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PubMed2005

Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immunoagglutination assay.

A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay. For this purpose a theoretical model of the initial agglutination kinetics, based on particle diffusion combined with a steric factor determined by the level of specific and nonspecific agglutination, was developed. The theoretical model combined with the experimental data can be used to optimize an agglutination-based assay with regard to sensitivity and dynamic range and to estimate the affinity, receptor surface density, molecular and binding site sizes, and level of nonspecific binding that is present in the assay. The experimental results are in good agreement with the theoretical model, indicating the usefulness of the model for immunoagglutination assay optimization.

Agglutination Tests↗
PubMed1986

Demonstration and determination of DNA in Pneumocystis carinii by fluorescence microscopy with 4', 6-diamidino-2-phenylindole (DAPI).

We have attempted to demonstrate DNA and to measure the amount of nuclear DNA in Pneumocystis carinii by fluorescence microscopy with 4', 6-diamidino-2-phenylindole (DAPI). Due to the high specificity of DAPI for DNA, only nuclei and mitochondria were stained. An uninucleate trophozoite and a mature cyst containing eight intracystic bodies were distinguished easily each other after staining with DAPI. The total amount of nuclear DNA of eight intracystic bodies in a cyst was approximately eight times that of the small type of trophozoite. When nuclei of uninucleate trophozoites were randomly examined, the distribution pattern of nuclear fluorescence intensity showed two peaks, one at 1C and the other at 2C. Moreover, it showed also a gradual transition in DNA values from 1C to 2C, and very few nuclei with polyploid DNA values. The primary advantage of this method with DAPI is that it allows a more efficient evaluation of the life cycle of P. carinii.

Animals↗
PubMed2003

Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues.

Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged proteins and transfected cells and is easily detected by fluorescence microscopy or flow cytometry in living cells. However, soluble EGFP molecules can be lost if cell integrity is disrupted by freezing, sectioning, or permeablization. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilize EGFP may also destroy its usefulness as a fluorescent reporter. Here we determined which methods of preparing murine lymphoid tissues immobilized soluble EGFP protein and retained its fluorescence while simultaneously maintaining the antigenicity of various immunologically important molecules and best preserving the overall morphology of the tissues. We found that EGFP could not be visualized in frozen sections of spleen that had not been fixed before freezing. However, robust EGFP fluorescence could be observed in frozen sections of tissues fixed under various conditions. Fixation was important to immobilize EGFP rather than to maintain conformation, because only minimal EGFP could be detected by immunofluorescence in unfixed frozen sections. Although it had little effect on EGFP fluorescence, the inclusion of sucrose during fixation better preserved the morphology of fixed tissues. These methods also preserved the antigenicity of a wide variety of molecules used to identify cell types in lymphoid tissues.

Animals↗
PubMed2003

Conventional, confocal and two-photon fluorescence microscopy investigations of polymer-supported oxygen sensors.

Luminescence-based, polymer-supported oxygen sensors, particularly those based on platinum group complexes, continue to be of analytical importance. Commercial applications range from the macroscopic (e.g. aerodynamic investigations in wind tunnels, monitoring of oxygen concentration during fermentation, and measurement of biological oxygen demand) to the microscopic (e.g. imaging of oxygen in blood, tissue, cells and other biological samples). Problems hindering the design of improved oxygen sensors include non-linear Stern-Volmer calibration plots and the multi-exponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule in the polymer support matrix. Conventional, confocal and two-photon fluorescence microscopy have proven to be invaluable tools with which the microscale heterogeneity and response of luminescence-based oxygen sensors can be investigated and compared to the macroscopic response. Results obtained for three ruthenium(II) alpha-diimine complexes in polydimethylsiloxane polymer supports indicate the presence of unquenched microcrystals within the polymer matrix that probably degrade oxygen quenching sensitivity and linearity of the Stern-Volmer quenching plot. Two-photon fluorescence microscopy proved most useful for imaging microcrystals within sensor films, and conventional microscopy allowed direct comparison between microscopic and macroscopic sensor response. The implications of the results in the rational design and mass production of luminescence-based oxygen sensors are significant.

Journal Article↗
PubMed2005

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy.

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies . Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Antigens↗
PubMed2001

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue.

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 degrees C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.

Animals↗
PubMed2002

New directions for fluorescent speckle microscopy.

Fluorescent Speckle Microscopy (FSM) is a technology for analyzing cytoskeleton dynamics, giving novel insight into their roles in living cells. New applications of FSM, together with the development of computer-based FSM image analysis, will make FSM the first microscopy-based method to deliver quantitative kinetic readouts at high spatial and temporal resolution for a wide variety of macromolecular systems. Here, we review the most recent applications and developments and give a glimpse of future directions and potentials of FSM.

Actins↗
PubMed1988

Adriamycin, aclacinomycin and thepirubicin intracardiac distribution examined by fluorescence microscopy.

Male Wistar rats received adriamycin, aclacinomycin or thepirubicin at a dose of 4 mg/kg b.w. by slow infusion. Cardiac tissue sections were examined by fluorescence microscopy to evaluate the distribution of the three anthracyclines. The nuclei regularly exhibited a stronger coloring with respect to the cytoplasm for all three drugs. Adriamycin cytoplasm fluoresced intensely, unlike aclacinomycin and thepirubicin. Our results indicate a lower uptake of these last two molecules into cardiac tissue, thus suggesting a different pharmacokinetic profile which might account for their lower cardiotoxicity.

Aclarubicin↗
PubMed2004

Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy.

Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 microM(-1) s(-1) and the dissociation rate constant is 0.89 s(-1). At the pointed end the association and dissociation rate constants are 0.56 microM(-1) s(-1) and 0.19 s(-1). When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s(-1) and from pointed ends at 0.16 s(-1) regardless of buffer nucleotide.

Actin Cytoskeleton↗
PubMed1987

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents.

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.

Adsorption↗
PubMed1994

Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane.

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.

Antibodies, Monoclonal↗
PubMed2003

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device.

A novel compact illumination device in variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell-substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four-layer model, as well as a Gaussian-shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell-substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time-resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).

2-Naphthylamine↗
PubMed1997

Direct visualization of individual DNA molecules by fluorescence microscopy: characterization of the factors affecting signal/background and optimization of imaging conditions using YOYO.

A new series of high-affinity cyanine dyes was tested for the visualization of the dynamics of single DNA molecules through a fluorescence microscope. In particular, YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)- bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)- 2-methylidene]-quinolinium tetraiodide) forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the images. We have characterized the factors affecting the signal/background in the imaging of single DNA molecules by fluorescence microscopy and compared the results obtained using YOYO-1 with those obtained using standard fluorescent dyes like ethidium bromide or acridine orange.

Acridine Orange↗
PubMed2002

Demonstration of immune complex deposits using fluorescence microscopy of hematoxylin and eosin-stained sections of Hollande's fixed renal biopsies.

A new method that may be useful in the evaluation of renal biopsies is described using fluorescence microscopy on standard hematoxylin and eosin-stained sections of kidney tissue fixed in Hollande's fixative. We describe brightly fluorescing immune complex deposits within glomerular basement membranes and mesangial matrices that correlate well with the results of standard direct immunofluorescence on frozen tissue and electron microscopy. In a blind analysis of 261 consecutive renal biopsies, we determine that this method has diagnostic utility for identification of immune complex glomerulonephritis and significantly extends the usefulness of standard histologic preparations before the use of special stains or procedures.

Antigen-Antibody Complex↗
PubMed2000

Time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin and benzoporphyrin derivative monoacid ring A in the hamster model: comparative fluorescence microscopy study.

The pharmacokinetics of the photosensitizer used play a key role in the understanding of the mechanism of photodynamic therapy-induced damage. Fluorescence microscopy was used to compare time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin (mTHPC) and benzoporphyrin derivative monoacid ring A (BPD-MA) in different hamster tissues, including an early, chemically induced, squamous cell carcinoma. Following injection of 0.5 mg/kg body weight of mTHPC and 2.0 mg/kg BPD-MA, groups of three animals were sacrificed at different time points and a series of fluorescence micrographs from different excised organs were analyzed. The highest fluorescence intensities of mTHPC were observed at 96 h for squamous epithelia and skin and at 48 h for smooth muscle. There is no real peak of BPD-MA fluorescence between 30 min and 3 h in the basal epithelial layers, fibroconnective tissue, muscles or blood vessels. At 4 h after injection, the fluorescence level of BPD-MA decreased and at 24 h it had returned to background level in all observed tissues. The significantly faster clearance of BPD-MA is the principal advantage as compared to mTHPC. However, similar localization patterns in different tissues with essentially vascular affinity represent a possible disadvantage for treating early malignancies with BPD-MA as compared to mTHPC, which is mainly localized in various epithelia. For both photosensitizers no significant selectivity between early squamous cell carcinoma and healthy mucosae is seen. Pharmacokinetic studies of different photosensitizers in an appropriate animal model are essential for selecting new-generation photosensitizers with the most favorable localization for photodynamic therapy of early malignancies in hollow organs.

Animals↗