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Microtubules contribute to the birefringence of the retinal nerve fiber layer.

PURPOSE: The retinal nerve fiber layer (RNFL) exhibits birefringence that is due to the oriented cylindrical structure of the ganglion cell axons. Possible birefringent structures include axonal membranes, microtubules (MTs), and neurofilaments. MTs are generally assumed to be a major contributor, but this has not been demonstrated. In this study, the MT depolymerizing agent colchicine was used to evaluate the contribution of MTs to RNFL birefringence. METHODS: Retinal nerve fiber bundles of isolated rat retina were observed through an imaging polarimeter set near extinction. Images were taken over an extended period. During baseline, the tissue was perfused with a physiological solution. During a treatment period, the solution was switched either to a control solution identical with the baseline solution or to a similar solution containing colchicine. The contrast of nerve fiber bundles was used to follow change of RNFL birefringence over time. RESULTS: When imaged by the polarimeter, birefringent retinal nerve fiber bundles appeared as either bright or dark stripes. Bundles displayed as bright stripes were used to follow changes in retardance. The contrast of nerve fiber bundles was stable in control experiments. However, in treatment experiments, bundles were bright during the baseline period, but the contrast of bundles decreased rapidly when the colchicine solution was applied; bundles were barely visible after 30 minutes of treatment. After 70 minutes, the bundle contrast was close to zero at all wavelengths studied (440-780 nm). CONCLUSIONS: MTs make a significant contribution to RNFL birefringence. The decrease of RNFL birefringence in glaucoma may indicate a loss of MTs.

Animals↗

[Two-dimensional reconstruction of birefringence map of the skeletal muscle sarcomere in relaxed and rigor states studied by interference microscopy].

A new method of automatized quantitative interferometry of skeletal muscle fibers was developed for the investigation of birefringence. A device based on the Linnic microscope was constructed to obtain phase images, which are two-dimensional pictures of birefringence. For the first time, two-and-three-dimensional maps of both total birefringence and birefringence for individual sarcomeres in the central part of muscle fiber were visualized using large databases. It was shown that total birefringence of fibers at rest length in the rigor state was lower as compared with the relaxed. Birefringence values from individual sarcomere interferograms revealed also that normalized A-disk birefringence was lower in the rigor state. The results obtained could be explained by a decrease of thick filaments anisotropy, due to the moving away of myosin heads from the rod during transition into the rigor state.

Animals↗

Individualized compensation of anterior segment birefringence during scanning laser polarimetry.

PURPOSE: To describe a method for assessment and individualized compensation of anterior segment birefringence with scanning laser polarimetry. METHODS: A scanning laser polarimeter (GDx Nerve Fiber Analyzer; Laser Diagnostic Technologies, Inc., San Diego, CA) was modified to accommodate a variable compensator. The magnitude and axis of anterior segment birefringence of normal eyes were determined from a polarimetry image of the Henle fiber layer. The variable compensator was then adjusted to minimize anterior segment birefringence. Retinal nerve fiber layer (RNFL) and macular measurements were then obtained. Macular images with individualized compensation served to verify the effectiveness of the compensation. To demonstrate individualized compensation, two sets of three images each were obtained from four eyes of four normal subjects. One set was obtained with individualized compensation and another with fixed compensation, as used in the commercial polarimetry system. RESULTS: In the tested eyes, the magnitude of anterior segment birefringence ranged from 21.7 to 86.3 nm, and the slow axis ranged from 5.7 degrees nasally upward to 54.3 degrees nasally downward. The maximum residual retardation resulting from compensation was 70 nm for fixed compensation and 11.5 nm for individualized compensation. The compensation residual directly affected the assessment of the RNFL by scanning laser polarimetry. RNFL images obtained with individualized compensation were more consistent with the expected anatomy of the eye. In the eyes measured, the range of RNFL thicknesses appeared to be narrower with the variable corneal and lens compensator (VCC) compared with the fixed corneal compensator (FCC). CONCLUSIONS: In eyes with a normal macula, the magnitude and axis of anterior segment birefringence can be determined from a polarimetry image of the Henle fiber layer. Individualized anterior segment compensation can be achieved with the described method so that the measured birefringence largely reflects the RNFL birefringence. Whether and how macular diseases affect this method remain to be investigated.

Anterior Eye Segment↗

Effect of caffeine on the birefringence signal in single skeletal muscle fibers and mammalian heart. Possible mechanism of action.

Effects of caffeine on single fibers of frog (Rana pipiens) toe muscle and rabbit atrial trabeculae were investigated by measuring action potential, tension and the E-C coupling-related birefringence signal. Caffeine in concentrations of 1.0 to 2.0 mmol/l potentiated the twitch, prolonged the action potential, delayed the onset and suppressed the rate of the birefringence signal in skeletal muscle fibers. In heart muscle, caffeine at concentrations of 2-10 mmol/l potentiated tension and suppressed the second component of the birefringence signal. Although theophylline also potentiated tension and suppressed the rate of rise of the birefringence signal, other diesterase inhibitors, Ro7-2956, or stimulators of adenylate cyclase system such as dibutyryl cAMP or adrenaline failed to alter the birefringence signal while potentiating tension. Since the second component of the birefringence signal has been associated with Ca2+-releasing activity of the sarcoplasmic reticulum, the suppression of this signal and the simultaneous potentiation of tension by caffeine suggests that either caffeine depresses the rate of Ca2+ release or that the birefringence signal is not associated directly with Ca2+ release, rather with Ca2+ uptake. In either case, the results suggest that potentiation of tension by caffeine may be mediated by suppression of the Ca2+ reuptake process.

Absorption↗

THE CHANGING PATTERN OF BIREFRINGENCE IN PLASMODIA OF THE SLIME MOLD, PHYSARUM POLYCEPHALUM.

Plasmodia of the acellular slime mold, Physarum polycephalum, reveal a complex and changing pattern of birefringence when examined with a sensitive polarizing microscope. Positively birefringent fibrils are found throughout the ectoplasmic region of the plasmodium. In the larger strands they may be oriented parallel to the strand axis, or arranged circularly or spirally along the periphery of endoplasmic channels. Some fibrils exist for only a few minutes, others for a longer period. Some, particularly the circular fibrils, undergo changes in birefringence as they undergo cyclic deformations. In the ramifying strand region and the advancing margin there is a tendency for fibrils of various sizes to become organized into mutually orthogonal arrays. In some plasmodia the channel wall material immediately adjacent to the endoplasm has been found to be birefringent. The sign of endoplasmic birefringence is negative, and its magnitude is apparently constant over the streaming cycle. The pattern of plasmodial birefringence and its changes during the shuttle streaming cycle of Physarum are considered in the light of several models designed to explain either cytoplasmic streaming alone or the entire gamut of plasmodial motions. The results of this and other recent physical studies suggest that both streaming and the various other motions of the plasmodium may very likely be explained in terms of coordinated contractions taking place in the fibrils which are rendered visible in polarized light.

Animals↗

Transient electric birefringence of agarose gels. I. Unidirectional electric fields.

The orientation of agarose gels in pulsed electric fields has been studied by the technique of transient electric birefringence. The unidirectional electric fields ranged from 2 to 20 V/cm in amplitude and 1 to 100 s in duration, values within the range typically used for pulsed field gel electrophoresis (PFGE). Agarose gels varying in concentration from 0.3 to 2.0% agarose were studied. The sign of the birefringence varied randomly from one gel to another, as described previously [J. Stellwagen & N.C. Stellwagen (1989), Nucleic Acids Research, Vol. 17, 1537-1548]. The sign and amplitude of the birefringence also varied randomly at different locations within each gel, indicating that agarose gels contain multiple subdomains that orient independently in the electric field. Three or four relaxation times of alternating sign were observed during the decay of the birefringence. The various relaxation times, which range from 1 to approximately 120 s, can be attributed to hierarchies of aggregates that orient in different directions in the applied electric field. The orienting domains range up to approximately 22 microns in size, depending on the pulsing conditions. The absolute amplitude of the birefringence of the agarose gels increased approximately as the square of the electric field strength. The measured Kerr constants are approximately 5 orders of magnitude larger than those observed when short, high-voltage pulses are applied to agarose gels. The increase in the Kerr constants in the low-voltage regime parallels the increase in the relaxation times in low-voltage electric fields. Birefringence saturation curves in both the low- and high-voltage regimes can be fitted by theoretical curves for permanent dipole orientation. The apparent permanent dipole moment increases approximately as the 1.6 power of fiber length, consistent with the presence of overlapping agarose helices in the large fiber bundles orienting in low-voltage electric fields. The optical factor is approximately independent of fiber length. Therefore, the marked increase in the Kerr constants observed in the low-voltage regime is due to the large increase in the electrical orientation factor, which is due in turn to the increased length of the fiber bundles and domains orienting in low-voltage electric fields. Since the size of the fiber bundles and domains approximates the size of the DNA molecules being separated by PFGE, the orientation of the agarose matrix in the applied electric field may facilitate the migration of large DNA molecules during PFGE.

Birefringence↗

A long-lasting birefringence change recorded from a tetanically stimulated squid giant axon.

A long-lasting birefringence change (the delayed response) was found to be produced in a tetanically stimulated squid giant axon. The change was independent of the concurrent membrane potential change, summated on repetitive stimulation, and always had a sign representing a decrease in resting birefringence. The axons was placed between a polarizer and an analyzer with their polarizing axes crossed, making an angle of 45 degrees with the longitudinal direction of the axon. The light beam that passed through the axon and the other optical elements was received by a photodiode. The change in light intensity evoked by repetitive stimulation was composed of brief initial responses, which took place in response to individual stimuli, and a delayed response, which developed gradually and lasted for several hundred msec. It was necessary to differentiate the effect of birefringence change from that of turbidity change. Formulas were derived on the assumption that the optical properties of the axon could be represented by a model of a uniaxial crystal that was not only birefringent but also dichroic, its extinction coefficients and the angle of retardation being changed independently on excitation. Calculations with them yielded the resting retardation, which agreed well with those obtained by the Senarmont's method, and the change in birefringence, which agreed well with the other calculated value derived from experiments using a quarter-wave plate. The results of the calculation confirmed the existence of the long-lasting birefringence change in the tetanically stimulated axon.

Action Potentials↗

A birefringence study of changes in myosin orientation during relaxation of skinned muscle fibers induced by photolytic ATP release.

The birefringence of isolated skinned fibers from rabbit psoas muscle was measured continuously during relaxation from rigor produced by photolysis of caged ATP at sarcomere length 2.8-2.9 microns, ionic strength 0.1 M, 15 degrees C. Birefringence, the difference in refractive index between light components polarized parallel and perpendicular to the fiber axis, depends on the average degree of alignment of the myosin head domain with the fiber axis. After ATP release birefringence increased by 5.8 +/- 0.7% (mean +/- SE, n = 6) with two temporal components. A small fast component had an amplitude of 0.9 +/- 0.2% and rate constant of 63 s-1. By the completion of this component, the instantaneous stiffness had decreased to about half the rigor value, and the force response to a step stretch showed a rapid (approximately 1000 s-1) recovery phase. Subsequently a large slow birefringence component with rate constant 5.1 s-1 accompanied isometric force relaxation. Inorganic phosphate (10 mM) did not affect the fast birefringence component but accelerated the slow component and force relaxation. The fast birefringence component was probably caused by formation of myosin.ATP or myosin.ADP.Pi states that are weakly bound to actin. The average myosin head orientation at the end of this component is slightly more parallel to the fiber axis than in rigor.

Adenosine Triphosphate↗

A NEW METHOD OF POLARIZATION MICROSCOPIC ANALYSIS. I. SCANNING WITH A BIREFRINGENCE DETECTION SYSTEM.

A new method of polarized light analysis is described in which a highly sensitive electronic detector specific for birefringence is used to identify the crystalline axes of an object and then measure its phase retardation due to birefringence. The microscopic system employed in the method consists of an electronic birefringence detection system (BDS), a microscope with strain-free lenses, and a driven stage for passing the specimen at appropriate velocities across the image of an aperture placed at the field stop and imaged in the specimen plane by the condenser. The detector registers retardations directly as voltage at a constant deflection sensitivity of ca. 1.1 v per angstrom unit over a range of 120 angstrom units. The basal rms noise level is 0.002 A for a spot 36 micro in diameter formed by a 95 x, N. A. 1.25 objective pair, and increases in proportion to the reciprocal of the diameter of the scanning spot. The increase in noise with high resolution scanning can be offset by increasing the instrumental time constant, which is adjustable in decades between 0.004 and 0.4 seconds. A number of difficult problems in high extinction polarization microscopy are avoided by the use of modulated light and a rapid electronic detector. For example: (a) The measured distribution of birefringence is unaffected by the usual diffraction anomaly; therefore polarization rectifiers are not required. (b) The detector is selective for birefringence, so that there is no problem in separating contrast due to different optical properties (e.g. dichroism, light scattering). (c) The speed and sensitivity are both increased by between one and two orders of magnitude over that attainable by visual or photographic methods, thereby rendering a vast number of weakly birefringent, light-scattering, and motile objects readily analyzable for the first time with polarized light.

Biophysical Phenomena↗

A melting point for the birefringent component of muscle.

The A filament of the striated muscle sarcomere is an ordered aggregate of one or a few species of proteins. Ordering of these filaments into a parallel array is the basis of birefringence in the A region, and loss of birefringence is therefore a measure of decreased order. Heating caused a large decrease in the birefringence of glycerinated rabbit psoas muscle fibers over a narrow temperature range ( approximately 3 degrees C) and a large decrease in both the birefringence and optical density of the A region of Drosophila melanogaster fibrils. These changes were interpreted as a loss of A filament structure and were used to define a transition temperature (T(tr)) as a measure of the stability of the A region. Since the transition temperature was sensitive to pH, ionic strength, and urea, solvent conditions which often affect protein structure, it is an experimentally useful indicator for factors affecting the structure of the A filament. Fibers from glycerinated frog muscle were less stable over a wide pH range than fibers from glycerinated rabbit muscle, a fact which demonstrates a species difference in structure. Glycerinated rabbit fibrils heated to 70 degrees C shortened to about 40% of their initial length. The extent of shortening was not correlated with the loss of birefringence, and phase-contrast microscopy showed that this shortening occurred in the I region as well as in the A region. This response may be useful for studying the I filament and actin in much the same way that the decrease in birefringence was used for studying the A filament and myosin. The observations presented show that some properties of muscle proteins can be studied essentially in situ without the necessity of first dispersing the structure in solutions of high or low ionic strength.

Animals↗

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle.

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.

Animals↗

Quantitative studies on the polarization optical properties of living cells II. The role of microtubules in birefringence of the spindle of the sea urchin egg.

Birefringence of the mitotic apparatus (MA) and its change during mitosis in sea urchin eggs were quantitatively determined using the birefringence detection apparatus reported in the preceding paper (Hiramoto el al., 1981, J. Cell Biol. 89:115-120). The birefringence and the form of the MA are represented by five parameters: peak retardation (delta p), through retardation (delta t), interpolar distance (D1), the distance (D2) between chromosome groups moving toward poles, and the distance (D3) between two retardation peaks. Distributions of birefringence retardation and the coefficient of birefringence in the spindle were quantitatively determined in MAs isolated during metaphase and anaphase. The distribution of microtubules (MTs) contained in the spindle is attributable to the form birefringence caused by regularly arranged MTs. The distribution coincided fairly well with the distribution of MTs in isolated MAs determined by electron microscopy. Under the same assumption, the distribution of MTS in the spindle in living cells during mitosis was determined. The results show that the distribution of MTs and the total amount of polymerized tubulin (MTs) in the spindle change during mitosis, suggesting the assembly and disassembly of MTs as well as the dislocation of MTs during mitosis.

Animals↗

Scanning laser polarimetry with variable corneal compensation: identification and correction for corneal birefringence in eyes with macular disease.

PURPOSE: In scanning laser polarimetry with variable corneal compensation (SLP-VCC), the macula is used as an intraocular polarimeter to calculate and neutralize corneal birefringence based on an intact Henle's layer. The purpose of this investigation was to validate this strategy in eyes with macular structural disease. METHODS: A nerve fiber analyzer was modified to enable the measurement of corneal polarization axis and magnitude so that compensation for corneal birefringence was eye specific. Normal subjects and patients with a variety of pathologic macular conditions underwent complete ocular examination, SLP-VCC, and direct measurement of the corneal polarization axis (CPA), with a slit-lamp-mounted corneal polarimeter. Macular birefringence patterns were classified as well defined, weak, or indeterminate bow ties. A new "screen" method is described that determines the anterior segment birefringence without relying on the presence of macular bow-tie patterns. RESULTS: Forty-seven eyes (20 normal, 27 with maculopathy) of 47 patients (mean age, 59.0 +/- 19.0 years; range, 24-88) were enrolled. The correlation between CPA measured with corneal polarimetry (CPA by P(IV) [fourth Purkinje image]) and SLP-VCC was less in eyes with macular disease (R(2) = 0.22, P = 0.024) compared with normal eyes (R(2) = 0.72, P < 0.0001). Eyes with macular disease had significantly (P = 0.007) more indeterminate macular bow ties (8/27; 29%) than did normal eyes (0/20). The magnitude of difference between CPA by P(IV) and CPA by SLP-VCC was significantly (P = 0.0007) greater in eyes with indeterminate bow-tie patterns than in weak and well-defined patterns. Although no relationship was observed between CPA and 12 retardation parameters obtained with SLP-VCC in normal eyes (P > 0.05), eyes with macular disease showed a significant association between CPA and average thickness (R(2) = 0.27, P = 0.005), ellipse average (R(2) = 0.24, P = 0.0085), superior average (R(2) = 0.24, P = 0.009), inferior average (R(2) = 0.28, P = 0.004), and superior integral (R(2) = 0.37, P = 0.0008), suggesting incomplete corneal compensation. Greater correlation between CPA by P(IV) and CPA derived by SLP-VCC was found by using the screen method (R(2) = 0.83, P < 0.0001) compared with the bow-tie method (R(2) = 0.22, P = 0.024) in eyes with maculopathy. CONCLUSIONS: Macular strategies for neutralization of corneal birefringence using SLP-VCC can fail if Henle's layer is disrupted by macular disease. The screen method provides a more robust measure of the anterior segment birefringence in some eyes with macular disease.

Adult↗

Variation of peripapillary retinal nerve fiber layer birefringence in normal human subjects.

PURPOSE: The retinal nerve fiber layer (RNFL) exhibits linear birefringence due to the oriented cylindrical structure of ganglion cell axons. The birefringence (Deltan) depends on the density and composition of axonal organelles. The purpose of this study was to evaluate the distribution of birefringence around the optic nerve head (ONH) in normal subjects. METHODS: Birefringence was calculated along circular scan paths around the ONH as Deltan = R/T, where R is RNFL retardance measured by scanning laser polarimetry (SLP) and T is RNFL thickness measured by optical coherence tomography (OCT). OCT scans on a 3.4 mm diameter circle were obtained from 26 normal subjects aged 18 to 53 years. Scans on circles with various diameters were obtained from 17 of these subjects. RESULTS: The average reproducibility of Deltan measured on three separate days in four subjects was +/- 0.05 nm/microm. In most subjects Deltan varied significantly along a circular path around the ONH, with maxima in superior and inferior bundles, minima temporally and nasally, and a mean of 0.32 +/- 0.03 nm/microm. Deltan profiles on circles of different diameter were similar, suggesting that Deltan did not vary along nerve fiber bundles. CONCLUSIONS: RNFL birefringence varies with position around the ONH. This variation may result from known structural differences among nerve fiber bundles that serve different retinal regions. Constant Deltan along bundles is consistent with this hypothesis. Measurements of RNFL birefringence may provide a means to detect early subcellular changes in glaucoma.

Adult↗

Spindle birefringence of isolated mitotic apparatus analysed by pressure treatment.

Sea-urchin zygote mitotic apparatus (MA) isolated in a glycerol/dimethylsulphoxide medium were treated with pressure. Pressure treatment had no effect on spindle birefringence when MA were in full-strength isolation medium. After placing MA in quarter-strength isolation medium, pressures of 4-0 X 10(3)-1-8 X 10(4) lbf in.-2 (2 X 76 X 10(4)-I X 24 X 10(5) k N m-2) for 15 min caused reduction of birefringence which occurred in 2 steps: firstly 20-30% of the birefringence was lost, and then, at higher pressures, the rest of the birefringence was lost. Electron microscopy suggested that pressure-induced changes were in non-microtubule material. Pressure treatment had no effect on MA isolated with hexylene glycol when the MA were pressurized in hexylene glycol; but pressure treatment did cause loss of birefringence when MA isolated in hexylene glycol were transferred immediately into glycerol/dimethylsulphoxide medium and were subsequently treated with pressure (after dilution into quarter-strength glycerol/dimethyl-sulphoxide). We discuss the differences in response between isolated MA and in vivo MA, and we discuss the possibility that 2 components contribute to MA birefringence.

Animals↗

Spindle birefringence of isolated mitotic apparatus analysed by treatments with cold, pressure, and diluted isolation medium.

Mitotic apparatus (MA) were isolated from sea-urchin zygotes using glycerol-dimethyl-sulphoxide. Cold treatment had no effect on MA birefringence when MA were in isolation medium, but caused a 10-15% reduction of MA birefringence when MA were in quarter-strength isolation medium. Pressure treatment also caused a reduction in MA birefringence, but the cold and pressure treatments were not additive, suggesting that both treatments affected the same MA component. MA were not stable in quarter-strength isolation medium, and birefringence gradually decayed, with a half-life of about 40 h. Electron microscopy after cold treatment, or after decay of 55% of the MA birefringence showed abundant, normal-looking microtubules, suggesting that alterations in non-microtubule components cause the reductions in birefringence. Addition of EGTA eliminates the effect of cold treatment, suggesting that Ca2+ has a role in maintenance of spindle structure. We discuss possible reasons why isolated MA do not respond to cold treatment like MA in vivo.

Animals↗

[Assessment of the viability of preserved hearts--birefringence test using a polarizing microscope].

The viability of preserved hearts was assessed using the quantitative polarizing microscopic technique or the birefringence test introduced by Braimbridge et al. To clarify the correlation between myocardial degeneration and the change of birefringence, twelve rat hearts under various preserved conditions were examined. To evaluate usefulness of the birefringence test in heart transplantation, thirteen pairs of mongrel dogs were subjected to heterotopic heart transplantation. Among these thirteen, seven were transplanted immediately and another six were transplanted following preservation in cold Collins-M solution for 12 hours. Biopsy specimens were taken at the time of harvest, after preservation, immediately after and 2 hours after transplantation and examined by a polarizing microscope which could measure the change in birefringence following addition of ATP. They were also examined electron-microscopically for degenerative changes. The results of the birefringence test correlated well with the change of the electron microscopic observation and the hemodynamic activities of the transplanted hearts. Thus, it was concluded that the birefringence test was a useful method to evaluate myocardial viability for transplantation within a short time.

Animals↗

Birefringence of actin.

The total strain birefringence of F-actin isolated from chicken gizzards was measured as a function of elongation in thin transparent films. Each film held at a certain elongation in a jig was allowed to swell in a penetrating but nondissolving liquid. Seven liquids with different refractive indices were employed. The thickness of the film in each swelling liquid was obtained once equilibrium was established. At each elongation, from 0 to 16%, a Wiener curve was obtained. The minima of the Wiener curves yielded the intrinsic birefringence of F-actin as a function of elongation. The intrinsic birefringence increases with elongation up to 16%, above which the thin films break. The form birefringence at a set refractive index also increases with elongation. The implication of the strain birefringence of F-actin is discussed as it affects the optical properties, mainly light scattering, of tissues such as the fiber cells of lens of the eye.

Actins↗