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Reflection contrast microscopy. Visualization of (peroxidase-generated) diaminobenzidine polymer products and its underlying optical phenomena.

Reflection contrast microscopy (RCM) has proven to be a useful tool for the study of living cells (Ploem 1975). Due to the effective suppression of aspecific reflected light by polarization optics combined with a quarter lambda plate at the front lens of the objective, low intensity reflection signals originating from minor amounts of precipitated diaminobenzidine (DABox) in immunocytochemically stained specimens, can be made visible. RCM has been successfully applied in demonstrating single copy nucleic acid sequences using in situ hybridization procedures (Landegent et al. 1984). We have systematically studied the aspects of image formation of DABox by RCM by using a model system consisting of glass slides coated with peroxidase containing protein layers to determine the conditions for optimal sensitivity of this detection method. Moreover, investigations were performed to study the relationship between the amount of reflected light and DABox depending on the thickness of the object. Both theoretical and practical evidence is obtained to show that DABox detection by RCM is based on interference phenomena occurring in the layer of DABox, and less on selective reflection. This restricts the type of specimen which can be used for sensitive detection of DABox by RCM. Consequently, in ultrathin (40 nm) sections osmificated DABox was visualized in peroxidatic positive cell organelles with high contrast and resolution. Similar results were obtained with immunoperoxidase stained material embedded in Lowicryl under conditions that did not allow visualization of the staining product by bright field microscopy.

3,3'-Diaminobenzidine

Continuous monitoring of gastroduodenal mucosal hemodynamics in rats by laser-Doppler flowmetry and reflectance spectrophotometry.

Gastroduodenal mucosal hemodynamics in rats was monitored continuously by laser-Doppler flowmetry (LDF) and reflectance spectrophotometry, and the validity of these techniques was determined. Corpus mucosal hemodynamics was recorded for 90 min, under stable conditions. In cases of graded hemorrhagic hypotension, corpus mucosal blood flow by LDF and hydrogen gas clearance, and potential differences showed a good correlation. Corpus, antral, and duodenal mucosal hemodynamics monitored by LDF, hydrogen gas clearance, and reflectance spectrophotometry reflected regional hemodynamic differences. In monitoring mucosal hemodynamics by LDF and reflectance spectrophotometry, regular oscillations (4-6 cycles/min) were observed in most animals. The characteristic change of oscillations during graded hemorrhagic hypotension was thus elucidated. In moderate hypotension (50-90 mmHg), high-amplitude and high-frequency (5-10 cycles/min) oscillations were observed, while in cases of severe hypotension (25-40 mmHg), the oscillations almost ceased. Observation of the oscillatory changes is thus a new application of LDF and reflectance spectrophotometry.

Animals

The thickness of monoolein lipid bilayers as determined from reflectance measurements.

Measurements of the reflectance of monoolein/n-alkane and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was 0.5 +/- 0.1 nm, independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When n-tetradecane and n-hexadecane were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.

Chemical Phenomena

Reflection and transmission of laser light from the esophagus: the influence of incident angle.

The application of lasers in gastrointestinal endoscopy is rapidly expanding. Because of the tubular configuration of the gastrointestinal tract, endoscopists often deliver laser energy at large angles of incidence. As incident angle affects the fraction of radiation reflected from the tissue surface, we measured the transmittance and reflectance of laser light from in vitro esophagus as a function of incident angle, using integrating sphere and goniometric techniques. At a wavelength of 633 nm and angles of incidence less than 50 degrees, the total transmittance of the esophagus is approximately 25% and the total reflectance is approximately 45%; both are isotropically distributed. At larger angles of incidence, a specularly reflected component becomes evident and the total reflectance increases. The absorbed light per unit area illuminated decreases with increasing angle, because the area illuminated by the laser beam is proportional to the secant of the incident angle. The data suggest that during endoscopic laser procedures the incident laser beam should be directed within 50 degrees of normal for optimal performance and safety.

Biophysical Phenomena

Photoplethysmographic evaluation of the relationship between skin reflectance and skin blood volume.

Although photoplethysmography can be used successfully to determine venous recovery time, it cannot be used in tests which rely on a determination of the blood volume change (e.g. maximum venous outflow). In seeking an explanation for this inadequacy it was shown that although the photoplethysmograph is able to indicate changes in reflectance, it is incapable of measuring reflectance, per se; information which is essential for an accurate calculation of venous volume decrease. A minor modification of the system enables relative reflectance measurements to be made. Measurements were performed under in vitro conditions to establish the skin-reflectance/skin-blood volume relationship; photon diffusion theory is then used to describe the skin-reflectance/skin-blood volume relationship and to determine the scattering and absorption coefficients of skin and whole blood.

Blood Volume

Fringe mode reflectance laser Doppler microscope system.

A reflectance fringe mode Laser Doppler microscope anemometer (LDMA) has been designed and tested. A novel beam splitter permits adjustment of the direction and separation of the two laser beams which form the fringe sensing volume, and furnishes the control needed to compensate for chromatic aberration and variation in optical quality. Tests conducted in a narrow 28:1 aspect rectangular flow channel filled with water showed close agreement between the predicted and measured velocity profiles. Velocity measurements, made alternately in the reflectance and transmittance modes using the same sensing volume in the channel, revealed deviations between the modes of less than 1.1% for water and 2.3% for whole blood. The deviation for the in vivo experiment was better than 2.8%. Velocity profiles were measured alternately in the same sensing volume, (dimensions were 10 x 12 microns) by reflectance and transmittance LDMA in 40 hamster cheek-pouch arterioles, ranging from 65 to 140 microns in diameter. These showed close agreement throughout the range of measured vessels. Reflectance LDMA makes non-invasive, high resolution velocity measurements in the range of 0.03 to 40 cm/s possible in small blood vessels near the surface of opaque organs and tissues. The accuracy and reproducibility of the readings were calculated to be better than 1.1%. Reflectance fringe mode LDMA greatly extends the applicability of LDMA because sites where transmittance LDMA can be applied are limited.

Animals

Reflectance of rat brain structures mapped by an optical fiber technique.

A method to detect differences in reflectance as a pair of glass fibers is advanced through various brain structures at a constant speed is described. The system has been used to develop a technique for accurate and easy placement of electrodes or cannulae into a limited brain region. The probe consisted of two thin (50 micrometer in diameter) glass optic fibers. A 5 W lamp was used to transmit light down one of these fibers. The relative intensity of light reflected from brain tissue into the other optic fiber was monitored with a photomultiplier and ink-writing recorder. Different brain structures were shown to vary in the amount of reflectance, with white matter having much higher reflectance than gray matter. It was demonstrated in a total of 239 penetrations that the patterns of successive changes in the reflected light response, as the probe was lowered into the brain, was characteristic for each frontal plane. The probe may additionally be itself used as an electrode if plated with silver.

Animals

Evoked otoacoustic emissions as cochlear Bragg reflections.

The reflection of cochlear waves is mathematically described for various assumptions about the dependence of the echo-generating mechanism on place and frequency, without requiring knowledge of the physical details of the mechanism. Any 'wave-related' reflections, caused by intrinsic features of the resonance or active-feedback mechanism, cannot explain long group delays in an approximately shift-similar basilar membrane (BM). Only 'BM related' scattering due to inhomogenities is a possible explanation then. Reflection at a fixed point of the BM could cause only narrow-band evoked otoacoustic emissions (EOAEs). Long group delays in a wider frequency band can be obtained by assuming Bragg reflection at an approximately periodic inhomogenity of BM parameters, possibly due to spatial variations of active undamping. This explains the long delays, about inversely proportional to frequency, the decreasing instantaneous frequency, the often found multi-packet or modulated structure of echoes (vanishing at higher SPL), the spectra of simultaneous and delayed EOAEs and their relation to the threshold of hearing. These phenomena do not necessarily require multiple reflection due to middle/inner-ear mismatch.

Auditory Threshold

Quantitative assessment of ultrasonic myocardial reflectivity in hypertrophic cardiomyopathy.

The purpose of this study was to investigate the relation between acoustic properties of the myocardium and magnitude of left ventricular hypertrophy in patients with hypertrophic cardiomyopathy. An on-line radio frequency analysis system was used to obtain quantitative operator-independent measurements of the integrated backscatter signal of the ventricular septum and posterior free wall in 25 patients with hypertrophic cardiomyopathy and 25 normal age-matched control subjects. The integrated values of the radio frequency signal were normalized for the pericardial interface and expressed in percent. Tissue reflectivity was significantly increased in the hypertrophied ventricular septum, as well as in the nonhypertrophied posterior free wall, in patients with hypertrophic cardiomyopathy (58 +/- 15% and 37 +/- 12%, respectively) compared with values in normal subjects (33 +/- 10% and 18 +/- 5%, respectively; p less than 0.001). Furthermore, measurements of reflectivity of the septum or posterior free wall, or both, were beyond 2 SD of normal values in greater than 90% of the patients and were also abnormal in each of the five study patients who had only mild and localized left ventricular hypertrophy. No correlation was identified between myocardial tissue reflectivity and left ventricular wall thickness in the patients with hypertrophic cardiomyopathy (correlation coefficient r = 0.4; p = NS). These findings demonstrate that myocardial reflectivity is abnormal in most patients with hypertrophic cardiomyopathy and is largely independent of the magnitude of left ventricular hypertrophy. Moreover, quantitative analysis of ultrasonic reflectivity can differentiate patients with hypertrophic cardiomyopathy from normal subjects independently of clinical features and conventional echocardiographic measurements.

Adolescent

Critical self-reflection and self-perceived altruism: when self-reward fails.

Social learning theory has produced a three-step model of prosocial development: In the young child, prosocial behavior is elicited by material rewards; in the older child, it is elicited also by social rewards; and in the morally mature adult, it is elicited by self-rewards based on an internalized perception of oneself as a kind, caring, altruistic individual. Self-perception theory has complicated this social learning model by demonstrating that once the third step is reached, the continued presence of material and social rewards may undermine intrinsic prosocial motivation based on self-rewards, producing moral regression. We propose a further complication. Critical self-reflection--the desire to know thyself, warts and all--may introduce a self-deprecating attributional bias that can undermine self-perceived altruism, even following helping for which one receives only intrinsic self-rewards. Two experiments are reported in which we manipulated critical self-reflection on one's reasons for helping. Results indicated that self-reflection undermined self-perceived altruism, especially when the salience of the self-rewards for helping was high. Experiment 2 also provided evidence that, as predicted, this self-reflection effect was most apparent for individuals who valued self-knowledge more highly than concern for others. Moral consequences of critical self-reflection are discussed.

Altruism

Reflectivity bias in picture-pointing grammatical comprehension tasks.

Two studies, one correlational and one experimental, supported the hypothesis that 4- and 5-year-olds' performance on a picture-pointing grammatical comprehension task administered under standard procedures is highly influenced by reflectivity style. In the first study, a substantial correlation was obtained between reflectivity and picture-pointing grammatical comprehension task performance. This correlation was significantly greater than that between reflectivity and object-manipulation grammatical comprehension task performance. In the second study, a reflectivity-promoting but grammatically irrelevant modification in the administration of the picture-pointing task resulted in significantly improved comprehension scores and a significantly lower correlation with reflectivity.

Age Factors

Reflection of CO2 laser radiation from typical surfaces encountered in hospital environments.

Measurements of the reflectivity of CO2 laser radiation (10.6 micron) from typical clinical surfaces are reported. The results obtained confirm that stray reflections from these surfaces could be a potential hazard to both patient and hospital staff. Some conducting surfaces, for example, reflect up to 95% of the radiation incident upon them and even dielectrics reflect over 50% in some cases. The results reported are for linearly polarised light only. Strong evidence is shown that the deposition of certain thin coatings dramatically reduces the percentage of 10.6 micron radiation reflected.

Carbon Dioxide

Dream self-reflectiveness as a learned cognitive skill.

This research was directed toward the contradiction sustained by cognitive dream psychology, which on the one hand regards dreaming as higher symbolic activity and, on the other, sees its organizational and functional characteristics as derivative and/or inferior to those of waking consciousness. Study 1 evaluates the degree of self-reflective meta-cognition in dreams from different sleep stages. Subjects were 24 college students selected such that half were self-reported high-frequency dream recallers and half were low-frequency recallers. Both groups were composed equally of men and women. Greater self-reflectiveness (SR) was found in REM dreams as compared with those from stages 2 and 4, which did not differ. High-frequency recallers showed more dream SR than did low-frequency recallers. Study 2 assessed the extent to which self-reflective and lucid dreaming can be learned as a cognitive skill by varying levels of intention and attention paid to dreaming. After 3 weeks of home dream collection, results showed that four experimental groups had greater dream SR than did a baseline group. The most effective treatment was the mnemonic, wherein attention patterning schemas learned in waking resulted in more self-reflective and lucid dreaming than did either baseline or attention-control conditions. These results provide evidence that dreaming is not single-minded but variable along a self-reflective process continuum, and suggest functional and organizational levels that are consistent with the conception of dreaming as higher order cognitive activity.

Adult

Capillary permeability in rat hindquarters as determined by estimations of capillary reflection coefficients.

Osmotic reflection coefficients (sigma) for a variety of solutes ranging from NaCl to albumin were determined in perfused maximally vasodilated rat hindquarters employing the osmotic transient method (Vargas & Johnson 1964). Measurements were performed at high flows and using short tubings with small volumes. Intracapillary solute concentrations of the osmotic transients were measured or estimated for solutes of the size of inulin or smaller. The PS for Cr-EDTA and cyanocobalamine were determined repeatedly in half of the experiments using an on-line modification of the single injection (indicator diffusion) method (Rippe & Stage 1978) and capillary filtration coefficients (CFC or LpS) were followed in all experiments. The capillary osmotic reflection coefficient was determined to 0.05 for NaCl, to 0.08 for sucrose, to 0.39 for inulin, to 0.57 for myoglobin and to 0.87 for albumin. These reflection coefficients were compatible with a 'small pore radius' of approximately 40 A (slit width (w) of approximately 50 A) according to modern hydrodynamic theories for the reflection coefficient and the parallel transcapillary pathway hypothesis. The best fit of the osmotic transient data to current theories for the reflection coefficient occurred if the major portion (86-87%) of the hydraulic conductivity (Lp) was accounted for by this paracellular 'small pore' (slit) pathway and if 3.0-4.1% of Lp could be ascribed to a transcellular pathway (sigma approximately I) while the remaining fraction (10%) of Lp was accounted for by a non-selective paracellular pathway (sigma approximately o); that is, by 'large pores'.

Animals

Some reflective properties of the tapetum lucidum of the cat's eye.

1. Observation of the fresh, isolated tapetum of the cat with a low-power microscope showed it to be composed of multilayer reflectors (domains) each being less than 6 mum in diameter. It is concluded that these are subdivisions of the intracellular bundles of rodlets seen in electron micrographs.2. Apparatus was devised for illuminating a single domain with a pencil of white light and observing the colour and direction of the reflected light.3. Those domains which lie near the vitreal surface of the tapetum tend to reflect blue light and deeper ones reflect longer wave-lengths; this is attributed to differences in the layer spacings of the domains.4. Each domain has a set of reflecting planes lying within 15 degrees of the general plane of the tapetal surface. In addition, many domains have surfaces inclined to these at angles greater than 20 degrees which also can reflect visible light.5. The mechanism of reflexion is discussed in relation to the latticelike structure of the domain.6. It is suggested that under some conditions the nature of the tapetum may lead to significant local variations in the spectral sensitivity of the visual system.

Animals

The left paratracheal reflection.

The left paratracheal reflection, which is found medial to the left subclavian artery reflection, was sought in 302 normal posteroanterior (PA) chest radiographs, 93 conventional chest tomograms, and 113 thoracic computed tomographic (CT) scans. The left paratracheal reflection was visible on 31% of normal PA chest radiographs. Conventional tomography and CT showed that this finding is produced by contact of the lung with the mediastinum anterior to the left subclavian artery. The CT scans studied showed that the left paratracheal reflection actually represents contact of the lung with left paratracheal mediastinal fat 94% of the time, with the proximal 1-2 cm of the lateral wall of the left common carotid artery 5% of the time, and with the left tracheal wall 1% of the time. A variety of entities may alter the left paratracheal reflection.

Adult

Propagation velocity and reflection of pressure waves in the canine coronary artery.

In this study the pressure wave velocity in the anterior descending branch of the left coronary artery (LADC) of the dog was measured by determining the delay time between pressure pulses along this artery. This method can only be applied if reflections of the pressure wave distal to the sites of pressure measurement are insignificant. From araldite casts of the coronary arteries the following relation between the diameter proximal to (dprox) and distal to (ddist 1, ddist 2) a bifurcation was found: dprox2.55 = ddist12.55 + ddist 22.55, indicating that reflections at a bifurcation areminimal. In dogs reflections were studied by inducing during diastole a pressure pulse in the aorta and measuring pressure and volume flow proximal to and pressure distal to a segment of the LADC at various levels of the coronary peripheral resistance. Reflection of high-frequency components (greater than 7 Hz) was found to be insignificant, allowing application of the above-mentioned method for measuring the wave-front velocity, which is insensitive to low-frequency reflection. At a pressure in the LADC of 13.3 kPa this velocity was 8.6 +/- 1.4 m.s-1 (mean +/- SD). The calculated dynamic cross-sectional stiffness (deltaP/(deltaA/A)) of the LADC was 97 +/- 11 kPa (mean +/- SE) at an arterial pressure of 13.3 kPa.

Animals

Reflectance measurements of hematocrit and oxyhemoglobin saturation.

Fiberoptic oximeters measure oxyhemoglobin saturation from the optical reflectance of whole blood, but the calibration of such oximeters is hematocrit dependent. Therefore, using photon-diffusion theory and an empirical approach, we have developed a new reflectance method that determines hematocrit and correspondingly corrects the oxyhemoglobin-saturation measurement. Our method employs four fiber-optic light guides, a photodetector, and three inexpensive light-emitting diodes (one with emissions at 660 nm and two at 813 nm). Hematocrit is determined from the ratio of reflectances from the differently spaced emitting fibers at 813 nm and is used to correct the 813-660 nm measurement of oxyhemoglobin saturation. In red cell suspensions, the mean difference between reflectance measurements of hematocrit and conventional determinations was only 2.09% (r = 0.99), and when compared with conventional gasometric measurements of oxyhemoglobin saturation, the reflectance method yielded the same calibration curve for different hematocrits and gave a mean difference of only 2.67%. Although the technique is demonstrated with a cuvette appropriate for an extracorporeal circulation in animal experiments, it could possibly be further developed for fiber-optic catheter oximeters.

Animals