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Results for “Fluorescence imaging”

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At least 145 records · Page 8Linked to original sources

PubMed2006

Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging.

A new phytotoxicity bioassay based on chlorophyll fluorescence imaging of algae suspensions in multiwell plates is introduced. Phytotoxicity is quantified via inhibition of photosystem II quantum yield, Y(II), assessed with the saturation pulse method. The basics of this approach as well as the factors enhancing and limiting its performance are outlined. Compared to other established techniques the new system allows exceptionally rapid and accurate measurements of phytotoxicity using pulse-amplitude-modulation (PAM) fluorometry. While instrument related errors are negligibly small, optimal performance depends on appropriate choice of algae and illumination conditions. Illustrative examples for the response of Phaeodactylum tricornutum to diuron are presented. The standard deviation involved in the Y(II) determination of a single well amounts to the equivalent of 44 ng/L diuron. A decisive role is played by the light (measuring light, saturation pulses, actinic light) to which samples are exposed during the bioassay: (1) the inhibitor response is enhanced at high measuring light intensity. (2) Saturation pulses may be considered non-invasive only, if applied at low frequency and as long as physiologically healthy algae cultures are used. (3) Continuous actinic light may be problematic, as it induces complex physiological reactions that limit the performance of the approach; it is not required for assessment of diuron-type inhibitors at high measuring light intensity.

Biological Assay↗

PubMed2004

Combined in vivo bioluminescence and fluorescence imaging for cancer gene therapy.

Nasopharyngeal carcinoma is intimately associated with the Epstein-Barr virus (EBV), which we have exploited therapeutically by constructing an EBV-specific synthetic enhancer sequence, within an adenoviral vector, denoted as adv.oriP. The achievement of tumor targeting provides therapeutic potential when delivered systemically, which could impact on distant metastases. We demonstrate here the feasibility and potential utility of combined, minimally invasive in vivo bioluminescence and fluorescence imaging to monitor adenoviral infection of subcutaneous C666-1 nasopharyngeal xenograft tumors stably expressing the DsRed2 gene. Fluorescence imaging was used to monitor the location and size of the C6661.DsRed2 tumors, whereas bioluminescence imaging demonstrated the distribution and specificity of a transcriptionally targeted adenoviral vector, adv.oriP.fluc, expressing the firefly luciferase gene. Fluorescence, bioluminescence, and photographic images were aligned using grids to examine colocalization of adenovirus and tumors. Bioluminescence and fluorescence co-localized in 92% (11/12) of tumors at 24 hr and 100% (12/12) at 96 hr after adv.oriP.fluc (10(9) ifu) was administered intravenously. Nonspecific luciferase signal was detected in the liver area. The combined imaging was therefore successful in monitoring the uptake of systemically administered adenovirus in implanted tumors. This may ultimately lead to an effective noninvasive method to monitor the response of metastases to adenoviral gene therapy.

Animals↗

PubMed2004

Remote fluorescence imaging of dynamic concentration profiles with micrometer resolution using a coherent optical fiber bundle.

Dynamic concentration profiles within the diffusion layer of an electrode were imaged in situ using fluorescence detection through a multichannel imaging fiber. In this work, a coherent optical fiber bundle is positioned orthogonal to the surface of an electrode and is used to report spatial and temporal micrometric changes in the fluorescence intensity of an initial fluorescent species. The fluorescence signal is directly related to the local concentration of a redox fluorescent reagent, which is electrochemically modulated by the electrode. Fluorescence images are collected through the optical fiber bundle during the oxidation of tris(2,2'-bipyridine)ruthenium(II) to ruthenium(III) at a diffusion-limited rate and allow the concentration profiles of Ru(II) reagent to be monitored in situ as a function of time. Tris(2,2'-bipyridine)ruthenium(II) is excited at 485 nm and emits fluorescence at 605 nm, whereas the Ru(III) oxidation state is not fluorescent. Our experiments emphasize the influence of two parameters on the micrometer spatial resolution: the numerical aperture of optical fibers within the bundle and the Ru(II) bulk concentration. The extent of the volume probed by each individual fiber of the bundle is discussed qualitatively in terms of a primary inner-filter effect and refractive index gradient. Experimentally measured fluorescence intensity profiles were found to be in very good agreement with concentration profiles predicted upon considering planar diffusion and thus validate the concept of this new application of imaging fibers. The originality of this remote approach is to provide a global view of the entire diffusion layer at a given time through one single image and to allow the time expansion of the diffusion layer to be followed quantitatively in real time.

Journal Article↗

PubMed2005

Digital fluorescence imaging of organic cation transport in freshly isolated rat proximal tubules.

The secretion of cationic drugs and endogenous metabolites is a major function of the kidney. This is accomplished by organic cation transport systems, mainly located in the proximal tubules. Here, we describe a model for continuous measurement of organic cation (OC) transport. In this model, organic cation transport in individual freshly isolated rat proximal tubules is investigated by use of digital fluorescence imaging. To directly measure organic cation transport across the basolateral membrane, the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+) is used with a customized perfusion chamber. ASP+ uptake in this model displayed the characteristics of organic cation transport. Over the tested range of 1 to 50 microM, it showed a concentration-dependent uptake across the basolateral membrane. In the presence of competitive inhibitors of OC transport such as N1-methylnicotinamide+, tetraethylammonium+, and choline+, a concentration-dependent and reversible inhibition of ASP+ uptake could be documented. In conclusion, continuous measurement of organic cation transport in freshly isolated rat proximal tubules by digital fluorescence imaging using ASP+ is a useful tool for investigation of drug transport and interactions and, furthermore, may be helpful for investigation of organic cation transport under pathophysiological conditions.

Animals↗

PubMed1996

Fluorescence imaging and spectroscopy of biomaterials in air and liquid by scanning near-field optical/atomic force microscopy.

We have developed scanning near-field optical/atomic force microscopy (SNOM/AFM). The SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Resonant frequency of the optical fiber cantilever is 15-40 kHz. Optical resolution of the SNOM/AFM images shows less than 50 nm. The SNOM/AFM system contains photon counting system and polychrometer/intensified coupled charge devise (ICCD) system to observe fluorescence image and spectrograph of micro areas, respectively. Cultured cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-keratin antibody or FITC-labeled phalloidin after treatment with Triton X-100. Fluorescence and topographic images were obtained in air and water. The fluorescence images showed clear images of keratin and actin filaments. The SNOM/AFM is perfect to observe biomaterials in liquid with a liquid chamber while the topographic Images showed subcellular structures which correspond to keratin and actin filaments.

Actins↗

PubMedDate not supplied

Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma.

Fluorescence imaging has been shown to be a potential complement to visual inspection for demarcation of basal cell carcinoma (BCC), which is the most common type of skin cancer. Earlier studies have shown promising results when combining autofluorescence with protoporphyrin IX (Pp IX) fluorescence, induced by application of delta-5-aminolaevulinic acid (ALA). In this work, we have tried to further improve the ability of this technique to discriminate between areas of tumor and normal skin by implementing texture analysis and Fisher linear discrimination (FLD) on bispectral fluorescence data of BCCs located on the face. Classification maps of the lesions have been obtained from histopathologic mapping of the excised tumors. The contrast feature obtained from co-occurrence matrices was found to provide useful information, particularly for the ALA-induced Pp IX fluorescence data. Moreover, the neighborhood average features of both autofluorescence and Pp IX fluorescence were preferentially included in the analysis. The algorithm was trained by using a training set of images with good agreement with histopathology, which improved the discriminability of the validation set. In addition, cross validation of the training set showed good discriminability. Our results imply that FLD and texture analysis are preferential for correlation between bispectral fluorescence images and the histopathologic extension of the tumors.

Adult↗

PubMed2000

Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin.

BACKGROUND AND OBJECTIVE: Fluorescence techniques can provide powerful noninvasive means for medical diagnosis, based on the detection of either endogenous or exogenous fluorophores. The fluorescence of delta-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) has already shown promise for the diagnosis of tumors. The aim of the study was to investigate the localization of skin tumors after the topical application of ALA, by detecting the PpIX fluorescence either in the spectral or in the time domain. STUDY DESIGN/MATERIALS N AND METHODS: Two fluorescence imaging systems were used to identify basal cell carcinomas of the skin in humans, after topical application of 20% ALA ointment. Both systems rely on the comparison between the exogenous and the endogenous fluorescence, performed either in the spectral domain or in the time domain. The first system works by using three images acquired through different spectral filters, whereas the second one measures the spatial map of the average fluorescence lifetime of the sample. RESULTS: A clear demarcation of skin malignancies was successfully performed in vivo noninvasively with both fluorescence imaging systems. CONCLUSION: The two complementary approaches considered in the present study show promise for skin tumor detection and delineation based on specific fluorescence features.

Adult↗

PubMed2005

[Establishment of a system for measuring blood flow velocity of rat microvessel using dark background fluorescent image analysis method].

Autologous red blood cells (RBC) labeled with fluorescence were immitted into microvessel of SD rat and observed under microscope. The movement of each individual labeled RBC was recorded by microscope video camera system. The recorded videotape is replayed to sample dark background fluorescent images through frame grabber. Sampled frame images were separated into odd and even field sequence images. Then these sequence images were analyzed to get the flow rate. The error between the actual flow velocity value and the flow velocity value of fluorescent globules in the chamber measured under the same system was below 7%. The upper limit was 9.6 mm/s. There are no obvious differences (P > 0.05). This system has been applied in the research of rat microcirculatory disturbance, and the temporal flow rate change in microvessel was obtained.

Animals↗

PubMed2003

Study on Schiff base complexes-cellular DNA interactions by a novel system of Hadamard transform fluorescence image microscopy.

A novel system of Hadamard transform microscopic fluorescence imaging for single cells is presented, based on which the DNA ploidy of rat hepatocyte was quantitatively measured. The result shows that diploid rat hepatocyte has a stable DNA content, thus diploid rat hepatocyte was used to investigate the binding of five clinical anticancer agents, vincristine, cyclophosphamide, nitrogen mustard, cis-diamminedichloroplatinum(n) (CDDP) and mitomycin-C, with cellular DNA when acridine orange (AO) was used as the competitive fluorescence probe. Based on this model, some Schiff base complexes-cellular DNA interactions were investigated. The results indicate that all the twenty-two compounds, including Schiff base ligands of N-2-hydroxy-naphthaldehyde with D-glucoamine (NG) and the complexes of 3d-transitional metals ions with NO and with D-glucoamine (Glu) and the mixed complexes of NG and Glu series with alpha-glycine (GNG), have the ability to enter the cell membrane and interact with cellular DNA. Four of the compounds, CuGlu, Fe(II)NG, Fe(III)NG and CuGluG can intercalate with DNA like AO does and depress AO-DNA fluorescence to 70% or lower. An in intro UV-visible spectroscopic study on the compound-DNA spectra testified the above results and suggests that diverse interaction mechanisms coexist for all these complexes except intercalating mode. This study presents a new in vitro method for initial screening of anticancer compounds.

Animals↗

PubMed2004

Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage.

Different biotic stresses yield specific symptoms, owing to their distinct influence on a plant's physiological status. To monitor early changes in a plant's physiological status upon pathogen attack, chlorophyll fluorescence imaging (Chl-FI) and thermography, which respectively visualize photosynthetic efficiency and transpiration, were carried out in parallel for two fundamentally different plant-pathogen interactions. These non-destructive imaging techniques were able to visualize infections at an early stage, before damage appeared. Under growth-room conditions, a robotized set-up captured time series of visual, thermal and chlorophyll fluorescence images from infected regions on attached leaves. As a first symptom of the plant-virus interaction between resistant tobacco and tobacco mosaic virus (TMV), thermal imaging detected a local rise in temperature while Chl-FI monitored a co-localized increase in fluorescence intensity. Chl-FI also revealed pre-symptomatic high-intensity spots for the plant-fungus system sugar beet-Cercospora beticola. Concomitantly, spots of lower temperature were monitored with thermography, in marked contrast with our observations on TMV-infection in tobacco. Knowledge of disease signatures for different plant-pathogen interactions could allow early identification of emerging biotic stresses in crops, facilitating the containment of disease outbreaks. Presymptomatic monitoring clearly opens perspectives for quantitative screening for disease resistance, either on excised leaf pieces or attached leaves.

Ascomycota↗

PubMed2005

Characterizing residue transfer efficiencies using a fluorescent imaging technique.

To reduce the uncertainty associated with current estimates of children's exposure to pesticides by dermal contact and indirect ingestion, residue transfer data are required. Prior to conducting exhaustive studies, a screening study to identify the important parameters for characterizing these transfers was designed. A fluorescence imaging system was developed (Ivancic et al., in press) to facilitate collection of surface residue transfer data for repeated contacts. Next, parameters that affect residue transfer from surface-to-skin, skin-to-other objects, and skin-to-mouth were evaluated using the imaging system and the fluorescent tracer riboflavin as a surrogate for pesticide residues. Riboflavin was applied as a residue to surfaces of interest. Controlled transfer experiments were conducted by varying contact parameters with each trial. The mass of a tracer transferred was measured and the contact surface area estimated using video imaging techniques. Parameters evaluated included: surface type, surface loading, contact motion, pressure, duration, and skin condition. Transfers both onto, and off of, the hand were measured. To efficiently identify parameter changes resulting in significant effects, the Youden ruggedness test was used to select the combination of parameters varied in each contact trial. In this way, more than one parameter could be varied at a time and the number of trials required was minimized. Results of this study showed that surface loading and skin condition (significant at alpha=0.05) are among the important parameters for characterizing residue transfers of riboflavin. Duration of contact within the time range investigated does not have a significant effect on transfer of this tracer. Results of this study demonstrate the potential for collecting dermal transfer data using the Ivancic et al. fluorescence imaging system and provide preliminary data to reduce uncertainty associated with estimating dermal exposures resulting from contact with residue-contaminated surfaces. These data will also aid in determining what additional residue transfer data should be collected and what type of microactivity data are needed to estimate dermal and indirect ingestion exposure to residues on household surfaces.

Administration, Cutaneous↗

PubMedDate not supplied

Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons.

We demonstrate how to construct calibrated, stable, and inexpensive tissue-like phantoms for near-IR (NIR) fluorescence imaging applications. The bulk phantom material is composed of gelatin, intralipid, hemoglobin, and indocyanine green (ICG). Absorbance, scatter, background fluorescence, and texture can be tuned as desired. NIR fluorescent inclusions are comprised of ICG-labeled polystyrene divinylbenzene beads and Pam78-labeled hydroxyapatite crystals. The former mimic tumor masses of controllable size and contrast agent concentration, and the latter mimic microcalcifications in breast cancer. NIR-fluorescent inclusions can be positioned precisely in phantoms, with one or more regions having different optical properties, and their position can be verified independently using microcomputed tomography. We demonstrate how these phantoms can be used to calibrate and compare imaging systems, and to train surgeons to operate under NIR fluorescence image guidance.

Animals↗

PubMed2001

Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging.

High resolution imaging of chlorophyll a fluorescence was used to identify the sites at which ozone initially induces perturbations of photosynthesis in leaves of Phaseolus vulgaris. Leaves were exposed to 250 and 500 nmol mol(-1) ozone at a photosynthetically active photon flux density of 300 micromol m(-2) s(-1) for 3 h. Images of fluorescence parameters indicated that large decreases in both the maximum and operating quantum efficiencies of photosystem II had occurred in cells adjacent to stomata in the upper, but not lower, leaf surfaces. However, this treatment did not produce any significant changes in the maximum or operating quantum efficiencies of photosystem II in the leaves when estimated from fluorescence parameters measured with a conventional, integrating fluorometer. The localized decreases in photosystem II photochemical efficiencies were accompanied by an increase in the minimal fluorescence level, which is indicative of photoinactivation of photosystem II complexes and a decrease in stomatal conductance. Perturbations of photochemical efficiencies were not observed in cells associated with all of the stomata on the upper leaf surface or within cells distant from the upper leaf surface. It is concluded that ozone penetrates the leaf through stomata and initially damages only cells close to stomatal pores.

Chlorophyll↗

PubMed2005

Intracellular trafficking pathways and drug delivery: fluorescence imaging of living and fixed cells.

Cellular processes depend on the fidelity of intracellular membrane traffic. Lipids, proteins, receptor ligands and solute molecules are trafficked to distinct compartments within the cell through both the biosynthetic and endocytic pathways. An appreciation of these pathways is vital for a complete understanding of intracellular drug delivery. Recent advances in fluorescence imaging have facilitated the analysis of these pathways in great detail. It is now possible to gain insight into the real-time dynamics of cellular components and macromolecular pharmacological agents as they are delivered into and traffic within single cells. Here, we discuss the analysis of intracellular drug delivery from the perspective of fluorescence imaging of both living and fixed cells. This review aims to cover trafficking pathways, markers for subcellular compartments, fluorescent labels for intracellular structures and pharmacological agents and relevant recent developments in imaging technology. In particular, we shall focus on the application of live cell imaging to the study of endocytic drug delivery.

Bacterial Toxins↗

PubMed2005

Virally directed fluorescent imaging improves diagnostic sensitivity in the detection of minimal residual disease after potentially curative cytoreductive surgery.

Completeness of cytoreduction is an independent prognostic factor after cure-intended surgery for peritoneal carcinomatosis. NV1066, a genetically engineered herpes simplex virus carrying the transgene for green fluorescent protein, selectively infects cancer cells. We sought to determine the feasibility of virally directed fluorescent imaging in the intraoperative detection of minimal residual disease after cytoreductive surgery. NV1066 infected human gastric cancer cells, OCUM-2MD3, and mesothelioma JMN cells at all doses. The infected cells expressed green fluorescent protein and were killed. OCUM-2MD3, and mesothelioma JMN cells at all doses. Peritoneal carcinomatosis was established in mice by injection of OCUM cells into the peritoneal cavity. Forty-eight hours after intraperitoneal injection of NV1066, two experienced surgeons resected all visible disease and identified mice free of disease. Eight of 13 mice thought to be free of disease were found to have residual disease as identified by green fluorescence (mean number of observations: 5; range: 1-9). Residual disease was most frequently observed in the retroperitoneum, pelvis, peritoneal surface, and liver. Specificity of NV1066 infection to tumor nodules was confirmed by immunohistochemistry and by polymerase chain reaction for viral gene. Virally directed fluorescent imaging, a novel molecular imaging technology, can be used for real-time visualization of minimal residual disease after cytoreductive surgery and can improve the completeness of cure-intended resection.

Animals↗

PubMed2004

FIZICS: fluorescent imaging zone identification system, a novel macro imaging system.

Constantly improving biological assay development continues to drive technological requirements. Recently, a specification was defined for capturing white light and fluorescent images of agar plates ranging in size from the NUNC Omni tray (96-well footprint, 128 x 85 mm) to the NUNC Bio Assay Dish (245 x 245 mm). An evaluation of commercially available products failed to identify any system capable of fluorescent macroimaging with discrete wavelength selection. To address the lack of a commercially available system, a custom imaging system was designed and constructed. This system provides the same capabilities of many commercially available systems with the added ability to fluorescently image up to a 245 x 245 mm area using wavelengths in the visible light spectrum.

Biological Assay↗

PubMed1999

In vivo application of intestinal pH measurement using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorescence imaging.

Measurement of gastrointestinal intramucosal pH (pHim) has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolutions. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). This study aimed to set up and validate a fluorescence imaging technique to measure in vivo the intramucosal pH (pHim) of the intestine. The intestine was inserted into an optical chamber placed under a microscope. Animals were injected intravenously with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pHim by constructing a calibration curve. The pHim controls were performed with a pH microelectrode and were correlated with venous blood gas sampling. Results show that pHim is determined with an accuracy of +/- 0.07 pH units and a response time of 1 min. In conclusion pHim mapping of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurements.

Animals↗

PubMed2002

Double-fluorescence image microscopy for quantitation of prostate-specific antigen in histologic sections of the prostate.

BACKGROUND: Although the assessment of serum prostate-specific antigen (PSA) has become a powerful instrument in the diagnosis and for prognosis of prostate carcinoma, there are few quantitative studies of PSA in tissue sections. METHODS: We developed a technique using double-fluorescence image microscopy for quantifying immunohistochemical reactions in tissue sections. PSA was stained by Texas Red and the cellular DNA was counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI). The fluorescence of Texas Red and DAPI was quantified separately after subtraction of background and shading correction. The amount of PSA related to the amount of DNA in identical tissue parts was studied in archival specimens from patients with hyperplasia and prostate carcinoma. RESULTS: The amount of tissue PSA decreased with the increase in tumor grade, Gleason score, and the change from diploid to aneuploid. CONCLUSION: Double-fluorescence image microscopy is a valuable technique for obtaining quantitative information of cellular constituents. For standardization of immunochemical reactions in tissue sections, cellular DNA seems to be most appropriate.

Humans↗