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PubMed1987

Fluorescence lifetime measurements using total internal reflection fluorimetry: evidence for a conformational change in albumin adsorbed to quartz.

We have modified the total internal reflectance fluorimetry (TIRF) method to determine, indirectly, quantum fluorescence yield and thus infer structural changes of adsorbed plasma proteins. We employed a frequency multiplied Nd:YAG pulse laser to excite tryptophan residues of bovine serum albumin (BSA), measuring the resulting fluorescence lifetimes. BSA adsorbed on quartz was investigated at normal pH and under acidic (pH 3.8) conditions at which albumin shape change has been established in bulk solution. Albumin adsorbed at pH 7.2 has a slightly lower lifetime than the bulk solution value, reflecting a population of both native and isomerized forms. Gently washing the adsorbate surface significantly reduces fluorescence lifetime, suggesting retention of more tenacious, conformationally altered molecules. Vigorous washing further reduces fluorescence lifetimes to values close to those obtained in bulk under acidic conditions. Similar alterations in fluorescence lifetimes were observed for heat-denatured albumin. These results support the hypothesis that adsorbed albumin exists in the form of a two-layer deposit, each with a possibly different structure. A loosely held layer, consisting of BSA microaggregates and including partially unfolded molecules, exists on the undisturbed surface and is partially removed by gentle washing. Vigorous washing removes all but the most tightly held protein, which exists in a possibly further unfolded configuration. The decrease in fluorescence lifetime of adsorbed BSA implies a corresponding change in quantum yield. A change in quantum yield of BSA tryptophan fluorophors implies that measuring adsorbed protein concentration by intrinsic fluorescence is unreliable.

Adsorption↗

PubMed1993

Measurement of intrinsic bone quality in vivo by reflection ultrasound: correction of impaired quality with slow-release sodium fluoride and calcium citrate.

The intrinsic (material) quality of cancellous and cortical bone was evaluated in vivo from the measurement of reflection ultrasound velocities in the ulna. In cancellous bone, the reflection ultrasound velocity was inversely correlated with age in normal women (r = -0.48, p = 0.001), with a significantly lower mean value in 32 normal postmenopausal women than in 14 premenopausal women (3124 versus 3341 m/s, p < 0.0001). In 32 untreated osteoporotic women the cancellous bone velocity was lower than in normal postmenopausal subjects (2906 versus 3124 m/s, p = 0.0001). Following treatment with slow-release sodium fluoride plus calcium citrate (mean 2.4 years in 33 osteoporotic patients with no fracture during treatment), the cancellous bone velocity was significantly higher than in untreated osteoporotic women (3082 versus 2906 m/s, p = 0.0002) and was not significantly different from that in normal postmenopausal women. The cortical bone velocity displayed similar trends, but the changes did not attain statistical significance. The measurements were repeated approximately 9 months later in 9 untreated and in 20 treated patients; in 5 additional patients, the measurements were made both before and after 9 months of treatment with slow-release sodium fluoride and calcium citrate. The cancellous bone velocity increased significantly (p = 0.046) in these patients, from 3008 m/s before treatment to 3112 m/s after the first 9 months of treatment. The velocity rose significantly from 3037 to 3167 m/s (p = 0.017) in patients treated for a short time (12-30 months at first measurement), but it did not change in untreated patients or those treated for more than 30 months. Thus, the material quality of cancellous bone decreases with normal aging and is reduced further with the osteoporotic process. This impaired quality may be corrected by treatment with slow-release sodium fluoride plus calcium citrate.

Analysis of Variance↗

PubMed1989

Experimental conditions for surface microanalysis with reflection electron energy-loss spectroscopy.

Experimental conditions for obtaining the optimum signal-to-background (S/B) ratio in reflection electron energy-loss spectroscopy (REELS) are investigated. It is shown that the S/B ratio can be improved by lowering the incident energy of the electrons. The spectra taken from the GaAs (660) specular reflection spot under the surface resonance condition is demonstrated to have the best S/B ratio and lowest surface plasma excitation, which is capable of providing structural information on the top few atomic layers.

Electron Probe Microanalysis↗

PubMed1990

Surface microanalysis by reflection electron energy-loss spectroscopy.

Several basic physical concepts of applying eq. Ik = I sigma Nxt to surface microanalysis by reflection electron energy-loss spectroscopy (REELS) are clarified. Here Ik and I are the integrated intensities of the core ionization edge and the low loss part, sigma is the scattering cross section of element x with atomic concentration Nx, and t is the specimen thickness. The reflected inelastic electrons are found to be distributed almost symmetrically around the Bragg spots and can be reasonably described by a Lorentzian function. EELS microanalysis can be performed by using the diffracted spots. The omega correction, arising from the angular contributions of the neighbouring spots into the spectrometer collecting aperture, is required to be considered.

Magnesium Oxide↗

PubMed1992

Reflection electron energy-loss spectroscopy and imaging for surface studies in transmission electron microscopes.

A review is given on the techniques and applications of high-energy reflection electron energy-loss spectroscopy (REELS) and reflection electron microscopy (REM) for surface studies in scanning transmission electron microscopes (STEM) and conventional transmission electron microscopes (TEM). A diffraction method is introduced to identify a surface orientation in the geometry of REM. The surface dielectric response theory is presented and applied for studying alpha-alumina surfaces. Domains of the alpha-alumina (012) surface initially terminated with oxygen can be reduced by an intense electron beam to produce Al metal; the resistance to beam damage of surface domains initially terminated with Al+3 ions is attributed to the screening effect of adsorbed oxygen. Surface energy-loss near-edge structure (ELNES), extended energy-loss fine structure (EXELFS), and microanalysis using REELS are illustrated based on the studies of TiO2 and MgO. Effects of surface resonances (or channeling) on the REELS signal-to-background ratio are described. The REELS detection of a monolayer of oxygen adsorption on diamond (111) surfaces is reported. It is shown that phase contrast REM image content can be significantly increased with the use of a field emission gun (FEG). Phase contrast effects close to the core of a screw dislocation are discussed and the associated Fresnel fringes around a surface step are observed. Finally, an in situ REM experiment is described for studying atomic desorption and diffusion processes on alpha-alumina surfaces at temperatures of 1,300-1,400 degrees C.

Aluminum Oxide↗

PubMed1992

Observation of surfaces by reflection electron holography.

Reflection electron holography is described as a method to observe sub-A surface morphology. Phase shift of a Bragg-reflected electron wave was measured by means of holographic interferometry using an electron microscope equipped with a field emission electron gun and an electron biprism. A short wavelength of high energy electrons is the essential key to the high vertical sensitivity of this method, since geometrical path differences produced by the surface topography are measured in units of wavelengths in interferometrical measuring. Phase shift at a monoatomic step and the displacement field around a dislocation emerging on the surface were observed.

Electrons↗

PubMed1995

Subchondral bone density in the human elbow assessed by computed tomography osteoabsorptiometry: a reflection of the loading history of the joint surfaces.

The functional adaptation of bone tissue to the mechanical stresses acting on it has been convincingly established. This association should apply as well to the subchondral bone, reflecting the long-term distribution of stress over the joint surfaces. Thirty-six specimens of the human elbow joint were investigated by computed tomography osteoabsorptiometry in order to assess the distribution of the subchondral mineralization. The distal surfaces usually were more highly mineralized than the proximal components of the joint, whereas the humeroulnar and the humeroradial parts exhibited a similar degree of mineralization. The fovea of the radial head always showed a central density maximum, and the trochlear notch usually presented a bicentric distribution pattern, with maxima beneath the ventral and dorsal regions of the articular surface. The different patterns of subchondral mineralization were shown to reflect the loading history of the overlying articular surfaces, which is determined mainly by geometrical factors. The flatter socket of the humeroradial joint leads to central load transmission, but the deeper socket of the humeroulnar joint will, by contrast, give rise to bicentric stress distribution.

Absorptiometry, Photon↗

PubMed1988

Studies on adsorptiochromism. II: Diffuse reflectance spectroscopy of adsorptiochromic spiropyrans adsorbed to some pharmaceutically useful solids.

The colored powders produced by the adsorption of four adsorptiochromic spiropyrans to many solids (silica gel, silicic acid, fumed silica, alumina, microcrystalline cellulose, talc, titanium dioxide) were examined by diffuse reflectance spectroscopy. The reflectance spectra were dominated by two bands, one at 550 nm and the other in the range 400-500 nm, often at 472 nm. Plots of the Kubelka-Munk function [F(R' infinity)] against g, the coverage expressed in nmol/m2, were linear at very low g and approached a limiting value independent of g at high coverage. The color formation upon adsorption terminates at coverages much lower than the maximum binding capacity of the solid. The slope of the plot of F(R' infinity) against g, at low g (denoted f0), appears to be sensitive to the scattering properties of the solid. For a single solid (silica gel), comparison of f0 for adsorptiochromic adsorbates with f0 for permanent dyes allowed estimates to be made of the fraction of adsorbed spiropyran in the colored form on the surface.

Benzopyrans↗

PubMed2002

Optimal methods for fluorescence and diffuse reflectance measurements of tissue biopsy samples.

BACKGROUND AND OBJECTIVE: In developing fluorescence spectroscopy systems for the in vivo detection of pre-cancer and cancer, it is often necessary to perform preliminary testing on tissue biopsies. Current standard protocols call for the tissue to be immediately frozen after biopsy and later thawed for spectroscopic analysis, but this process can have profound effects on the spectroscopic properties of tissue. This study investigates the optimal tissue handling methods for in vitro fluorescence spectroscopy studies. STUDY DESIGN/MATERIALS AND METHODS: The epithelial tissue of the Golden Syrian hamster cheek pouch was used in this study. Three specific experiments were carried out. First, the fluorescence properties of tissues in vivo and of frozen and thawed tissue biopsies were characterized at multiple excitation wavelengths spanning the ultraviolet-visible (UV-VIS) spectrum. Next, comparison of tissue fluorescence emission spectra in vivo, ex vivo (immediately after biopsy), and after the freeze and thaw process were systematically carried out at the excitation wavelengths corresponding to the previously identified fluorescence peaks. Lastly, intensities at the excitation and emission wavelength pairs corresponding to the fluorescence peaks were measured as a function of time after biopsy. Diffuse reflectance measurements over the UV-VIS spectrum were also made to evaluate the effects of oxygenation, blood volume, and scattering on the tissue fluorescence at these different excitation-emission wavelengths. RESULTS: This study indicates that the freezing and thawing process produces a significant deviation in intensity and lineshape relative to the in vivo fluorescence emission spectral data over the entire UV-VIS range between 300 and 700 nm. By contrast, examination of ex vivo emission spectra reveals that it closely preserves both the intensity and lineshape of the in vivo emission spectra except between 500 and 700 nm. The observed deviations can be explained by the diffuse reflectance measurements, which suggest increased hemoglobin deoxygenation and wavelength dependent changes in scattering in ex vivo tissues, and increased total hemoglobin absorption in the frozen and thawed samples. Furthermore, it was found that over a time window of 1.5 hours, spectroscopic changes brought about by degradation of the tissue due to biopsy or other factors are significantly smaller (10-30% variations in intensity) than those associated with the freezing and thawing process (50-70% decrease in intensity). CONCLUSIONS: It was found that the effects of freezing and thawing on the fluorescence properties of tissue are greater than any changes brought about by degradation of tissue over a time frame of 90 minutes after biopsy. Performing ex vivo fluorescence measurements within a reasonable time window has the advantage of more accurately reproducing the clinically relevant in vivo conditions in the case of the hamster cheek pouch tissue. Therefore, in tissue biopsy studies, the tissue sample should ideally be maintained in an unfrozen state prior to measurement.

Animals↗

PubMed2002

Dynamic oxygen-enhanced MRI reflects diffusing capacity of the lung.

The purpose of this study was to demonstrate the feasibility of dynamic oxygen-enhanced MRI in a clinical setting. We hypothesized that dynamic oxygen enhancement can reflect the regional diffusing capacity of the lung. Ten patients with pulmonary emphysema and seven healthy volunteers were examined with a respiratory-synchronized inversion recovery single-shot turbo spin-echo sequence (TR = 3200-5000 ms, TE = 16 ms, TI = 720 ms, ETS = 4 ms) following 100% oxygen inhalation, using a 1.5 T whole-body scanner. Maximum mean relative enhancement ratios calculated by averaging six defined regions of interest (ROIs) in both lungs were statistically compared between healthy volunteers and patients, and were correlated with diffusing lung capacity (%DL(CO)). In patients with pulmonary emphysema, maximum mean relative enhancement ratios were significantly decreased compared to those in healthy volunteers (P = 0.0008). Maximum mean relative enhancement ratio had excellent correlation with % DL(C0) (r(2) = 0.83). Dynamic oxygen-enhanced MRI may reflect the diffusing capacity of the lung; therefore, imaging of oxygen enhancement with MRI may provide maps of the diffusing capacity.

Adult↗

PubMed1980

Influence of aqueous fixation on articular surface morphology. A reflected light interference microscope study.

Fresh, unfixed temporomandibular fibrous articular surfaces from baboons were examined by reflected light interference microscopy. The appearances, recorded photographically, were compared with those of joints subjected to aqueous fixation in buffered glutaraldehyde and formalin. Fixation caused readily detectable, measureable changes in articular surface morphology. Reflected light interference microscopy is a valuable technique for the demonstration of the effects of fixation on articular surface morphology.

Aldehydes↗

PubMed1990

Do AgNOR counts reflect cellular ploidy or cellular proliferation? A study of trophoblastic tissue.

There is considerable debate as to whether AgNOR counts reflect cellular ploidy or cellular proliferation. Trophoblastic tissue from hydropic abortions and from hydatidiform moles offers an excellent model for analysing this problem. Thus, complete moles show considerable cell proliferation but are diploid, whilst partial moles show markedly less cell proliferation but are triploid: hydropic abortuses are diploid and show no cellular proliferation. AgNOR counts in villous cytotrophoblastic cells from hydropic abortions and from complete hydatidiform moles are similar, but those in partial hydatidiform moles are 50 per cent higher than in either hydropic abortions or complete moles. Thus, in non-neoplastic trophoblastic tissue AgNOR counts are clearly a reflection of ploidy rather than of cell proliferation.

Abortion, Spontaneous↗

PubMed2007

Memory deficits in long-term survivors of childhood brain tumors may primarily reflect general cognitive dysfunctions.

BACKGROUND: To analyze the impact of potential predictors on memory performance in survivors of childhood brain tumors and to examine whether deficits in memory after radiotherapy (RT) should be considered part of a more global mental dysfunction. PROCEDURE: We studied 126 patients with brain tumors diagnosed before the age of 15 years and treated January 1970 through February 1997 in Eastern Denmark. Sixty-nine of the 126 patients had received RT. In addition to working memory and long-term memory (LTM), general intelligence (IQ) was assessed. RESULTS: The mean test scores in nearly all memory tests were lower than expected from available norms. In multiple linear regression, treatment with RT, hemisphere tumor location, and hydrocephalus treated with a shunt at the time of diagnosis were found to be significant risk factors for memory deficits. When IQ was included as a covariate, RT did not significantly predict memory performance, while shunt and tumor location remained significant for several but not all tests. CONCLUSION: These results suggest that in patients treated with RT, memory deficits primarily reflect a general cognitive dysfunction, and it is likely that these intelligence deficits may compromise the chances of success of special memory training. In contrast, memory deficits in non-irradiated patients may to some degree reflect specific cognitive dysfunctions being most pronounced for patients treated with a shunt and for patients with hemispheric tumors.

Adolescent↗

PubMed2006

Plasma concentrations of soluble CTLA-4, CD28, CD80 and CD86 costimulatory molecules reflect disease severity of acute asthma in children.

Recent studies have demonstrated an elevation of plasma soluble costimulatory molecules B7.1 (CD80) and B7.2 (CD86), and their T lymphocyte counter receptors cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and CD28 in asthmatic patients. We hypothesized that these costimulatory molecules may reflect the severity of asthma and investigated the longitudinal changes of these soluble costimulatory molecules, and their clinical significance in children with an asthmatic exacerbation. Sixteen children hospitalized for asthmatic exacerbation were recruited and treated with systemic corticosteroid (CS) for 5 days. Plasma total Immunoglobulin E (IgE) and eosinophil cationic protein (ECP) concentrations were measured by microparticle immunoassay and fluorescence enzyme immunoassay, respectively. Soluble CTLA-4 (sCTLA-4), sCD28, sCD80, and sCD86 concentrations in plasma were measured by enzyme-linked immunosorbent assay, and their relationships with asthma severity, total IgE, ECP concentrations, and blood eosinophil count were analyzed. Plasma sCTLA-4, sCD28, sCD80, and sCD86 concentrations in patients were highest during the acute attack. They decreased significantly with a parallel increase of peak expiratory flow rate (PEFR) after CS treatment (all P < 0.05). Plasma sCTLA-4, sCD28, and sCD86 concentrations of patients at recruitment were inversely correlated with PEFR, whereas plasma sCD28 and sCD86 concentrations correlated positively with eosinophil count and plasma ECP concentration (all P < 0.05). Although there was no such correlation with IgE concentration, sCD28 was correlated very significantly with eosinophil count (r = 0.83, P < 0.0001). In conclusion, plasma sCTLA-4, sCD28, sCD86, and sCD80 concentrations may reflect the severity of acute asthma and more studies on larger cohorts are needed to assess whether these markers are useful for assessing asthmatic exacerbation in children.

Acute Disease↗

PubMed1999

Interaction between patient and physiotherapist: a qualitative study reflecting the physiotherapist's perspective.

BACKGROUND AND PURPOSE: The interaction between patient and physiotherapist (PT) is central in physiotherapeutic treatment. The purpose of this study was to investigate expert physiotherapists' perception of important factors influencing the quality of the interaction in physiotherapeutic treatment. METHOD: Ten experienced PTs working in primary healthcare were interviewed twice, triangulating a sort of important events, an exemplar and a key informant interview. The data were transcribed, coded and analysed for main themes and categories according to the qualitative technique described by Shepard et al. (1993) and Merriam (1988). RESULTS: Important factors in interaction could be divided into the 'Prerequisites dimension' and the 'Interaction dimension'. In the former, the most dominant theme was 'Internal prerequisites of the PT'. The most-cited category in this theme was 'Practical professional skills and patient experience'. In the latter, the themes 'Establishing contact', 'Ways of contact', 'Frames' and the 'Therapeutic process' emerged. Here, the most-cited categories were 'Establishing contact and confidence', having a 'Therapeutic role', 'Being sensitive and intuitive', 'Encountering', 'Listening' and 'Identification of patient resources'. These factors were seen as essential for promoting a positive patient outcome. Interaction skills were reinforced by reflection on patients' experiences. CONCLUSIONS: Interaction skills of the expert PT were thought to enhance the resources of the patient and lead to a positive patient outcome. Within professional development more emphasis ought to be put on reinforcing the reflective process of the PT, for instance by the use of supervision by an experienced colleague.

Adult↗

PubMed1996

Comparison of reflectance and transmission densitometry, using document and laser scanners, for quantitation of stained Western blots.

The quantitation of stained Western blots by reflectance and transmission scanning was explored using a blot of monoclonal immunoglobulin G probed with anti-mouse antibody. The so-called "scanned absorbance" output from a document scanner was found to be directly proportional to the fraction of light absorbed rather than obeying the logarithmic relationship expected for true spectrophotometric absorbance. This explains observations in the literature of a strongly curved relation between loading and "absorbance." Laser transmission densitometry of a blot immersed in a clarifying solvent mixture showed that peak area was linearly related to loading over a wide range. In reflectance mode the document scanner also gave equally linear quantitation of dry blots, providing that a logarithmic correction curve was applied during scanning. It was found advantageous to interpose a red acetate filter sheet between the blot and the scanning table to aid detection of weakly stained bands. The document scanner gave less satisfactory results when used in transmission mode on a clarified blot because weak bands were poorly quantitated.

Animals↗

PubMed1997

Two-photon excitation by the evanescent wave from total internal reflection.

We report the first observation of two-photon excitation of fluorescence using the evanescent wave from total internal reflectance (TIR). The evanescent wave at 770 nm from a fs Ti:Sapphire laser was used to excite the calcium probe Indo-1 at a quartz-water interface. The emission intensity of Indo-1 depended quadratically on the incident power at 770 nm, when incidence angles were above and below the critical angle (theta c) for TIR. The time-resolved intensity and anisotropy decays with TIR at 770 nm demonstrated the origin of the signals as Indo-1 and eliminated the possibility of scattered light contributing to the signal. The emission from Indo-1 was further demonstrated to be due to two-photon excitation by the increased anisotropy observed both from the steady-state and time-resolved data. Comparison of the intensities for one-photon and two-photon evanescent wave excitation revealed a smaller effective excited volume for two-photon excitation, indicating that the excited fluorophores are located closer to the interface with two-photon excitation. These results suggest that total internal reflectance can be combined with two- or multiphoton excitation for studies of surface absorption, immunoassays, or pattern photobleaching.

Fluorescence↗

PubMed2001

Detection of submicroscopic magnetite particles using reflectance mode confocal laser scanning microscopy.

Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy. We have used reflectance mode confocal laser scanning microscopy to detect single-domain magnetite crystals in both live and resin-embedded preparations of magnetotactic bacteria. We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria. En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles. The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue.

Bacteria↗