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Results for “Fluorescence imaging”

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At least 37 records · Page 2Linked to original sources
PubMed2007

Case study of combinatorial imaging: what protocol and what chlorophyll fluorescence image to use when visualizing infection of Arabidopsis thaliana by Pseudomonas syringae?

Localized infection of a plant can be mapped by a sequence of images capturing chlorophyll fluorescence transients in actinic light. Choice of the actinic light protocol co-determines fluorescence contrast between infected leaf segment and surrounding healthy tissue. Frequently, biology cannot predict with which irradiance protocol, in which fluorescence image of the sequence, and in which segment of the image there will be the highest contrast between the healthy and infected tissue. Here, we introduce a new technique that can be applied to identify the combination of chlorophyll fluorescence images yielding the highest contrast. The sets of the most contrasting images vary throughout the progress of the infection. Such specific image sets, stress-revealing fluorescence signatures, can be found for the initial and late phases of the infection. Using these signatures, images can be divided into segments that show tissue in different infection phases. We demonstrate the capacity of the algorithm in an investigation of infection of the model plant Arabidopsis thaliana by the bacterium Pseudomonas syringae. We show that the highest contrast is found with transients elicited by fluctuating, harmonically modulated irradiance with long periods.

Arabidopsis↗
PubMed2004

Development and evaluation of a quantitative video-fluorescence imaging system and fluorescent tracer for measuring transfer of pesticide residues from surfaces to hands with repeated contacts.

A video imaging system and the associated quantification methods have been developed for measurement of the transfer of a fluorescent tracer from surfaces to hands. The highly fluorescent compound riboflavin (vitamin B2), which is also water soluble and non-toxic, was chosen as the tracer compound to simulate the transfer from surfaces to hands of pesticide residues deposited on carpeted and laminate surfaces of a residence. The system was designed around the unique properties of riboflavin. Excitation energy was centered near 440 nm (in the blue region of the visible spectrum); emitted energy was measured at 600 nm (in the red/orange region), well beyond the significant fluorescence peak maximum of natural skin. A video camera system with an image intensifier was interfaced to an image processing analysis software system. Quantification utilized chemometric techniques to account for the non-linearity of pixel detectivity and non-linear excitation strength. Method quantification and detection limits were approximately 0.1 and 0.02 micro g/cm(2), respectively. The relative error was approximately 100% at the quantification limit, but <20% at higher levels. Transfer of riboflavin to hands, resulting in dermal loadings in the range 0.1-2.0 micro g/cm(2), were measured with this system from surfaces whose loadings approximated the pesticide levels that occur in homes after broadcast application.

Calibration↗
PubMed1996

Effects of topoisomerase I inhibition on the expression of topoisomerase II alpha measured with fluorescence image cytometry.

A fluorescence image cytometry technique was developed to measure the effects of topotecan, a topoisomerase I inhibitor, on the nuclear expression of topoisomerase II alpha in a series of patients with refractory or relapsed acute myeloid leukemia (AML). We used a commercially available affinity-purified rabbit polyclonal antibody and a fluorescein-conjugated secondary antibody. By using DAPI as a DNA counterstain and dual wavelength excitation, it was possible to measure enzyme expression in the cell nucleus, and to examine its cell cycle phase distribution. In human acute leukemia cell lines, topoisomerase II alpha expression was greatest in late S and G2 phases, but in leukemia patient samples the enzyme expression appeared to be much less cell cycle dependent. There was considerable interpatient variation in the effects of topotecan on topoisomerase II alpha expression in the leukemia patients, with a threefold increase in the median value after 48 h followed by a decline to pretreatment levels after 5 days of treatment with the topoisomerase I inhibitor. Although these findings should be treated with caution because of the small number of cases studied, they support the prediction that topoisomerase I inhibitors might be capable of increasing sensitivity to topoisomerase II active drugs such as anthracyclines and epipodophyllotoxins by upregulating topoisomerase II expression. They also illustrate the potential value of fluorescence image cytometry for making sequential measurements of the effects of drug resistance modulating agents in cancer patients.

Animals↗
PubMed2006

Monitoring and screening plant populations with combined thermal and chlorophyll fluorescence imaging.

Thermal and chlorophyll fluorescence imaging are powerful tools for the study of spatial and temporal heterogeneity of leaf transpiration and photosynthetic performance. The relative advantages and disadvantages of these techniques are discussed. When combined, they can highlight pre-symptomatic responses not yet apparent in visual spectrum images and provide specific signatures for diagnosis of distinct diseases and abiotic stresses. In addition, their use for diagnosis and for selection for stomatal or photosynthetic mutants, these techniques can be applied for stress tolerance screening. For example, rapid screening for stomatal responses can be achieved by thermal imaging, while, combined with fluorescence imaging to study photosynthesis, they can potentially be used to derive leaf water use efficiency as a screening parameter. A particular advantage of imaging is that it allows continuous automated monitoring of dynamic spatial variation. Examples of applications include the study of growth and development of plant lines differing in stress resistance, yield, circadian clock-controlled responses, and the possible interactions between these parameters. In the future, such dual-imaging systems could be extended with complementary techniques such as hyperspectral and blue-green fluorescence imaging. This would result in an increased number of quantified parameters which will increase the power of stress diagnosis and the potential for screening of stress-tolerant genotypes.

Chlorophyll↗
PubMed1997

Distribution of aluminum phthalocyanine disulfonate in an oral squamous cell carcinoma model. In vivo fluorescence imaging compared with ex vivo analytical methods.

Photosensitizer-induced fluorescence is studied as a technique for the detection of cancer. Therefore we investigated the ability of a photosensitizer, aluminum phthalocyanine disulfonate (AIPcS2), to localize in tumor tissue. In vivo endoscopic fluorescence imaging, fluorescence microscopy, conventional spectrofluorometry and high performance liquid chromatograpy combined with diode laser-induced fluorescence (HPLC-Dio-LIF) were used. Squamous cell carcinomas were induced with 4-nitro-quinoline-1-oxide (4NQO) in the mucosa of the palate of the rat. In vivo fluorescence images, taken after injection of 1.5 mumol/kg AIPcS2 intravenously, showed that 4NQO-treated palates had higher fluorescence signals than normal palates. Areas displaying locally high amounts of AIPcS2 fluorescence (hot spots) were present only in 4NQO-treated rats 2-8 h but had disappeared 24 h after injection. However, HPLC-Dio-LIF showed that the relative AIPcS2 content was highest at 24/48 h in biopsies taken in the areas of the hot spots. Fluorescence microscopy revealed that AIPcS2 was present only between 2 and 8 h in the epithelial layer, while in biopsies the connective tissue contained large quantities of AIPcS2 at 24/48 h. In vivo fluorescence imaging appears to show mainly fluorescence from the epithelial layer and the ex vivo analytical techniques mainly show the connective tissue fluorescence. Care should be taken when interpreting data using one technique only.

Animals↗
PubMed1991

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

3T3 Cells↗
PubMed2003

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging.

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.

Animals↗
PubMedDate not supplied

Planar fluorescence imaging using normalized data.

Fluorescence imaging of tissues has gained significant attention in recent years due to the emergence of appropriate reporter technologies that enable noninvasive sensing of molecular function in vivo. Two major approaches have been used so far for fluorescence molecular imaging, i.e., epi-illumination (reflectance) imaging and fluorescence molecular tomography. Transillumination is an alternative approach that has been employed for imaging tissues in the past and could be similarly beneficial for fluorescence molecular imaging. We investigate data normalization schemes in reflectance and transillumination mode and experimentally demonstrate that normalized transillumination offers significant advantages over planar reflectance imaging and over nonnormalized methods. Our observations, based on phantoms and on postmortem and in vivo mouse measurements display image quality improvement, superior depth sensitivity, and improved imaging accuracy over the nonnormalized methods examined. Normalized planar imaging retains implementation simplicity and could be used to improve on standard fluorescence reflectance imaging and as a simplified alternative to the more integrated and accurate tomographic methods.

Algorithms↗
PubMed1997

Simultaneous topographic and fluorescence imagings of recombinant bacterial cells containing a green fluorescent protein gene detected by a scanning near-field optical/atomic force microscope.

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.

Escherichia coli↗
PubMed1998

Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog.

Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis.

4-Chloro-7-nitrobenzofurazan↗
PubMed2006

Ultraviolet fluorescence imaging of fingerprints.

We studied fluorescence imaging of fingerprints on a high-grade white paper in the deep ultraviolet (UV) region with a nanosecond-pulsed Nd-YAG laser system that consists of a tunable laser and a cooled CCD camera. Clear fluorescence images were obtained by time-resolved imaging with a 255- to 425-nm band-pass filter, which cuts off strong fluorescence of papers. Although fluorescence can be imaged with any excitation wavelength between 220 and 290 nm, 230 and 280 nm are the best in terms of image quality. However, the damage due to laser illumination was smaller for 266-nm excitation than 230- or 280-nm excitation. Absorption images of latent fingerprints on a high-grade white paper are also obtained with our imaging system using 215- to 280-nm laser light. Shorter wavelengths produce better images and the best image was obtained with 215 nm. Absorption images are also degraded slightly by laser illumination, but their damage is smaller than that of fluorescence images.

Absorption↗
PubMed2002

Localization and staging of cervical intraepithelial neoplasia using double ratio fluorescence imaging.

A phase zero evaluation of a new fluorescence imaging technique for diagnosing cervical intraepithelial neoplasia (CIN) was performed. The fluorescence imaging prototype performed quantitative imaging of Protoporphyrin induced by a topically applied aminolevulinic acid using double ratio (DR) fluorescence imaging technique developed by our group. A total of 38 patients were in the protocol, with 16 colposcopically selected for biopsy. Fluorescence images of these 16 patients were taken, 19 sites were biopsied, and the disease was staged histopathologically. DR fluorescence imaging of the cervix using our general purpose prototype appeared to be cumbersome but feasible. In four cases strongly localized fluorescent hotspots were observed at the location where the disease was colposcopically visible. In the other cases the fluorescence showed a more diffuse multifocal image. The value of the DR determined at the site of biopsy correlated in a statistically significant way with the histopathologically determined stage of the disease [Spearman rank correlation, r=0.881, p<0.001 (confidence interval 0.7044-0.9552)]. This suggests that noninvasive staging of CIN using this technique is feasible. We believe that the results of this study justify the development of a dedicated device that combines regular white light colposcopy with DR fluorescence imaging.

Adult↗
PubMed2004

[Simultaneous Fluorescent Imaging of Mg2+ and Ca2+].

Simultaneous fluorescent imaging technique with several indicators is a powerful bio-imaging tool for a comprehensive survey about molecular functions in living cells. There are several interactions between Mg(2+) and Ca(2+) such as a counter transport or a competition at binding sites of divalent cation. To measure Mg-Ca images simultaneously, indicators with high selectivity to each divalent cation and multi-color fluorescent imaging system are essential. Here we introduce a setup for Mg-Ca fluorescent imaging we construct, and some reports about Mg-Ca imaging.

Animals↗
PubMed2004

Biomedical applications of fluorescence imaging in vivo.

Optical imaging can advance knowledge of cellular biology and disease at the molecular level in vitro and, more recently, in vivo. In vivo optical imaging has enabled real-time study to track cell movement, cell growth, and even some cell functions. Thus, it can be used in intact animals for disease detection, screening, diagnosis, drug development, and treatment evaluation. This review includes a brief introduction to fluorescence imaging, fluorescent probes, imaging devices, and in vivo applications in animal models. It also describes a quantitative fluorescence detection method with a reconstruction algorithm for determining the location of fluorophores in tissue and addresses future applications of in vivo fluorescence imaging.

Algorithms↗
PubMed2002

Noninvasive native fluorescence imaging of head and neck tumors.

Fluorescence images were acquired from ex vivo head and neck tumor specimens. The excitation and emission wavelength combinations were selected to image emission from native tissue fluorophores (collagen, tryptophan, elastin and nicotinamide adenine dinucleotide). Two dimensional intensity ratio maps were generated from the fluorescence images and compared to histology images. The ratio maps accurately distinguished the regions of tumor from normal tissue in the specimens. Additional features, such as collagen capsules, blood vessels, mucus glands and muscles were evident in the fluorescence images.

Carcinoma, Papillary↗
PubMed2005

Fiber-optic fluorescence imaging.

Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components.

Animals↗
PubMed2005

Fluorescence imaging of tumors in vivo.

We review recent progress in tumor imaging in vivo using fluorescent tags, highlight the problems of fluorescence imaging in small animals, discuss recent advances in near-infrared fluorochromes and quantum dots, and point to some future possibilities. GFP-based fluorescence imaging is briefly discussed. The authors believe that improvements in near-infrared fluorochromes are required to enable practical imaging in tissues at centimeter depths.

Animals↗