhodamine to the 48/80 polymer (R-48/80). The histamine liberating characteristics of this analog were similar to those of the parent compound. The binding characteristics of R-48/80 to rat peritoneal mast cells were then studied using fluorescence microscopy. At concentrations that caused minimal secretory stimulation (less than 1.0 microgram/ml), R-48/80 bound to the mast cell surface in a diffuse manner, with no indication of patching or capping. When the cells were incubated at higher concentrations, where non-cytotoxic histamine secretion was stimulated, the drug bound heavily to the exposed granules, but not to unexposed granules or other cell organelles. At cytotoxic concentrations, R-48/80 caused extensive cell clumping, with the drug bound to masses of cell debris and released granules. Therefore, although R-48/80 binds initially to the cell membrane, its primary binding site at concentrations that induce secretion becomes the mast cell granule. The properties of these granules should thus be considered when studying the binding of compound 48/80 or other cationic drugs to rat peritoneal mast cells.

Animals↗
PubMed1996

[Ca2+]i in rat afferent arteriole during constriction measured with confocal fluorescence microscopy.

Using confocal fluorescence microscopy, we followed the time course of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) and endothelial cells (EC) of microperfused afferent arterioles isolated from rat juxtamedullary nephrons. Measurements were made while arterioles were exposed to pharmacological agonists or myogenic stimulation. Luminal addition of acetylcholine triggered an immediate increase of [Ca2+]i in EC and vasodilation. Addition of norepinephrine to the bath solution constricted the vessel and initiated periodic oscillations of [Ca2+]i in VSMC and synchronized vasomotion. Increase of perfusion pressure from 80 to 120 mmHg induced an immediate 9.6 +/- 2.9% (P < 0.05, n = 9) increase of [Ca2+]i in VSMC that was sustained. The arteriole dilated transiently when the perfusion pressure was increased, and persistent vasoconstriction to reduced diameter was observed after 35 s. When nitric oxide (NO) production in the perfused vessel was blocked with nitro-L-arginine methyl ester prior to the pressure step, similar profiles of change in VMSC [Ca2+]i were observed and persistent vasoconstriction began after 28 s. The same pressure step triggered an increase of [Ca2+]i in EC by 11.3 +/- 1.1% (P < 0.05, n = 5). These observations suggest that myogenic constriction of afferent arterioles was associated with an increase of [Ca2+]i in VSMC, and the constriction was delayed by endogenous NO production.

Animals↗
PubMed1986

A new and permanent staining method for starch granules using fluorescence microscopy.

A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.

Fabaceae↗