Bleaching Light Research Articles

A fluorescence photobleaching study of the microsecond reorientational motions of DNA

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.

Guanosine 3',5'‐cyclic monophosphate‐activated conductance studied in a truncated rod outer segment of the toad.

1. In darkness, a single rod outer segment isolated from the toad retina was sucked partially, tip first, into a tight-fitting, Ringer solution-filled glass pipette for recording membrane current. The basal end of the outer segment outside the pipette was sheared off with a probe to allow internal dialysis. The potential between the inside and the outside of the pipette was held at 0 mV. 2. With cyclic GMP and IBMX (isobutylmethylxanthine) in the dialysis solution, a large inward current appeared across the plasma membrane of the outer segment; this current saturated at around 1 mM-cyclic GMP. IBMX by itself was ineffective. 3. The saturated cyclic GMP-induced current recorded varied in size with the length of outer segment (L) within the suction pipette. For L less than 25 micron, the relation was linear, with a current density of 4-20 pA micron-1. 4. At short L (less than 25 micron), the dose-response relation between current magnitude and cyclic GMP concentration was sigmoidal, with a Hill coefficient (n) of 1.8-3.1 and a half-saturating cyclic GMP concentration (K1/2) of 30-85 microM. 5. In the presence of IBMX and the absence of GTP, the dose-response relation was the same in continuous bleaching light as in darkness. This indicates that both the characteristics of cyclic GMP binding and the intrinsic conduction properties of the open conductance are not affected by light. 6. Removing IBMX from the dialysing solution had little effect on the saturated current, but substantially reduced the current induced at low concentrations of cyclic GMP. When the analogue 8-bromo cyclic GMP was used instead, however, the presence of IBMX was relatively unimportant even at low agonist concentrations. These observations indicated that significant phosphodiesterase activity was present within the truncated outer segment. 7. In the absence of IBMX and the presence of GTP, the cyclic GMP-induced current could be suppressed by light. When ATP was also present in the dialysing solution, the effect of light was significantly reduced and the suppression also became more transient. 8. We conclude from the above results that the cyclic GMP-gated conductance is indeed present in the plasma membrane of the rod outer segment, and that this conductance and the light-sensitive conductance are one and the same entity. 9. From the results, we estimate that only about 1% of the conductance is normally open in darkness. This fraction of open conductance corresponds to a free cyclic GMP concentration of a few micromolar.

Sources of noise in photoreceptor transduction.

Photoreceptors are surprisingly noisy, and the properties of this noise are providing clues to the molecular mechanisms underlying phototransduction. Three distinct components of noise arise from excitation of the rhodopsin photopigment molecule: the first is activation by photons, and the second is thermal activation in darkness. The third component, induced following intense bleaching lights, probably reflects reversibility in the reactions that inactivate isomerized rhodopsin; in this way a small degree of reactivation of the excited form of rhodopsin is generated from the vast amounts of bleached photoproduct that exist in the photoreceptor following intense exposures. A fourth component of noise, the continuous component present in darkness, probably arises from stochastic fluctuations in the number of excited molecules of a biochemical intermediate, either transducin (G-protein) or phosphodiesterase. This fourth component of noise appears to be much more prominent in cones than in rods. A fifth (and smaller) noise component is caused by the random opening and closing of light-sensitive channels in the outer segment.

Infrared studies of the dynamics of transformations between normal and metastable state of the EL2 center in GaAs

The bleaching of the EL2 absorption and its thermal regeneration are typical time-dependent processes. Compared to the conventional time transient analysis, faster and more accurate data acquisition is described. This technique is based on the combined use of optical absorption and its time derivative, measured while either the energy of the bleaching light or the sample temperature is varied. The peak of the spectral response for bleaching shifts to lower energies in the temperature range 10 to 130 K. The activation energy for thermal regeneration can decrease by a factor of 6 between semi-insulating and n-type samples. We report the first optical identification of persistent electrons, which are photogenerated during EL2 bleaching at 10 K. The thermal disappearance of persistent electrons and holes has also been studied.

The equivalent background of bleaching.

Stiles and Crawford proposed that a retinal region bleached by preexposure to intense light behaves as if it were illuminated by some steady veiling or background luminance. We test this notion by comparing the afterimage of a bleaching light with a steady (and retinally stabilized) light of adjustable intensity, in the manner of Barlow and Sparrock. With their matching procedure, and also with a new procedure, we find as they did that during the rod phase of recovery the afterimage does look like a stabilized field of an intensity which, presented as a background, brings visual sensitivity to the same level. It is as if the two conditions produce equal signals at some stage of the visual pathway. Liked Barlow and Sparrock we observe a rod-cone break in the afterimage matches. However, we argue that the appearance of the rod-cone break presents a paradox and we show a way to resolve it.

Photodestruction of Propionibacterium acnes porphyrins.

The fluorescence spectra of colonies of Propionibacterium acnes were studied under various experimental conditions. The spectra contained peaks at 580 nm and 620 nm. These bands were due to two different components; the 580 nm component was likely to be a metalloporphyrin, and there are indications that the 620 nm component could be a coproporphyrin. The 580 nm fluorescence was destroyed by the combined action of light and oxygen (no destruction under strict anaerobic conditions). A dark period interrupting the bleaching light stopped the destruction of this component for the time of the dark period. The initial production of the 620 nm component was due to the oxygen exposure. Upon light irradiation this component was later destroyed by the combined action of oxygen and light.

Prolonged rod dark adaptation in retinitis pigmentosa.

The time course of rod dark adaptation was measured in 12 patients and carriers of retinitis pigmentosa (RP). In contrast to previous studies the rod absolute threshold was determined prior to any exposure to the bleaching light. For seven of the patients and carriers the recovery of rod sensitivity to the prebleach level was prolonged, with the delay ranging from mild to severe. The prolongation appeared to be limited to the late phase of bleaching recovery; the early portion of rod dark adaptation, measured following a weak bleach, was normal. These results suggest a selective impairment in the processes underlying rod dark adaptation in some patients with RP.

Oxygen vacancies in lithium niobate

Optical absorption peaks at 760 and 500 nm in LiNbO3 are assigned to oxygen vacancies containing one and two electrons, respectively. The 500-nm band appears after annealing above approximately 500 °C in a vacuum, i.e., a reducing atmosphere, and continues to grow with increasing anneal temperature. Optical bleaching near 77 K destroys the band at 500 nm and produces both a band at 760 nm and a Nb4+ electron spin resonance spectrum. The spectral dependence of the bleaching light required to convert the 500-nm band to the 760-nm band is determined.

Modeling the rod outer segment birefringence change correlated with metarhodopsin II formation

A rapid birefringence loss associated with metarhodopsin II formation, delta (delta n) MII, is produced when frog rod outer segments are exposed to a bleaching light flash. To analyze the nature of the underlying structure change, measurements of delta (delta n) MII were made in rod outer segments perfused with glycerol solutions to increase the refractive index of the cytoplasmic and intradisk spaces. Comparisons of experimental results with computed changes in the form birefringence component using two- and three-dielectric outer segment models for several putative structure changes were made. It is concluded that delta (delta n) MII can be due to either a change in the intrinsic birefringence component caused by the reorientation of anisotropic molecules, or to a change in the form birefringence component caused by small changes in the cytoplasmic and/or intradisk volumes.

The estimation of cone spectral sensitivities

AbstractMethods for estimating the cone spectral sensitivities are reviewed (selectively). Techniques applied to the normal eye include measurement of flicker photometric sensitivity against colored backgrounds; creation of temporary artificial dichromacy by colored bleaches; observations of the afterimages of colored bleaching lights; and transient photopic saturation by flashed backgrounds. Results from these techniques agree well with cone sensitivities estimated from studies of congenital dichromats, and these estimates are further supported by appropriately comparing normal with anomalous trichromatic matches. The indicated sensitivities for red‐ and green‐sensitive cones peak at 566 and 543 nm, respectively. Factor analysis of color‐matching functions indicates normal variation of the order of 1–1.5‐nm standard deviation.

Signal transmission from rods to ganglion cells in rat retina after bleaching a portion of the receptive field.

1. Recordings from single axons of retinal ganglion cells in the rat's optic tract in response to small flashing test lights were used to follow the course of dark adaptation after exposing half of the receptive field to a bleaching light. 2. The recovery of log sensitivity followed an exponential time course in the exposed and unexposed half-fields. The curves had different time constants, with the exposed side taking longer to recover. 3. The time constants of recovery were increasing functions of exposure, but the rate of increase was different in the exposed and the unexposed half-fields. Direct exposure increased the time constant at a greater rate than did indirect exposure. 4. Comparison of the time constants of recovery in the exposed half-fields with those for pigment regeneration suggests that sensitivity recovers with the time course of rhodopsin regeneration. 5. Increment thresholds were determined using steady backgrounds which illuminated half of the receptive field. A greater threshold elevation was produced in the directly illuminated half-field compared with the half-field illuminated only by scattered light. Comparisons of the threshold-raising capacity of direct and indirect illumination were used to establish an 'upper bound' on the magnitude of light scatter. The time courses of the recovery of sensitivity after two different bleaches were compared. First, thresholds were measured in the unexposed half-field after a half-field bleach. Secondly the recovery of sensitivity after direct bleaching-exposure to the predetermined scatter upper bound' was measured. Recovery was more rapid in the latter case than the former, thus indicating that adaptation spreads laterally via some process other than light scattering.

The involvement of rod photoreceptors in dark adaptation

From analysis of previously reported scotopic dark adaptation curves ( Pugh, 1975b), evidence has been found for the existence following a bleach of three separate components in the decline of the “equivalent background intensity”, with decay time constants of about 5 sec, 100 sec and 7 min. The results are consistent with the idea that the equivalent background is caused by photon-like events in the rods as a result of intermediates produced by the bleaching light, and a model of the removal of these intermediates provides a good description of the psychophysical results. The ideas are consistent with the recent demonstration of an increased rate of occurrence of quantal events in rods following a bleach ( Lamb, 1980).

A continuously recording retinal densitometer

A densitometer is described which enables continuous monitoring of the radiances of three different light beams reflected from the fundus of the eye. The optical system is designed such that within a 30 msec period a deep red reference light, a measuring light, a bleaching light and a dark period are sequentially presented to the eye. A microprocessor based electronic system counts, sorts and averages the output pulses of the photomultiplier that receives the reflected light. A theoretical relationship is derived which describes the fluctuations in the output of the densitometer as a function of the radiance of the measuring light. The uncertainty of measurements with an artificial eye is about 0.002 density units. The maximum change in measured density between a fully bleached and a fully dark adapted retina is for most subjects between 0.25 and 0.40. Twenty measurements on one subject show a value of 0.295 ± 0.021. These density changes are nearly twice as high as those hitherto reported in the literature and are attributable to optimization of the optical system. We discuss the relation between observed density changes and changes in the visual photopigments and argue that either the amount of intra ocular stray light in our measurements must be low or the time constant of cone regeneration must be much longer than 120 sec.

Spontaneous quantal events induced in toad rods by pigment bleaching.

Barlow proposed that absolute visual threshold is limited by photon-like noise events in the rod photoreceptors, and he later extended the idea to explain the elevation of threshold following bright bleaching lights in terms of increased noise in the photoreceptors. Rushton, on the other hand, has proposed that the threshold elevation during dark adaptation involves changes not within the photoreceptors themselves but rather in the gain of a subsequent 'pool'. I report here measurements of outer segment current in individual rod photoreceptors which demonstrate that spontaneous fluctuations occur at a greatly increased rate following bleaches of around 1%, and that these fluctuations have the form expected for random occurrences of the single photon events. This is consistent with Barlow's ideas but does not indicate whether a gain change subsequent to the receptors also occurs.

Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method.

The Fc receptors for IgE on rat basophil leukemia 2H3 cells were labeled with either rhodamine- or fluorescein-conjugated IgE and then with Fab fragments of anti-IgE conjugated with the other dye. Fluorescence photobleaching recovery measurements of lateral diffusion were performed with one of the dyes before and after extensive bleaching of the other dye over an entire cell. Bleaching one dye did not affect subsequent measurements made with the other dye. We thus detect no evidence for photoinduced artifacts in fluorescence photobleaching recovery experiments.

The structure of colour centers in photochromic glass

An ellipsoidal model of colour centers in photochromic glass was proposed to explain an additional absorption spectrum. Absorption spectra of small ( R ⪅ 100 A ̊ ) involving oblong and oblate ellipsoids of rotation have been analysed. The splitting of the absorption spectrum of the small silver particles with non-spherical form resulted in an absorption spectrum different from that for spherical particles. Calculation of the absorption spectra of the system of oblate ellipsoids with considerable dispersion in eccentricity and of about 20 Å in size was in good agreement with experiment. Absorption spectra of the system of oblong ellipsoids differed significantly from experimental findings, indicating that oblong particles were absent. To verify the basic theory of the colour center model, advanced experiments have been carried out on the bleaching of photochromic glass by monochromatic polarized light. The so called photo-adaptation effect has been found, that is, the absorption decreased faster at the wavelength of the bleaching light. The photo-adaptation spectral width indicated that the particle size was nearly 20 Å. The ratio of changing absorption for perpendicular and parallel orientations of bleaching and reading polarization vectors was about 0.7 in the longwave visable range, indicating that the colour were substantially anisotropic. In the thermal recovery of photochromic glass the shortwave and longwave absorptions were bleached faster, indicating the lower stability of the strong oblate particles, which specifies absorption in those parts of the spectrum.

Lateral interactions in human cone dark adaptation.

1. The course of cone dark adaptation after exposure to a strong bleaching light depends on the size of the bleached region. Threshold for brief, tiny test flash centred in the bleached region is elevated more, and recovery is retarded by a small bleach. This effect has its parallel in the sensitization effect observed with steady backgrounds. 2. Previous results, that a similar sensitization effect is not observed in rod dark adaptation, are confirmed. 3. This sensitization effect in cone dark adaptation does not transfer binocularly, and is unaffected by pressure blinding during the bleaching exposure. 4. Threshold following a small bleach may be lowered by adding a steady annular background to the region surrounding the bleached patch. Conversely, bleaching the area surrounding a small, steady background can lower threshold for a test flash centred on the background. 5. These interactions between backgrounds and bleaches may be explained if bleaches produce long-lasting signals from neurones in the bleached area, which then lead into a spatially opponent stage of processing. 6. It is likely that the persisting signals come from the cone receptors, since the Bunsen-Roscoe Law (intensity-time reciprocity) holds for small bleaches as well as large, for durations up to about 3 sec.

After-effects of small adapting fields.

1. Sensitivity to a small test probe in the centre of a small, steady background is less than when the background is large (sensitization). When an equiluminous steady annulus is added to the region surrounding a small background, rod threshold takes several minutes to stabilize at its new, lower level. The after-effects of the small background follow a time course characteristic of cortical adaptation. 2. The sensitivity loss and time course of recovery after intense bleaching lights in the cone system depend markedly on the size of the retinal region bleached, although no such effect is observed in the rod system. If a steady annular surround is added to the region surrounding the bleached patch, threshold falls rapidly to the value it would have after a large-area bleach of the same intensity. 3. The interaction between bleaches and steady surrounds suggests that bleaches produce long-lasting signals in the cone receptors. 4. The different temporal properties of sensitization on rod backgrounds and sensitization after cone bleaches suggest that different mechanisms underlie the two phenomena. 5. In cone vision, if light is added to the area surrounding a small, steady background, the subsequent readjustment takes minutes to complete, as it does in rod vision. But in addition, for cones, a large proportion of the sensitivity loss caused by the small background can be rapidly restored, as it is with cone bleaches. 6. The above results, together with the known absence of sensitization in rod dark adaptation, are consistent with the hypothesis that sensitization occurs at least partly at the retinal level in the cone system, but not (or only weakly) in the rod system, and that there is an additional, probably cortical elevation, common to rod and cone systems, for small backgrounds, but not for small, brief bleaches.

Trapped hydrogen atoms in irradiated frozen solutions. Isotope effects

Isotope effects in the yields of H- and D-atoms in acid, neutral and alkaline matrices have been studied by EPR methods. In acid matrices the isotope effect is the same in UV- and X-irradiated matrices, but in neutral and alkaline matrices the effect is larger in UV-irradiated matrices. Furthermore, when the H- and D-atoms are formed by photobleaching of trapped electrons the isotope effect is strongly dependent on the wavelength of the bleaching light, increasing with increasing wavelenghts. The isotope effect in sulphuric acid matrices is dependent on the concentration of the acid. The observation are briefly discussed.

Light-initiated changes of cyclic guanosine monophosphate levels in the frog retina measured with quick-freezing techniques.

Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.