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Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis.

Two-photon molecular excitation microscopy has several advantages over conventional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photolysis. We describe and examine the benefit of incorporating non-descanned fluorescence detection in our microscope system. In a scattering sample where almost no signal could be obtained at a depth of 50 microm with confocal detection, non-descanned detection resulted in an improvement of signal strength by more than an order of magnitude at depths >40 microm. The spatio-temporal properties of stationary spot two-photon excited flash photolysis (TPEFP) in drops of test solutions and cardiac myocytes were also examined. At input powers that produce >10% of the maximum rate of DM-nitrophen photolysis, serious photodestruction of the reporter fluorochrome (Fluo-3) at the photolysis spot occurred. At power levels of approximately 4 mW for periods <50 ms, we were able to produce small repeatable calcium release events using DM-nitrophen in cardiac myocytes, which were similar to naturally occurring calcium sparks. The properties of these artificial calcium sparks were very similar to signals obtained from drops of test solutions, suggesting that the apparent rate of calcium diffusion in myocytes is similar to the rate of diffusion of Fluo-3 in solution. Using TPEFP, we also examined the ability of a combination of EGTA and a low-affinity calcium indicator to track the time course of calcium release. Although the addition of EGTA improved the temporal fidelity of the rise of the calcium signal, it did not significantly reduce the spread of the fluorescence signal from the photolysis spot.

Acetates↗

A comparative FLASH and EPI study of repetitive and sustained visual activation.

Functional responses to either brief repetitive or sustained activation of the human visual cortex (movie presentation) were monitored using both fast low angle shot and echo planar imaging sequences. To allow for proper comparisons, native image contrasts were equally sensitized to changes in cerebral blood oxygenation with other experimental conditions matched as much as possible. Putative influences of receiver bandwidth and absolute voxel size were specifically addressed. In all cases resulting correlation maps and regional signal intensity time courses showed excellent spatial and temporal congruence, respectively. In particular, for a 6 min protocol of sustained activation, both FLASH and EPI yielded an initial signal increase (oxygenation overshoot), a subsequent signal decrease during ongoing stimulation, and a marked signal drop (oxygenation undershoot) after the end of stimulation. These findings exclude technical differences between FLASH and EPI as the source of previous contradictory observations more likely to be explained by differences in stimulus design.

Adult↗

One-electron redox processes during polyoxometalate-mediated photocatalytic reactions of TiO(2) studied by two-color two-laser flash photolysis.

The one-electron redox processes of several compounds during polyoxometalate (POM)-mediated photocatalytic reactions of TiO(2) were investigated using the two-color two-laser flash-photolysis technique. The efficiency of the one-electron oxidation of aromatic sulfides by the trapped hole (h(tr) (+)) or the surface-bound OH radical (OH(s) (.)) is found to be significantly enhanced due to electron transfer from the conduction band (CB) of TiO(2) to the POM. The efficiency of the electron transfer from the CB of TiO(2) to the POM decreases in the order H(2)W(12)O(40) (6-) < SiW(12)O(40) (4-) < PW(12)O(40) (3-), that is, it depends on the reduction potential (E(red)) of the POMs. Electron injection from PW(12)O(40) (4-) in the excited state (PW(12)O(40) (4-*)) to the CB of TiO(2) was clearly observed using the two-color two-laser flash-photolysis technique. Storage of electrons in the TiO(2)/PW(12)O(40) (3-)/methyl viologen (MV(2+)) ternary system was also achieved upon two-color two-laser irradiation.

Catalysis↗

Clinical comparison of three-dimensional MP-RAGE and FLASH techniques for MR imaging of the head.

Three-dimensional (3D) MP-RAGE (magnetization-prepared rapid gradient-echo) imaging was evaluated as a high-resolution 3D T1-weighted brain imaging technique for patients with suspected neurologic disease. Fourteen patients were studied. In five, 3D MP-RAGE images were compared with 3D FLASH (fast low-angle shot) images. Signal difference--to-noise ratios and T1 contrast were not statistically different for 3D MP-RAGE images as opposed to 3D FLASH images. Advantages intrinsic to the application of 3D MP-RAGE sequences include decreased imaging time and decreased motion artifact. With this technique, it is possible to perform a relatively motion-insensitive, T1-weighted screening brain study with voxel resolution of 1.0 x 1.4 x 2.0 mm or smaller, in an imaging time of 5.9 minutes or less--permitting offline (poststudy) reconstruction of high-resolution images in any desired plane.

Brain↗

Bowel disease: prospective comparison of CT and 1.5-T pre- and postcontrast MR imaging with T1-weighted fat-suppressed and breath-hold FLASH sequences.

The potential of new high-field-strength magnetic resonance (MR) imaging sequences to evaluate bowel disease was investigated and compared with computed tomographic (CT) studies. Thirty-two patients were studied, 14 with known or suspected gastrointestinal tumors and 18 with known or suspected bowel inflammatory conditions. T1-weighted fat-suppressed spin-echo and breath-hold FLASH (fast low-angle shot) images were obtained before and after intravenous injection of 0.1 mmol/kg gadopentetate dimeglumine. Pathologic confirmation was obtained by biopsy (n = 18), surgical excision (n = 8), or endoscopy (n = 6). CT and MR images were analyzed separately in a prospective fashion and reviewed by consensus. Information from CT and MR images was comparable in cases of confirmed bowel neoplasia. CT scans had better spatial resolution, while fat-suppressed gadolinium-enhanced MR images had better contrast resolution. In the 18 cases of bowel inflammation, CT scans showed concentric wall thickening in 16, while MR images showed concentric wall thickening in 14 and increased contrast enhancement in 17. Contrast enhancement was better appreciated on fat-suppressed images than on FLASH images. The results suggest that MR imaging may play a role in the evaluation of bowel disease.

Adipose Tissue↗

Use of three-dimensional segmented FLASH sequence with magnetization transfer contrast to improve Gd-DTPA-enhanced intrahepatic MR portography.

Twenty healthy volunteers underwent gadopentetate dimeglumine (gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA))-enhanced MR angiography (MRA) using three-dimensional-segmented fast low angle shot images (FLASH) with magnetization transfer contrast (MTC) pulses. MRA was obtained at 75 seconds (early phase) and 135 seconds (late phase) after bolus injection of Gd-DTPA (MTC+ group) during one period of breath-holding. Within 1 week. MRA without MTC was performed under the same scanning conditions. Visualization of intrahepatic portal branches with these methods was compared in both phases. Portal vein-liver contrast-to-noise ratios were significantly higher in the MTC+ group in both phases. For third- and fourth-order portal branches, visualization was significantly better in the MTC+ group in both phases. Use of three-dimensional-segmented FLASH shortened acquisition time and facilitated imaging during breath-holding and also reduced whole-body average specific absorption rate values. Visualization of intrahepatic portal vein branches was improved by MTC pulses, and effective imaging time was prolonged.

Adult↗

Quantitative flash-methylation analysis of phenobarbital.

In phenobarbital measurement by GLC with the flash-methylation technique, using trimethylanilinium hydroxide as a methylating reagent, a small amount of water decomposed phenobarbital and interfered with the quantitative analysis. Thus, both the sample and the methylating reagent must be sufficiently dehydrated to attain quantitative analyses. The hydrolysis decomposition product of phenobarbital was N-methyl-2-phenylbutyramide, as shown by its mass spectrum. The sum of methylated phenobarbital and N-methyl-2-phenylbutyramide (if observed in the spectrum) can be used for an accurate phenobarbital assay in the present flash-methylation technique.

Chromatography, Gas↗

600 nm flash pumped dye laser for fragile telangiectasia of the elderly.

Since 1984, a continuum of controlled studies have been done on the use of the fragile, superficial telangiectasia of the elderly, which were utilized as a model for some of the basic studies of the effects of lasers on the cardiovascular system. We report the use of the 600 nm flash pumped dye laser for the fragile telangiectasia of the elderly in our classification of the Class 2 group. In some vessels studied, the special Teflon-insulated, intravenous electric needle of Kobayashi-Yamada was used. For the bluish vessels, the 600 nm flash pumped dye laser penetrated deeper into the vessel than the 585 nm, but multiple and repeated treatments were needed for clinical effect. Preliminary studies have also shown some initial response in port wine marks and facial telangiectasia.

Aged↗

Evaluation of a novel flash lamp system (FLS) incorporating optimal spectral filtration for the treatment of photoaging.

BACKGROUND AND OBJECTIVES: Improvement in photoaging using laser and Intense Pulsed Light (IPL) modalities is well documented in the literature. We report a prospective study evaluating the safety and efficacy of a novel flash lamp IPL system incorporating a spectral filtration system designed to maximize improvement of facial dyschromias, telangiectases, and skin texture. The device was a prototype xenon flashlamp pulsed light. The novel features of this IPL device are the extended pulse duration and smooth temporal pulse profile. STUDY DESIGN/MATERIALS AND METHODS: We enrolled 23 patients (22 female, 1 male) of Fitzpatrick skin type I-IV with evidence of photoaging (lentigenes, ephelides, telangiectases, rhytides). Each patient underwent test sites on their back to establish safe treatment parameters. Using treatment fluences of 20-37 J/cm(2), each patient was given 2-4 full-face treatments separated by 3-4 weeks. RESULTS: Test sites revealed that shorter wavelength filters and shorter pulse durations increased the risk of epidermal injury. Objective assessment by evaluation of pre and post treatment photographs of the face showed an average improvement of 53% in hyperpigmentation, 39% in telangiectases, and 8% in wrinkles. There were no significant adverse effects. CONCLUSIONS: The novel flash lamp IPL system in this study was safe and effective in reducing vascular and pigment dyschromias in patients with skin types I through IV.

Adult↗

Rapid NMR imaging of dynamic processes using the FLASH technique.

FLASH (Fast Low-Angle SHot) imaging is a new method for rapid NMR imaging which has been demonstrated to provide abdominal images without artifacts due to respiratory or peristaltic motions. The sequence typically employs 15 degrees radiofrequency excitation pulses and acquires a free induction decay signal in the form of a gradient echo. Here FLASH images are recorded in the presence of dynamic processes with time constants even smaller than the measuring time of about 2 s for an image with a 128 X 128-pixel resolution. Experiments are carried out on flow phantoms and on rabbits yielding heart images without gating of the cardiac motion.

Animals↗

Flow suppression in rapid FLASH NMR images.

Rapid FLASH (fast low angle shot) NMR images are very sensitive to flow phenomena. In particular, a steady reflow of unsaturated spins from outside the imaging plane results in high image intensities which depend on the flip angle, the repetition time, and the flow velocity. Here we describe a technique that suppresses these signals by saturating remote spins prior to entering the imaging plane, e.g., by intercalating the FLASH sequence with slice-selective 1-2-1 pulse packages. Experiments have been carried out on phantoms and human extremities. Flow artifacts in the phase-encoding direction of the images are strongly reduced. Vascular structures may be easily delineated using difference images obtained with and without flow suppression.

Arm↗

Direct FLASH MR imaging of magnetic field inhomogeneities by gradient compensation.

MR images based on gradient echoes are sensitive to artifacts caused by inhomogeneities of the static magnetic field. This paper describes the effects of local gradients in rapid FLASH MR images and presents a way of directly imaging affected areas. The idea is to compensate for signal losses due to mutual cancellation of dephased magnetizations by deliberate "misadjustments" of the refocusing part of the slice selection gradient. In contrast to conventional field imaging techniques no three-dimensional data acquisition or subsequent Fourier analysis is required to obtain images at a particular gradient strength. Conventional as well as inhomogeneity compensated FLASH images have been obtained on phantoms and human heads using a 2.35-T 40-cm magnet and a 1.5-T whole-body system, respectively.

Head↗

31P FLASH NMR imaging.

A method of 31P FLASH NMR imaging is described using a low flip angle excitation pulse and three-dimensional Fourier transform spectroscopic imaging (3DFT). In vivo high-resolution 31P NMR spectra are obtained for each of 32 x 32 image elements using a minimum measuring time of 1.5 min. 31P FLASH images of a rat showing the spatial distribution of phosphocreatine, alpha-, beta-, and gamma-ATP have been measured in a 4.7-T magnet with a spatial resolution of 6 mm.

Adenosine Triphosphate↗

Elimination of transverse coherences in FLASH MRI.

Fast low-angle shot (FLASH) imaging enables T1-weighted scans to be acquired in a few seconds. However, the diagnostic image quality is severely compromised by the appearance of artifactual bands parallel to the frequency encode direction. We show that the band structure arises from differences in the ability of the phase encode gradient to spoil transverse coherences that build up between successive repetition intervals. A theoretical understanding of the mechanisms involved leads to a comparison between various methods of spoiling the unwanted echoes throughout the whole field of view. Spoiler gradients whose amplitudes change linearly with phase encode step number are treated in detail. The theory predicts that the spoilers will rotate and rescale the band structure and these results are confirmed experimentally. The effect of the spoilers at a given location along the gradient is equivalent to the effect on the entire field of view of an incremented phase shift applied to the radiofrequency pulse. An appropriate rf phase shift scheme should therefore provide ideal spoiling characteristics for FLASH imaging.

Magnetic Resonance Imaging↗

Multislice snapshot FLASH using SIMUSIM.

A combination of the SIMUSIM technique and snapshot FLASH which allows efficient multislice snapshot FLASH imaging is described. Simultaneously acquired images of deliberate slice position and thickness are obtained with an increased SNR compared to standard time sequential multislice experiments. Experimental results show four coronal cross sections through the human head measured in four acquisitions.

Brain↗

3-dimensional functional imaging of human brain using echo-shifted FLASH MRI.

A 3-dimensional MRI method has been developed for functional mapping of the human brain, based on blood oxygenation level dependent (BOLD) contrast mechanisms. The method uses recently introduced principles of echo-shifted FLASH to acquire a single 3D data set in 20 s. The technique was tested on a conventional 1.5 Tesla clinical scanner with a standard head coil using visual stimulation with a 8 Hz flashing white light, or a varying checkerboard pattern. Areas of increased signal intensity were identified in the visual cortex, consistent with the known functional organization.

Brain↗

Echo train shifted multi-echo FLASH for functional MRI of the human brain at ultra-high spatial resolution.

This paper describes the development of a novel technique for functional MRI of the human brain at 0.135 microL resolution for a whole brain section. In comparison with conventional studies at 3 mm isotropic resolution or 27 microL voxel size, the method yields an improvement by a factor of 200. To achieve optimum image quality, the approach is based on a multi-echo fast low-angle shot (FLASH) sequence with unipolar traversals of k-space in the frequency-encoding dimension and echo train shifting to avoid amplitude discontinuities in the phase-encoding dimension. These strategies ensure a smooth point-spread function and eliminate image ghosting artifacts without the need for any phase correction or other post-processing. Signal-to-noise losses due to the considerably reduced voxel sizes are compensated for by single slice acquisitions, optimized bandwidths and an experimental four-channel shoulder coil matched to the posterior portion of the head. Multi-echo FLASH studies of the human brain (2.9 T, seven echoes, 200 Hz/pixel bandwidth, effective echo time 36 ms, acquisition time 6 s) at 300 microm resolution (no interpolation) and 1.5 mm slice thickness revealed robust activations in primary visual areas in response to binocular stimulation. The new method holds promise for refined studies of the columnar organization of specific brain systems and for functional assessments of the gray matter at laminar resolution.

Animals↗

Investigation of the rate limiting step for electron transfer from NADPH:cytochrome P450 reductase to cytochrome b5: a laser flash-photolysis study.

The reduction kinetics of the one-electron-reduced cytochrome P450 reductase:cytochrome b5 complex (P450R1e:b5ox) has been investigated by the laser flash-photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Investigation of the kinetic properties of the individual components at 470 nm indicated that P450R1e and b5ox were reduced via second-order kinetics by 5-dRfH. with rate constants of 1 x 10(8) M-1 s-1 and 4.2 x 10(8) M-1 s-1, respectively. Intramolecular electron transfer from (laser reduced) FADH. to FMNH. was measured at 585 nm and a first-order rate constant of 36 s-1 was obtained for this process. Reduction of the preformed P450R1e:b5ox complex by 5-dRfH. was biphasic and second-order rate constants of 5.4 x 10(8) M-1 s-1 and 7.5 x 10(7) M-1 s-1 were obtained for the fast and slow phases of reduction. The time-resolved flash-induced difference spectrum was consistent with the simultaneous direct reduction (by 5-dRfH.) of protein-bound flavin and heme, followed by an additional slower first-order intracomplex electron transfer (kLim = 37 s-1) from protein-bound flavin semiquinone to heme. The results indicate that the (laser-generated) two-electron-reduced form of cytochrome P450 reductase is catalytically competent in the transfer of reducing equivalents to oxidized cytochrome b5 and suggest that formation of FMNH2 as a result of internal electron transfer from FADH. to FMNH. within P450R1e is the rate limiting step in the reduction of cytochrome b5 by cytochrome P450 reductase. The Kd of the cytochrome P450 reductase:cytochrome b5 complex was estimated to be approximately 1.5 x 10(-6) M.

Animals↗