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PubMed1965

LOCAL REDUCTION OF SPINDLE FIBER BIREFRINGENCE IN LIVING NEPHROTOMA SUTURALIS (LOEW) SPERMATOCYTES INDUCED BY ULTRAVIOLET MICROBEAM IRRADIATION.

Irradiation of the mitotic spindle in living Nephrotoma suturalis (Loew) spermatocytes with an ultraviolet microbeam of controlled dose produced a localized area of reduced birefringence in the spindle fibers. The birefringence was reduced only at the site irradiated, and only on the spindle fibers irradiated. Areas of reduced birefringence, whether produced during metaphase or during anaphase, immediately began to move toward the pole in the direction of the chromosomal fiber, even though the associated chromosomes did not necessarily move poleward. Both the poleward and the chromosomal sides of the area of reduced birefringence on each chromosomal fiber moved poleward with about the same, constant, velocity. On the average, the areas of reduced birefringence moved poleward with about the same velocities as did the chromosomes during anaphase. The area of reduced birefringence was interpreted as a region in which most, though not necessarily all, of the previously oriented material was disoriented by the irradiation. The poleward movement of the areas of reduced birefringence indicates that the spindle fibers are not static, nonchangeable structures. The poleward movement possibly represents the manner in which the birefringent spindle fibers normally become organized. All the experiments reported were on primary spermatocytes which completed the second meiotic division subsequent to the experimentation. Since both the irradiated and the control cells completed the two meiotic divisions, the movement and irradiation effects studied in the first division were nondegenerative.

Anaphase↗
PubMed1985

Higher order structure of chromatin: influence of ionic strength and proteolytic digestion on the birefringence properties of polynucleosomal fibers.

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential. On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments. When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone H1 and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.

Animals↗
PubMed1967

Ultrastructure and birefringence of the isolated mitotic apparatus of marine eggs.

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.

Animals↗
PubMed1976

Quantitative studies on the polarization optical properties of striated muscle. I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxed state.

The changes in birefringence in the rigor to relax transition of single triton-extracted rabbit psoas muscle fibers have been investigated with quantitative polarized light techniques. The total birefringence of rest lenght fibers in rigor was (1.46 +/- 0.08) x 10(-3) and increased to (1.67 +/- 0.05) x 10(-3) after Mg-ATP relaxation. Pyrophosphate relaxation increased the total birefringence only slightly, whereas subsequent Mg-ATP relaxation elicited the maximum increase in birefringence. Changes in lattice spacing did not account for the total increase in birefrigence during relaxation. Moreover, the increase in total birefringence was attributable to increases in intrinsic birefringence as well as form birefringence. No change in birefringence was exhibited upon exposure to a relaxation solution after myosin extraction. Synthetic myosin filaments were prepared and treated with relaxation and rigor solutions. The negatively stained filaments treated with a rigor solution had gross irregular projections at either end, while the filaments treated with a relaxing solution were more spindle shaped. The results are compatible with the view that the subfragment-2 moieties of myosin angle away from the myosin aggregates (light meromyosin) to permit the attachment of the subfragment-1 moieties to actin.

Adenosine Triphosphate↗
PubMed1983

Comparison of birefringence signals and calcium transients in voltage-clamped cut skeletal muscle fibres of the frog.

The characteristic features of birefringence and calcium transients were compared in voltage-clamped cut skeletal muscle fibres: Birefringence signals were measured by introducing crossed polarizers above and below the fibres (+/- 45 degrees to the fibre axis) and using light of 790 nm. Calcium transients were monitored by the metallochromic indicator dye, Antipyrylazo III recording the changes in fibre absorbance at 720 nm. The dye entered the myoplasmic space by diffusion through the cut end. The early large birefringence signals, related to excitation-contraction coupling had a time course similar to that of calcium transients. The two signals had superimposable onset but the change in optical retardation peaked later and declined more slowly than the calcium signal. Using depolarizing pulses with increasing amplitudes both transients showed the same voltage dependence in the rate of rise, the time-to-peak and the peak amplitude. Birefringence signals recorded at different voltages along the strength-duration curve for contraction threshold had the same amplitudes and similar time constants for the falling phase comparable to the properties of the calcium transients. After applying dantrolene sodium both signals were reduced to the same extent. A shift in the contraction threshold was found towards more positive membrane potential values. The birefringence and calcium transients recorded at the new contraction threshold during the dantrolene treatment showed nearly the same size and time course as threshold transients obtained before the treatment. A subthreshold concentration of caffeine increased the peak amplitude of birefringence signals at a given voltage and decreased the latency of the signals. Birefringence transients at the new contraction threshold under caffeine were smaller than controls. Both effects are very similar to the changes in calcium transients due to caffeine treatment as previously reported. Consequently the voltage-dependent properties of birefringence and calcium transients and their responses to caffeine and dantrolene treatment are nearly the same. These results support the view that the changes in optical retardation of the fibres reflect calcium bound to some sarcoplasmic binding site rather than a potential change of the sarcoplasmic reticulum.

Animals↗
PubMed2004

Thickness and birefringence of healthy retinal nerve fiber layer tissue measured with polarization-sensitive optical coherence tomography.

PURPOSE: Thinning of the retinal nerve fiber layer and changes in retinal nerve fiber layer (RNFL) birefringence may both precede clinically detectable glaucomatous vision loss. Early detection of RNFL changes may enable treatment to prevent permanent loss of vision. Polarization-sensitive optical coherence tomography (PS-OCT) can provide objective information on RNFL thickness and birefringence. METHODS: PS-OCT scans around the optic nerve head (ONH) of two healthy young volunteers were made using 10 concentric circles of increasing radius. Both the mean RNFL thickness and mean retinal nerve fiber birefringence for each of 48 sectors on a circle were determined with data analysis. RESULTS: Both the RNFL thickness and birefringence varied as a function of sector around the ONH. The RNFL became thinner with increasing distance from the ONH. In contrast, the birefringence did not vary significantly as a function of radius. CONCLUSIONS: Birefringence of healthy RNFL is constant as a function of scan radius but varies as a function of position around the ONH, with higher thickness values occurring superior and inferior to the ONH. Measured double-pass phase retardation per unit depth around the ONH ranged between 0.10 and 0.35 deg/microm, equivalent to birefringences of 1.2 x 10(-4) and 4.1 x 10(-4) respectively, measured at a wavelength of 840 nm. Consequently, when a spatially constant birefringence around the ONH is assumed, the conversion of scanning laser polarimetry (SLP) phase-retardation measurements to RNFL thickness may yield incorrect values. The data do not invalidate the clinical value of a phase-retardation measurement, but affect the conversion of phase retardation to RNFL thickness.

Adult↗
PubMed1983

Birefringence of articular cartilage and the distribution on collagen fibril orientations.

Birefringence of the deep zone of pig tibial plateau cartilage has been measured by polarized light microscopy using monochromatic light and a Sénarmont compensator. Thin (20 micrometers) sections of the tissue were imbibed with mixtures of physiological saline and glycerol to separate the effects of form and intrinsic birefringence. Measured birefringence was plotted against liquid refractive index and the experimental points defined a parabola whose minimum corresponded to a refractive index for collagen of 1.48. When the tissue was imbibed with glycerol, the birefringence measurements provided a good estimate of intrinsic birefringence. The orientation distribution function, g(phi), of collagen fibrils in this zone was then determined by X-ray diffraction. Thus X-ray diffraction yielded the form of g(phi) which corresponded to the measured intrinsic birefringence. Using this result the intrinsic birefringence of a hypothetical cartilage site with perfectly oriented collagen fibrils was calculated to be (6 +/- 1) x 10(-4). Birefringence measurements on further sites could then be converted into standard deviations of g(phi), assuming a Gaussian distribution.

Animals↗
PubMed1997

Quantitative measurements of linear birefringence during heating of native collagen.

BACKGROUND AND OBJECTIVE: Linear birefringence is an anisotropic property of rat tail tendon, which is largely composed of collagen. Our goal is to show that the dynamic range and sensitivity of the linear birefringence loss of collagen during heating are sufficient for kinetic modeling of the reaction. STUDY DESIGN, MATERIALS AND METHODS: The linear birefringence loss was quantified for tendon denatured via both a heated-isotonic-saline bath and a heated stage. All measurements were made with a polarizing transmission microscope equipped with a Berek compensator. RESULTS: The data show that the loss of linear birefringence is a first-order kinetic reaction. The native rat tail tendon birefringence, delta n = 3.0 +/- 0.6 x 10(-3) (mean +/- std. err.), is lost after denaturation occurs (delta n = 0). Application of the Arrhenius equation to the linear birefringence data yields the activation energy (Ea = 89 +/- 1 kcal/mole), pre-exponential coefficient (A = e130 +/- 1 s-1), enthalpy (delta H = 88 +/- 1 kcal/mole) and entropy (delta S = 197 +/- 2 cal/degree K.mole). CONCLUSION: This study shows that dynamic changes in linear birefringence can be used to monitor thermally induced changes in collagen.

Animals↗
PubMed1989

Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements.

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mM ionic strength, 15 degrees C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.

Adenosine Triphosphate↗
PubMed2005

Birefringence of the primate retinal nerve fiber layer.

The purpose of this study was to measure the peripapillary retinal nerve fiber layer (RNFL) thickness, phase retardation (PR), and depth-resolved birefringence (Deltan) of the normal primate eye using Enhanced Polarization-Sensitivity Optical Coherence Tomography (EPS-OCT). Both eyes of two rhesus monkeys were imaged with EPS-OCT. A multiple incident polarization state nonlinear fitting algorithm was used to determine RNFL phase retardation. RNFL thickness (RNFLT) was determined from the corresponding EPS-OCT intensity image and phase retardation per unit depth (PR/UD, proportional to Deltan) was calculated by dividing PR by RNFLT. Peripapillary area maps consisting of pixels uniformly distributed along a radius from 0.8 to 1.8 mm from the center of the optic nervehead were constructed for RNFLT, PR, and PR/UD. Average PR/UD in the superior and inferior quadrants was 18 degrees /100 mivrom (Deltan=4.2 x 10(-4)) and average PR/UD in the nasal and temporal quadrants was 6.3 degrees /100 microm (Deltan=1.5 x 10(-4)). Relative magnitude of PR radial gradient is similar to that of RNFLT radial gradient and no radial gradient was observed for PR/UD. Polarization-dependent amplitude attenuation per unit depth (PDAA/UD) was 0.02 rad/100 microm in thick RNFL regions. RNFL birefringence was higher in the arcuate bundles compared to nasal and temporal fibers (P=0.001). Birefringence was nearly equal in nasal and temporal quadrants. No statistically significant (P=0.01) radial gradient of birefringence was observed in any quadrant. RNFL birefringence is believed to originate from anisotropic structures within the cytoskeleton of the parallel axons. Birefringence differences presented in this study cannot be explained by the known axon diameter distribution around the optic nervehead and suggest other sources of the birefringence signal including neurotubules and neurofilaments.

Algorithms↗
PubMed1963

Birefringence of protein solutions and biological systems. II. Studies on TMV, tropocollagen, and paramyosin.

The intrinsic birefringences of TMV, tropocollagen, and paramyosin were calculated from flow birefringence measurements using the theory of Peterlin and Stuart. The values are -0.029, -0.029, and -0.030, respectively. The intrinsic birefringences of TMV and tropocollagen were measured as a function of the refractive index of the solvent in glycerol-water mixtures. In both cases the values were not constant and became less negative as the refractive index increased. Theoretical calculations showed that the large solvent effect could not be caused by a hydration shell of index different from that of the bulk solvent. It is concluded that either (a) the intrinsic birefringence calculated from the Peterlin-Stuart theory is incorrect or (b) the intrinsic birefringence depends markedly on the solvent. These results are of importance to the problem of quantitative polarized light microscopy since the separation of form and intrinsic birefringence contributions is based on the assumption that intrinsic birefringence is independent of solvent.

Biophysical Phenomena↗
PubMed1989

Form birefringence of muscle.

We investigate the sensitivity of measurements of muscle birefringence to cross-bridge dynamics in the resting, active, and rigor states. The theory of form birefringence is reviewed, and an optical model is constructed for the form birefringence of muscle. Values for the parameters in the model are selected or deduced from the literature. As an illustration of the use of the model, plausible distributions for the orientations of cross-bridges in the resting, active, and rigor states are constructed using a model for cross-bridge dynamics suggested by Huxley and Kress (1985). The general magnitude of the predictions of our model is comparable with that of published measurements of muscle birefringence. However, the precise values of the predicted birefringence for the resting, active, and rigor states are sensitive to the assumed orientations of cross-bridges. We also investigate the dependence of muscle birefringence on sarcomere length and on disorder in the orientation of the myofilament array. We conclude that measurements of muscle birefringence can play a useful role in distinguishing between proposed models of cross-bridge dynamics.

Actin Cytoskeleton↗
PubMed1985

Transient electrical birefringence characterization of heavy meromyosin.

Heavy meromyosin (HMM) and myosin subfragment 1 (S1) were prepared from myosin by using low concentrations of alpha-chymotrypsin. The light chain distribution in HMM was identical with that of myosin, within experimental error, when analyzed on 12% polyacrylamide gels after electrophoresis. Specific birefringences and birefringence decay times were measured by transient electrical birefringence in 5 mM KCl, 5 mM tris(hydroxymethyl)aminomethane (pH 7), and 1 mM MgCl2 at 4 degrees C under gentle conditions that reduced the CaATPase activity by less than 10%. For solutions of HMM, by use of electric field pulses shorter than 0.5 microseconds, the birefringence decay signal from the S1 portions of HMM could be resolved and the rotational motions of the S1 moieties observed directly. The rotation relaxation time, adjusted to 20 degrees C, was 0.34 microseconds; this is in quantitative agreement with previous hydrodynamic results obtained by using covalently attached probes. The assignment of the fast decay time obtained with HMM to the S1 portions was confirmed by birefringence decay measurements on free S1, for which the relaxation time was 0.13 microseconds, corrected to 20 degrees C. The specific birefringences for S1 and HMM, respectively, were 0.37 X 10(-6) and 12.8 X 10(-6) (cm/statvolt)2. Thus, for much longer electric field pulses, the signal from HMM is due almost entirely to its subfragment 2 (S2) portion, and its rotational dynamics can also be monitored directly by using electrical birefringence. The decay of the signal from the S2 portion could be adequately fit without evoking bending of the S2 portion of HMM other than at its junction with S1.

Birefringence↗
PubMed1990

Electric birefringence of dilute agarose solutions.

The technique of transient electric birefringence was used to investigate the orientation of agarose solutions in pulsed electric fields. If the agarose was dissolved in deionized water, the sign of the birefringence was positive when the electric field was small, indicating that the agarose molecules were orienting parallel to the electric field lines. The decay of the birefringence was rapid, consistent with the orientation of individual agarose helices. The amplitude of the birefringence, but not the birefringence decay times, increased as the agarose solution aged, suggesting that the helices formed slowly from the sol state. Increasing the amplitude or duration of the pulsed electric field caused additional negative, and then positive, birefringence signals to appear, characterized by much slower rise and decay times, consistent with the formation of aggregates. The slowest decay times ranged from 7.5-9.0 s, suggesting that the aggregates were several microns in size. When agarose was dissolved in dilute Tris buffer instead of deionized water, the fast positive birefringence signal was not observed, suggesting that individual helices were not present in solutions containing dilute buffer.

Birefringence↗
PubMed2006

Development of a real-time corneal birefringence compensated glucose sensing polarimeter.

BACKGROUND: As is well known, in order to optimally manage diabetes mellitus, monitoring blood glucose levels several times daily is recommended so appropriate actions can be taken to maintain these levels within a near-normal physiologic range. One technique that shows promise is the use of optical polarimetry. This technique has the potential to noninvasively measure physiological glucose levels in the eye that are correlated to blood glucose concentrations. To date, the main factor limiting in vivo polarimetric glucose measurements is corneal birefringence, which tends to mask the glucose signature. In this investigation, a method to compensate for the effects of corneal birefringence is demonstrated, thus allowing for polarimetric glucose measurements in samples with time-varying birefringence contributions. METHODS: In this paper, using a custom-designed laser-based optical polarimetry system with an integrated birefringence compensator, noninvasive glucose measurements in the physiological range are accurately measured within various birefringent samples similar in structure to the eye. RESULTS: Using the laser-based polarimetric approach, it is shown that glucose levels within the physiological range in the presence of significant varying birefringence can be accurately predicted with as low as 13.84 mg/dL error. CONCLUSIONS: The ability to compensate for corneal birefringence effects provides promise for the eventual development of a commercial home-based noninvasive polarimetric glucose monitor.

Aqueous Humor↗
PubMed1989

Changes in birefringence as markers of thermal damage in tissues.

Light microscopy using polarized transmission illumination of routinely stained histologic sections shows changes of the native birefringence of certain tissue constituents when heated by laser irradiation or electrosurgical current. The naturally occurring birefringence of cardiac muscle disappears permanently when the muscle is frozen, thawed, and heated to temperatures in excess of 42 degrees C in vitro. This loss of birefringence is produced with temperatures at which other morphologic thermal changes are hard to detect; thus, it is a low-temperature tissue marker which can be used to observe the extent of thermal damage in tissues. Partial loss of the native birefringence of collagen occurs in canine urinary bladder coagulated by laser irradiation and pericardium heated with electrodes. In addition, thermally coagulated collagens have variable birefringence color shifts when compared to the adjacent unaffected collagens in stained histologic sections. The gradual birefringence color changes are seen at tissue temperatures higher than those at which the thermally induced hyalinization (coagulation) of collagen usually occurs (about 60-70 degrees C), but below those at which carbonization is seen (200+ degrees C). Birefringence changes can be measured to test mathematical models of thermal damage necessary for development of dosimetry models in medical applications of laser irradiation.

Animals↗
PubMed1984

Birefringence signal and early mechanical changes at normal and increased tonicities in frog skeletal muscle.

Simultaneous measurements of the time course of early mechanical events and the early large birefringence signal were performed during activation of isolated frog skeletal muscle fibres bathed in iso- and hypertonic media. A piezo-electric transducer was used which had a resonance frequency of 3.3 kHz and a sensitivity of 740 mV/mN (0.5 mV noise peak to peak) and a compliance of 0.6 mu/mN. The delay from stimulus onset to the onset of the birefringence signal was estimated to be 0.78 +/- 0.03 ms in normal Ringer solution at room temperature (20-23 degrees C). The beginning of latency relaxation appeared subsequent to the birefringence signal after a delay of 0.36 +/- 0.03 ms. Increasing the tonicity retarded the time course of both birefringence signal and latency relaxation. In solutions of twofold greater tonicity an increase in tension (denoted pre-relaxation contraction) was observed to precede latency relaxation. Pre-relaxation contraction became more prominent with increasing tonicity and appeared to coincide with its onset with the onset of the birefringence signal. The pre-relaxation contraction was shown not to be a stimulus artifact and was not reduced in amplitude by D 600 or calcium-poor solutions. Lowering the temperature to 2.2 degrees C in normal Ringer solution delayed the onsets of the birefringence signal and latency relaxation, and increased the interval between the two signals but did not result in a tension increase before latency relaxation. The coincidence of the birefringence signal onset with the onset of pre-relaxation contraction suggests a common aetiology. The aetiology remains speculative but seems to require hypertonicity for expression of the mechanical signal.

Action Potentials↗
PubMed1990

Monitoring rejection after heart transplantation: cytoimmunological monitoring on blood cells and quantitative birefringence measurements on endomyocardial biopsy specimens.

Cytoimmunological monitoring and quantitative birefringence measurements were used as potential aids in diagnosing acute rejection after heart transplantation instead of histopathological assessment of the endomyocardial biopsy specimen alone. Cytoimmunological monitoring was based on morphological inspection and quantitation of mononuclear cells, particularly activated lymphoid cells. Quantitative birefringence measurements comprise a variable for myocyte contractile function. Its read out is the ratio of the degree of birefringence before contraction to that after. Cytoimmunological monitoring indicated significantly higher concentrations of activated lymphocytes in moderate or severe acute rejection, and quantitative birefringence measurements indicated decreased myocyte function during severe and resolved or resolving rejection. Cytoimmunological monitoring and quantitative birefringence measurements were diagnostically most useful in terms of sensitivity, specificity, and predictive value, when only data gathered before the first episode of acute rejection were considered. For cytoimmunological monitoring, diagnostic relevance was optimal when the data were expressed as relative proportions of activated lymphocytes. The quantitative birefringence measurements correlated best with analysis of the endomyocardial biopsy specimen when a cut off value of 1.25 was used. When both methods for diagnosing acute rejection were analysed together, no improvement in sensitivity (value 0.44) was found, but the specificity increased to 0.98 and the predictive value to about 0.80. It is concluded that cytoimmunological monitoring is a useful, non-invasive additional method for diagnosing the first period of acute rejection after heart transplantation and that quantitative birefringence measurements give valuable information on the extent of myocyte damage.

Birefringence↗