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PubMedDate not supplied

Fluorescence imaging with a laser trapping scanning near-field optical microscope.

We investigated fluorescence imaging using a near-field scanning optical microscope which uses a laser-stabilized gold nanoparticle as a near-field probe. This microscope is suitable for observations of biological specimens in aqueous solutions because the probe particle is held by a noncontact force exerted by a laser beam. Theoretical calculations based on Mie scattering theory are presented to evaluate the near-field enhancement by a gold particle of 40 nm diameter. We also present fluorescence images of a single fluorescent bead and discuss the near-field contribution to the fluorescence image in this type of microscope.

Journal Article↗

PubMed2005

A comparison of transit-time flowmetry and intraoperative fluorescence imaging for assessing coronary artery bypass graft patency.

BACKGROUND: Intraoperative graft patency assessment during coronary artery bypass grafting enables detection and immediate correction of graft failure. Currently transit-time flowmetry is used to assess graft patency on the basis of mean graft flow and derived values, such as the pulsatility index. Intraoperative fluorescence imaging, based on the fluorescence of indocyanine green dye, provides direct visual images to confirm graft patency. METHODS: We performed a prospective observational study to assess intraoperative graft patency in patients undergoing coronary artery bypass grafting, by using an intraoperative fluorescence imaging system (SPY) and transit-time flowmetry (BF 2004). Poor flow with the intraoperative fluorescence imaging system was defined if there was an absence of fluorescence or if it did not appear within 15 seconds in the graft. A persistent mean graft flow value less than 5 mL/min and a pulsatility index greater than 5 with transit-time flowmetry were considered unacceptable and prompted graft revision. RESULTS: We assessed the intraoperative patency of 266 grafts in 100 coronary artery bypass grafting patients. Intraoperative fluorescence imaging and transit-time flowmetry confirmed adequate flow in 241 (91%) grafts in 75 patients (75%). Transient poor flow was detected with both intraoperative fluorescence imaging and transit-time flowmetry in 7 (2.6%) grafts in 7 (7%) patients. This subsequently proved to be adequate on repeat testing and hence did not necessitate graft revision. Both intraoperative fluorescence imaging and transit-time flowmetry confirmed persistent poor flow in 8 (3%) grafts in 8 (8%) patients that necessitated graft revision. However, in a further 10 (3.8%) grafts in 10 (10%) patients, transit-time flowmetry indicated persistently poor flows on the basis of mean graft flow and pulsatility index values, whereas the intraoperative fluorescence imaging system demonstrated satisfactory flow. These grafts were not revised. CONCLUSIONS: In most patients, both intraoperative fluorescence imaging and transit-time flowmetry are useful to confirm intraoperative graft patency. However, in a small proportion of patients (10%), graft patency assessment with transit-time flowmetry alone might prompt unnecessary graft revision.

Aged↗

PubMed2001

Fast and noninvasive fluorescence imaging of biological tissues in vivo using a flying-spot scanner.

We have developed a flying-spot scanner (FSS), for fluorescence imaging of tissues in vivo. The FSS is based on the principles of single-pixel illumination and detection via a raster scanning technique. The principal components of the scanner are a laser light source, a pair of horizontal and vertical scanning mirrors to deflect the laser light in these respective directions on the tissue surface, and a photo multiplier tube (PMT) detector. This paper characterizes the performance of the FSS for fluorescence imaging of tissues in vivo. First, a signal-to-noise ratio (SNR) analysis is presented. This is followed by characterization of the experimental SNR, linearity and spatial resolution of the FSS. Finally, the feasibility of tissue fluorescence imaging is demonstrated using an animal model. In summary, the performance of the FSS is comparable to that of fluorescence-imaging systems based on multipixel illumination and detection. The primary advantage of the FSS is the order-of-magnitude reduction in the cost of the light source and detector. However, the primary disadvantage of the FSS its significantly slower frame rate (1 Hz). In applications where high frame rates are not critical, the FSS will represent a low-cost alternative to multichannel fluorescence imaging-systems.

Animals↗

PubMed2006

Subnanosecond-resolution phase-resolved fluorescence imaging technique for biomedical applications.

Characterization of fluorescence emissions from cells often leads to conclusive results in the early detection of cellular abnormalities. Cellular abnormalities can be characterized by their difference in the fluorescence lifetime, which may be less than nanoseconds. A sensitive frequency domain technique, also called a phase-resolved fluorescence imaging technique, is proposed in which fluorescence emissions at the same wavelengths can more effectively be separated with subnanosecond resolution in their lifetime difference. The system configuration is optimized by incorporating even-step phase shifting in the homodyne-assisted signal-processing concept along with the phase-resolved fluorescence technique to eliminate the dc offsets of emission. Experiments are carried out with simulated samples composed of two fluorescence emissions of the same wavelength but with different lifetime values. Suppression of either of the fluorescence emissions by selective imaging of the other validates the superiority of the proposed technique. Hence, this technique can potentially be applied in the early detection of cellular abnormalities.

Biomedical Engineering↗

PubMed1992

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.

Base Sequence↗

PubMedDate not supplied

Fluorescence imaging method for in vivo pH monitoring during liposomes uptake in rat liver using a pH-sensitive fluorescent dye.

Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6-carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.

Animals↗

PubMed2004

[Fluorescence imaging technique: diagnostic and therapeutic interest in gynecology].

Fluorescence techniques are presently used by several medical and surgical disciplines (dermatology, pneumology, urology, gastrointestinal surgery) for the diagnosis of pre-cancerous and cancerous lesions. The technique is based on the application of 5-aminolevulinic acid (5-ALA) which induces the production of an endogen photosensitizer: protoporphyrin IX (PpIX). Fluorescence detection of the infraclinical dysplasia lesion is based on the contrast between fluorescent pathological tissue and non-fluorescent healthy tissue. Moreover, there is a correlation between the degree of tissue dysplasia and fluorescence intensity. Fluorescence imaging could allow easier detection of infraclinical lesions enabling more well-targeted treatment. In gynecology, many experimental and clinical studies have been conducted on the detection and treatment of cervical dysplasia and the diagnosis and treatment of vaginal and vulvar diseases as well as the diagnosis of peritoneal micrometastasis from ovarian cancer, the diagnosis and treatment of endometrial alterations, and the treatment of locoregional recurrent skin breast cancer. The aim of this work is to present the fundamental principles of fluorescence imaging technical and to expose the diagnostic and therapeutic prospects in gynecology.

Aminolevulinic Acid↗

PubMed2003

An operational near-infrared fluorescence imaging system prototype for large animal surgery.

Near-infrared (NIR) fluorescence imaging has the potential to revolutionize human cancer surgery by providing sensitive, specific, and real-time intraoperative visualization of normal and disease processes. We have previously introduced the concept of a low-cost, safe, and easy-to-use NIR fluorescence imaging system that permits the surgeon to "see" surgical anatomy and NIR fluorescence simultaneously, non-invasively, with high spatial resolution, in real-time, and without moving parts. In this study, we present an operational prototype designed specifically for use during large animal surgery. Such a system serves as a foundation for future clinical studies. We discuss technical considerations, and provide details of the implementation of subsystems related to excitation light, light collection, computer, and software. Using the prototype, and the clinically available NIR fluorophore indocyanine green, we demonstrate vascular imaging in 35 kg pigs. Cancer-specific applications of this imaging system include image-guided cancer resection with real-time assessment of surgical margins, image-guided sentinel lymph node mapping, intraoperative mapping of tumor and normal vasculature, image-guided avoidance of critical structures such as nerves, and intraoperative detection of occult metastases in the surgical field. Taken together, this study describes an optical imaging system engineered for eventual translation to the clinic.

Animals↗

PubMed1987

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope.

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.

Animals↗

PubMed2000

[Autofluorescence bronchoscopy: Laser imaging fluorescence endoscope].

The LIFE (laser imaging fluorescence endoscope) system has been shown to increase the diagnosis of dysplasia and carcinoma in situ when used in combination with conventional bronchoscopy. A doubling to tripling of the rate of early centrally located lung cancer diagnosis is a step forward in the detection of early lung cancer. A wide spectrum of interventional procedures for endoluminal treatment of lung cancer in functionally inoperable patients makes it possible to treat this group of patients. The LIFE system works without exogenous sensitisers, with no increase in complications as compared to conventional bronchoscopy, and takes only a little longer in examination time.

Bronchitis↗

PubMed2004

Fluorescence imaging in vivo: raster scanned point-source imaging provides more accurate quantification than broad beam geometries.

Two fluorescence imaging systems were compared for their ability to quantify mean fluorescence intensity from surface-weighted imaging of tissue. A broad beam CCD camera system was compared to a point sampling system that raster scans to create the image. The effects of absorption and scattering in the background tissue volume were shown to be similar in their effect upon the signal, but the effect of the three-dimensional shape of the tissue was shown to be a significant distortion upon the signal. Spherical phantoms with Intralipid and blood for absorber and scatterer were used with a fixed concentration of aluminum phthalocyanine fluorophore to illustrate that the mean intensity observed with the broad beam system increased with size, while the mean intensity observed with the raster scanned system was not as significantly affected. Similar results were observed in vivo with mice injected with the fluorophore and imaged multiple times to observe the pharmacokinetics of the drug. The fluorescence in the tumor observed with the broad beam system was higher than that observed with the raster scanned system. Based upon the phantom and animal observations in this study, it should be concluded that using broad beam fluorescence imaging systems to quantify fluorescence in vivo may be problematic when comparing tissues with different three dimensional characteristics. In particular, the ratio of fluorescence from tumor to normal tissue can yield inaccurate results when the tumor is large. However, similar measurements with a narrow beam system that is raster scanned to create the images are not as significantly affected by the three dimensional shape of the tissue. Raster scanned imaging appears to provide a more uniform and accurate way to quantify fluorescence signals from distributed tissues in vivo.

Animals↗

PubMed1991

Fluorescent image cytometry: from qualitative to quantitative measurements.

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.

Analog-Digital Conversion↗

PubMed2000

Fluorescence imaging of pyrene-labeled lipids in living cells.

Microscopic imaging of fluorescent lipid derivatives is a powerful tool to study membrane organization and lipid trafficking but it is complicated by cellular autofluorescence background and photobleaching of the fluorophore as well as by the difficulty to selectively image membranes stacked on top of each other. Here we describe protocols that strongly alleviate such problems when pyrene-labeled lipids are being used. First, photobleaching of these lipids is virtually eliminated when oxygen is depleted from the medium by using a gentle and simple enzymatic method. Second, an image practically free of cellular autofluorescence contribution can be obtained simply by subtracting from the pyrene image the background image obtained at a slightly different excitation wavelength. This type of background subtraction more properly accounts for the typically uneven distribution of cellular background fluorescence than other, commonly used methods. Third, it is possible to selectively image the pyrene lipids in the plasma membrane by using plasma membrane-specific quencher trinitrophenyl lysophosphatidylethanolamine and image subtraction. Importantly, either the outer or the inner leaflet can be selectively imaged by labeling the cells with pyrene phosphatidylcholine or phosphatidylserine, respectively. These protocols should be of considerable help when studying organization of the plasma membrane or intracellular lipid trafficking.

Cell Membrane↗

PubMed2001

Fluorescent imaging in living systems.

The use of fluorescent imaging techniques in the study of living biological systems has become an important experimental tool in modern biology. Over recent years novel imaging technologies have been developed and older techniques refined. New fluorescent probes continue to become available and the ways in which they are used is increasingly creative. Commonly used imaging methods such as confocal and multiphoton microscopy, when combined with techniques such as fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM), can provide powerful strategies with which to study molecular events in intact cells.

Animals↗

PubMed1991

[Spatial fluorescence imaging of atherosclerotic plaque: contrast enhancement by 2 wavelength laser stimulation, digital image processing and dye marking].

A new video-enhanced fluorescence imaging technique has been used for the first time for in vitro differentiation of human atherosclerotic plaques vs normal arterial wall. Laser-induced superficial tissue fluorescence of specimens from human aorta was documented after alternating excitation with violet (405 nm +/- 5 nm) and blue (470 nm +/- 10 nm) krypton-ion laser light. Subsequent digital subtraction of the corresponding fluorescence images allowed to differentiate areas of atherosclerosis from normal intima. Fluorescence intensity was correlated with the morphological aspect of samples and histology of the plaque. Dihematoporphyrin-ether/ester (DHE) incubation enhanced fluorescence contrast of plaques in comparison to normal artery vessel wall. Depending on the concentration of the incubation solution (10, 20, and 40 micrograms DHE/ml NaCl solution), fluorescence increased. Fluorescence intensity was highest in fatty plaque areas, while calcific lesions showed no substantial DHE uptake.

Angioplasty, Laser↗

PubMed2006

Assessment of unspecific near-infrared dyes in laser-induced fluorescence imaging of experimental arthritis.

OBJECTIVE: The aim of the study is to evaluate in vivo fluorescence imaging of experimental inflammatory joint disease by applying two different near-infrared (NIR) dyes in a model of Borrelia-induced Lyme arthritis. MATERIALS AND METHODS: Forty mice, 20 with Lyme arthritis and 20 controls, were examined. Two nonspecific NIR carbocyanine dyes, indocyanine green (ICG) and a hydrophilic carbocyanine derivative (1,1'-bis-[4-sulfobutyl] indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium salt [SIDAG]), were administered intravenously at two doses. Fluorescence images were acquired before and during 120 seconds after injection of cyanine dyes. For both dyes, the area under the curve (AUC) was determined for the interval between 40 and 80 seconds after injection. In addition, the slope of the signal decrease was compared among animal groups. Results were compared with histological findings. RESULTS: The general temporal fluorescence intensity course for ICG was characterized by a rapid increase, with a peak at 40-50 seconds followed by a decrease; conversely for SIDAG, by a slow increase. AUC analysis for both dyes showed that the fluorescence signal differed significantly between controls and arthritic animals (P < .05). Within these groups, there were significant differences between the two doses investigated. ICG differed significantly between control and arthritic animals in the slope of the signal decrease for both doses investigated (P < .05). Histological examination showed early stages of inflammation in arthritic animals. CONCLUSIONS: NIR fluorescence imaging based on the pharmacokinetic behavior of ICG or SIDAG is a promising approach to detect inflammatory joint changes of experimental arthritis. Moreover, SIDAG is suited to differentiate inflammatory and noninflammatory joints 24 hours after dye application.

Animals↗

PubMed2006

Transcranial fluorescence imaging of auditory cortical plasticity regulated by acoustic environments in mice.

Functional brain imaging using endogenous fluorescence of mitochondrial flavoprotein is useful for investigating mouse cortical activities via the intact skull, which is thin and sufficiently transparent in mice. We applied this method to investigate auditory cortical plasticity regulated by acoustic environments. Normal mice of the C57BL/6 strain, reared in various acoustic environments for at least 4 weeks after birth, were anaesthetized with urethane (1.7 g/kg, i.p.). Auditory cortical images of endogenous green fluorescence in blue light were recorded by a cooled CCD camera via the intact skull. Cortical responses elicited by tonal stimuli (5, 10 and 20 kHz) exhibited mirror-symmetrical tonotopic maps in the primary auditory cortex (AI) and anterior auditory field (AAF). Depression of auditory cortical responses regarding response duration was observed in sound-deprived mice compared with naïve mice reared in a normal acoustic environment. When mice were exposed to an environmental tonal stimulus at 10 kHz for more than 4 weeks after birth, the cortical responses were potentiated in a frequency-specific manner in respect to peak amplitude of the responses in AI, but not for the size of the responsive areas. Changes in AAF were less clear than those in AI. To determine the modified synapses by acoustic environments, neural responses in cortical slices were investigated with endogenous fluorescence imaging. The vertical thickness of responsive areas after supragranular electrical stimulation was significantly reduced in the slices obtained from sound-deprived mice. These results suggest that acoustic environments regulate the development of vertical intracortical circuits in the mouse auditory cortex.

Acoustic Stimulation↗

PubMed2004

Analysis of hyperspectral fluorescence images for poultry skin tumor inspection.

We present a hyperspectral fluorescence imaging system with a fuzzy inference scheme for detecting skin tumors on poultry carcasses. Hyperspectral images reveal spatial and spectral information useful for finding pathological lesions or contaminants on agricultural products. Skin tumors are not obvious because the visual signature appears as a shape distortion rather than a discoloration. Fluorescence imaging allows the visualization of poultry skin tumors more easily than reflectance. The hyperspectral image samples obtained for this poultry tumor inspection contain 65 spectral bands of fluorescence in the visible region of the spectrum at wavelengths ranging from 425 to 711 nm. The large amount of hyperspectral image data is compressed by use of a discrete wavelet transform in the spatial domain. Principal-component analysis provides an effective compressed representation of the spectral signal of each pixel in the spectral domain. A small number of significant features are extracted from two major spectral peaks of relative fluorescence intensity that have been identified as meaningful spectral bands for detecting tumors. A fuzzy inference scheme that uses a small number of fuzzy rules and Gaussian membership functions successfully detects skin tumors on poultry carcasses. Spatial-filtering techniques are used to significantly reduce false positives.

Animals↗