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At least 1,585 records · Page 88Linked to original sources
PubMed1998

Application of fluorescence microscopy to measure apoptosis in Jurkat T cells after treatment with a new investigational anticancer agent (N.C.1213).

1. Apoptosis as the mechanism of cell death induced by a new cytotoxic and anticancer agent (N.C.1213) was investigated by morphological and biochemical criteria in human Jurkat T leukemia cells. 2. The effect of N.C.1213 on the survival of Jurkat T, LV-50, H-9, and Molt-3 cells was measured. Jurkat T cells exhibited the highest response, with less than 10% of the cells remaining viable after exposure to 10 microM N.C.1213 for a 24 hr period. All other cell cultures were also affected but to a lesser extent. 3. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in Jurkat T cells after exposure to N.C.1213 and melphalan. The results indicated that melphalan was more cytotoxic than N.C.1213 as shown by the dye exclusion test. However, N.C.1213 showed a greater apoptotic index than melphalan. The IC50 of N.C.1213 in Jurkat T cells was determined to be 3.5 microM. 4. A DNA ladder (fragmentation of DNA into multimers of approximately 200 base pairs), which is one characteristic feature of apoptosis, was not detected when Jurkat T cells were exposed to N.C.1213. Hence it is probable that the key morphological events in apoptosis observed in the present experimental conditions precede the internucleosomal cleavage of DNA.

Antineoplastic Agents↗
PubMed2001

Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence microscopy.

Because small intestine submucosa (SIS) is a bioscaffold for tissue regeneration, we describe a method to analyze the material for growth peptides and for structural molecules. Immunofluorescence methods are described for relative quantification of abundant structural proteins. Additionally, a quantitative technique for comparison of the content of less abundant proteins in SIS was developed using the tyramide signal amplification (TSA) system that is applicable to paraffin-preserved tissue blocks. Frozen sections generally shredded when cut thinly enough to permit entry and washout of reagents. Five micrometer sections cut from paraffin blocks were immunolabeled for collagen, heparan sulfate proteoglycans (HSPG), FGF2, TGFbeta, and VEGF. Images of tissue sections were acquired by a linear image camera and quantified by densitometry after thresholding the signal to minimize nonspecific fluorescence. Immunohistochemistry was used to confirm the immunofluorescence methods. HSPG was widely distributed but concentrated in vessels. FGF2 was distributed diffusely and was associated with fibrous structures. VEGF was distributed mainly around vessels. TGFbeta was barely detectable above background. Collagen fibrils were distinctly present, and with a two-color fluorescence system, the distribution of components relative to collagen can be assessed. The anatomic structure of SIS is likely to play an important role in the regeneration of tissues, and factors in remnant vessels may facilitate penetration of the matrix along these avenues.

Animals↗
PubMed1975

Immunoreactive LH-RH neurons in the hypothalamus identified by light and fluorescent microscopy.

The sites of luteinizing hormone-releasing hormone (LH-RH) neurons and their axonal pathways in the hypothalmi of rats and mice were studied by the immunoglobulin-peroxidase bridge technique and b) the immunofluorescent isothiocyanate technique, using rabbit antiserum to synthetic LH-RH. Both of these techniques give similar results concerning LH-RH localization both in rats and mice. The immunoreactive LH-RH positive neurons are about 18-26 mu in diameter, mostly angular, pyriform or spindle shaped and very rarely oval or round. Most of these neurons are generally scattered in the hypothalamus. In the preoptic and medial preshiasmatic areas, ventromedial, arcuate nuclei and ventrolateral-premammillary body, the neurons are numerous and mostly in groups of 3-6. In other areas of the hypothalamus surrounding the suprachiasmatic area, supraoptic area, paraventricular nuclei and the anterior hypothalamic area, the LH-RH neurons are few and scattered. Very few neurons occur in the median eminence or in the infundibular stem. Major projections of LH-RH neurons apparently originate from preoptic, prechiasmatic and surrounding suprachiasmatic area, and from the anterior hypothalamic area. The fibres run in the fabrillar zone, descend in the external infundibular zone, and finally terminate for the most part around the capillaries of the primary portal plexus of the hypophysis. Other major groups of fibres apparently originate from the arcuate and ventromedial neurons. These fibres bend above the fibrous zone for some distance, ultimately descending in the zona palisadica and zona granulosa of the median eminence, towards the capillaries. The fibers of the ventrolateral-premammillary neurons are seen on the floor of the premammillary recess of 3rd ventricle and apparently terminate close to the capillaries in the infundibular region. Most of the LH-RH nerve fibres terminate near the primary portal plexus of the hypophysis, while some terminate in the pars tuberalis and a few seem to terminate in the 3rd ventricle. The LH-RH antiserum is ineffective after absorption with the antigen, so the staining reaction appears to be specific for LH-RH.

Animals↗
PubMed1989

Visualization of cyclic nucleotide binding sites in the vertebrate retina by fluorescence microscopy.

Cyclic nucleotides play a major role in cell signaling, especially in the nervous system. They act as cytoplasmic messengers in a wide range of physiological responses, but the spatial distribution of their sites of action within cells and tissues is not well-known. In the vertebrate retina, there is a class of well-characterized cGMP binding sites which control the permeability of cation channels in the rod outer segments (ROS), while cAMP is involved in several other systems in the inner retina. Biochemical studies of the cGMP-activated permeability in ROS have not distinguished between the subcellular compartments of disk and plasma membrane. By a new method using fluorescein-conjugated cyclic nucleotides, we have found strong cyclic GMP binding to the plasma membrane of the ROS, both on frozen sections of retina and in freshly isolated, leaky ROS. We also found a high density of cGMP binding sites on structures resembling the inner segment calycal processes. Little specific binding could be detected on the disk membranes or on any other retinal layer. In contrast, fluorescent cAMP did not label ROS, but gave a striking pattern of labeling on several deeper layers of the retina. These results suggest that the ROS plasma membrane has a much higher density of cGMP-controlled cation channels than the disk membranes, and point to other retinal layers where cAMP is likely to shape cellular responses. This method opens up novel morphological approaches to the study of cyclic nucleotide regulation.

Animals↗
PubMed1979

Immune deposits in human glomerulopathy. Fluorescent microscopy findings in 366 kidney biopsies correlated to symptoms, clinical course and immunosuppressive therapy.

366 kidney biopsies from 339 patients, 290 of which were classified on combined light microscopy (LM) and clinical criteria as glomerulonephritis (GN) and 49 as non-glomerulonephritis nephropathy, were examined by immunofluorescent microscopy (IFM) without knowledge of the clinical or LM findings at the time of examination. The IFM findings in the glomeruli were correlated to (l) the LM diagnosis, (2) the clinical symptoms, (3) the clinical course, and (4) the effect of immunosuppressive treatment. A few IFM results were found to correlate significantly with the LM diagnosis and clinical symptoms, but not to the effect of immunosuppressive treatment. It was impossible using IFM alone to group patients into any specific categories with uniform symptomatology and prognosis. Defined by IFM "immune complex nephritis" was demonstrated in 72%, and linear nephritis was found in 2% of the patients with GN. IgA occurred more frequently in GN secondary to systemic disease, particularly in SLE (60%) and HSP (88%). IgA was demonstrated in only 10% of patients with nonglomerulonephritic nephropathy. Demonstration of IgA is therefore a good indicator for corroborating the LM diagnoses of GN. Demonstration of IgG and/or IgM in GN was not found to be sufficient evidence for GN because these deposits also appeared in 40% of patients with non-glomerulonephritic nephropathy. An immunopathological classification based solely on glomerular deposits of immunoglobulin/C3 appears to have no practical importance. The demonstration of glomerular deposits of immunoglobulin/C3, however, showed to be a necessary supplement to clinical and morphological findings in some instances, in order to attain practical diagnostic boundaries within the very ill-defined concept which today constitutes GN.

Adult↗
PubMed1996

3D resolved two-photon fluorescence microscopy of living cells using a modified confocal laser scanning microscope.

Non-linear 3D imaging of fluorophore-labelled vital cells has been performed by femtosecond near infrared (NIR) microscopy. Ultraviolet and visible transitions of intracellular fluorophores, such as Fura-2, Calcium Green, Rhodamine 123 and fluorescent microspheres, were excited via simultaneous absorption of two 780 nm photons provided by a tunable Ti:Sapphire laser. The femtosecond laser was coupled to a conventional upright Zeiss confocal laser scanning microscope expanding its one-photon capabilities to 3D resolved two-photon microscopy. Pinhole-free non-linear 3D imaging was possible with 400 nm lateral and approximately 1 micron axial resolution. Axial resolution could be further improved by using an additional detection pinhole. The NIR average power and pulse width at the sample were adjusted to be 1-4 m W and 150-200 fs, respectively. Higher power levels resulted in cell damage as demonstrated by photoinduced lysis of human erythrocytes. The powerful capabilities of this universal microscope were demonstrated by 3D imaging of two-photon excited fluorophore-labelled macrophages during phagocytosis of fluorescent microsized beads.

Erythrocytes↗
PubMed1997

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin.

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.

Biophysical Phenomena↗
PubMed1994

Detection of oxidation products in individual neurons by fluorescence microscopy.

In the study of the central nervous system, it is necessary to address mechanisms by which cells are injured. In vitro investigations using cells in culture allow sharply focused mechanistic questions to be addressed; however, these studies have often been limited by the sensitivity constraints of assays. Many assays for oxidative products require large amounts of cells that must be disrupted. The extent of oxidation in individual cells is therefore unknown and the results yield an average among different cell types. This is inconvenient in cultures of the nervous system which often have multiple cell types. Using a newly developed method for visualizing oxidation products in individual cells, we have examined oxidation in neurons in culture. The method uses a hydrazide, biotin-4-amidobenzoic hydrazide, to bind carbonyls generated from oxidation. Biotin is detected by streptavidin conjugated with a fluorescent dye. Neurons in culture were exposed to 0.1 to 100 microM ferrous sulfate and fluorescence was visualized and quantitated using confocal laser microscopy. Low levels of oxidation (0.1 microM ferrous sulfate) were easily detected with this method. Iron concentration and fluorescence intensity correlated highly (r = 0.991). As an indicator of the sensitivity of this new method, carbonyl content in the cultures was also quantitated using the 2,4-dinitrophenylhydrazine assay (DNPH). The DNPH assay failed to detect the low levels of oxidation which were detected by the biotin-4-amidobenzoic hydrazide method. Fluorescence intensity partially paralleled loss of neuronal viability. Low concentrations of iron (0.1 and 1.0 microM) did not produce significant neuronal death; however, higher concentrations (10 and 100 microM) produced 19 and 53% neuronal loss, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Intra-vital fluorescence microscopy for intra-myocardial graft detection following cell transplantation.

INTRODUCTION: The aim of our study was the development of a potentially clinically applicable approach, which allows for intra-myocardial detection of the transplanted cells without the need for collection of tissue samples. Intra-myocardial transplantation of myocytes and bone marrow derived cells is currently under clinical evaluation as a therapy of heart failure. A major limitation of all clinical studies for myocardial restoration through cell transfer is the inability to track the fate of the transplanted cells. METHODS: Fetal canine cardiomyocytes were labelled with the non-toxic fluorescent membrane dye Vybrant CM-DiI and injected into the free wall of the left ventricle of six adult mongrel dogs. For subsequent tracking of the cellular graft, the dogs were re-operated and an intra-vital microscope was mounted above the exposed heart within the thorax. RESULTS: Two months following cell transplantation, the fluorescent graft was visible by intra-vital microscopy using a 10x magnification. Histological studies served as microscopic control and confirmed the presence of DiI-labelled cells at the site of injection. Connexin 43 immunoreactivity was visible at junctional complexes between donor and recipient cells, suggesting morphologic coupling as a result of gap junction formation. CONCLUSIONS: Our results demonstrate that in vivo detection of transplanted cells in the heart is feasible. Further technical adjustments will facilitate thoracoscopic and therefore less invasive application of this method.

Animals↗
PubMed1998

Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell.

Confocal fluorescence correlation spectroscopy (FCS) and other confocal spectroscopic techniques are ideally suited for the analysis of molecular interactions at the subcellular level. However, one requires exact positioning in three dimensions within the cell. Our instrument integrates FCS with high sensitivity digital imaging microscopy and high precision positioning. We present first measurements of intracellular FCS, with specification of the instrumental requirements and methods of data analysis. We propose the term fluorescence correlation microscopy (FCM) for this extended modality of FCS.

Dextrans↗
PubMed1999

Imaging of renal medullary interstitial cells in situ by confocal fluorescence microscopy.

Renal medullary interstitial cells are a prevalent and characteristic feature of the inner medulla of the kidney, but the physiological significance of this is unclear. We have developed a method for imaging renal medullary interstitial cells in situ by loading the cells with fluorescent dyes and monitoring their distribution using confocal microscopy. The pH-sensitive probe 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester was used as a marker of cytoplasmic volume and therefore of cell morphology. Nile Red was used to demonstrate the presence of renal medullary interstitial cell lipid droplets. Papillae were excised from 100 g Sprague-Dawley rats and loaded with the appropriate dye. The papillae were then examined using a Leica TCS 4D confocal microscope and oil immersion lenses. Fluorescence was excited (488 nm) using an argon laser and emission wavelengths above 515 nm collected using a long pass filter. Images of papillae loaded with 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester clearly demonstrate a ladder-like arrangement of renal medullary interstitial cells. More detailed examination revealed the presence of cytoplasmic extensions that appear to make close contact with adjacent loops of Henle. Three-dimensional reconstructions of serial sections revealed spiral arrangements in some ladders of renal medullary interstitial cells. Nile Red-labelled lipid droplets of 0.5-1.0 microm diameter were located throughout the cytoplasm of renal medullary interstitial cells and especially within the cytoplasmic extensions. These experiments highlight the ability of confocal microscopy to allow investigation of renal medullary interstitial cells in situ.

Animals↗
PubMed1981

Use of fluorescence microscopy for monitoring periodontal disease state.

Samples of subgingival plaque from patients with periodontal disease and control subjects were stained with the Fluoretec Fluorescent test kits (Pfizer Inc., New York) developed for the rapid detection of members of the Bacteroides fragilis and B. melaninogenicus groups of anaerobes. The same fluorescent fields were also examined by dark-field microscopy for the total count of bacteria. Bacteroides fragilis and B. melaninogenicus were found in plaque samples of healthy subjects and periodontally diseased patients with no significant difference in percent of total flora. Oral spirochetes also fluoresced with the antisera used. Samples from healthy sites showed virtually no spirochetes; spirochetes were present in diseased sites. Tests with other antisera also showed that fluorescein-labelled antibodies can be adsorbed nonspecifically to the surface of spirochetes. Such a phenomenon can be used to monitor an individual's periodontal disease state.

Bacteroides↗
PubMed2006

Intravital fluorescence microscopy in pulmonary research.

Over the last several years, microscopy as a scientific tool has reinvented itself evolving from a group of principally descriptive methodologies to encompass a wide range of primary tools and techniques to investigate the molecular organization of organs, tissues and cells. Advances in microscope and camera design, fluorescent dye technology, the development of fluorescent proteins as well as the advent of inexpensive powerful computers, has led to the feasibility of simultaneous sub micron resolution and quantitation of multiple concurrent molecular markers for both protein and DNA. Confocal microscopy has allowed optical sectioning and reconstruction of tissues in three dimensions. Finally, the development of multiphoton methodologies as an extension of optical sectioning microscopy has further improved the potential utility of this technology when examining living or light scattering tissues such as the lung. In order to illustrate the utility of two-photon methods in pulmonary biology, we present the application of this approach to the study of cellular trafficking in situ and to the study of pulmonary vasoregulation in an ex vivo rodent model.

Animals↗
PubMed2005

Observing three-dimensional human microvascular and myogenic architecture using conventional fluorescence microscopy.

Microangiography and vascular casting have previously been used to demonstrate the three-dimensional architecture of human uterine microvasculature. However, a limitation of these perfusion-dependent techniques is the difficulty in identifying surrounding tissue components. We have previously shown that it is possible to visualise microvascular networks on the cut surfaces of fresh tissue specimens by diffusive labelling of vascular endothelium with fluorescently conjugated UEA-1 lectin. Unlike perfusion methods that are limited to accessible vascular networks, diffusive fluorescence labelling (DFL) allows additional visualisation of extravascular cellular components, such as smooth muscle. Following UEA-1 DFL, smooth muscle-myosin and -actin were then visualised by immunolocalisation on the acetone-fixed tissue pieces. This allowed clear three-dimensional distinction between the vascular and muscle architecture of the myometrium and endometrium. This method can also be applied for studying the relative distribution of microvascular and muscle architecture in leiomyomas (fibroids). The techniques described in this methodological study provide a simple way of directly examining the uterine vasculature in three dimensions using conventional microscopy, while also distinguishing myometrial from endometrial parts of the network.

Endometrium↗