Multiple excitations in photosynthetic systems.

The yield of fluorescence in Chlorella from a 7 ns pulse of light is found to decrease gradually as a function of the number of hits in the photosynthetic units. The fivefold decrease in yield is spread over some three orders of magnitude of pulse energy and strongly suggests another random process in addition to that of photon absorption. Evidence supports the view that this random process is not in the time but in the spatial domain. The model used to fit the data is that of a unit with multiple traps for the singlet excitation. An excitation is captured by an open trap or destroyed by a filled trap with equal probability. These studies give evidence for the connectivity of the photosynthetic energy transfer apparatus on the short time scale. The short fluorescence lifetimes following picosecond pulse excitation of photosynthetic systems reported by several laboratories may be explained by the effect of multiple excitations.     

Time-resolved spectroscopy of hemoglobin and myoglobin in resting and ischemic muscle

Difficulties of quantitation of hemoglobin/myoglobin absorption changes in muscle have led to the development of a new approach using short pulses of light. This method uses input light pulses sufficiently short so that the time course of travel of light through the brain can be precisely measured. The time of arrival of light at the detector gives the optical path length, given the velocity of light in tissues. The intensity profile of photon migration in tissues permits determination of the path length that the exiting photons have traveled and the concentration change of the pigments. A cavity-dumped liquid dye laser illuminates the tissue with 130-ps pulses detected as 600-ps duration at a half height at 3.0-cm distance from the input point. The decay of intensity from the 50% point onward to 0.1% follows a logarithmic function of slope mu which is attributed to the total absorption coefficient of the tissue. Increments of mu due to deoxyhemoglobin absorption at 760 and 630 nm are used to calculate the concentration change. This permits the calculation of the path length for continuous light measurements of 2 cm for a particular geometry. Variation of the wavelength of the laser affords determination of a spectrum of changes in the tissue.     

Chemical excitation of Limulus photoreceptors. II. Vanadate, GTP-gamma- S, and fluoride prolong excitation evoked by dim flashes of light

We treated Limulus ventral photoreceptors with the phosphatase inhibitors fluoride, vanadate, and GTP-gamma-S [guanosine-5'0-(3-thiotriphosphate)] under various conditions of illumination and external calcium concentrations. In the dark in low-calcium (1 mM) artificial seawater (ASW), fluoride-induced discrete waves cluster together in time. Under these conditions, the intervals between waves were found to be correlated, and there were excess short intervals beyond the number expected from an exponential interval distribution. To assess the effects of the inhibitors on the light response, we stimulated ventral receptors with a series of dim flashes and averaged the current response under voltage clamp. In ASW, vanadate and GTP-gamma-S prolong the decay of the averaged response to dim test flashes, but prolongation does not always accompany the induction of discrete waves in the dark. Prolongation induced by vanadate in normal-calcium (10 mM) ASW was enhanced in low-calcium (1 mM Ca2+) ASW. Many individual response records suggest that prolongation results from extra discrete waves late in the light response, whereas others reveal long-lasting complex waveforms that cannot easily be resolved into discrete waves. The apparent effect of the inhibitors on the light response is to allow a single photoactivated rhodopsin molecule to produce multiple discrete waves and complex long-lasting events. We suggest that both prolongation of the light response and clustering of waves in the dark result from inhibition of a step in the pathway of visual transduction, in which GTP hydrolysis normally helps to turn off the production of both light-evoked and spontaneous waves.     

Theory of photoselection by intense light pulses. Influence of reorientational dynamics and chemical kinetics on absorbance measurements.

The theory of absorbance measurements on a system (e.g., chromophore(s) in a protein) that undergoes a sequence of reactions initiated by a linearly polarized light pulse is developed for excitation pulses of arbitrary intensity. This formalism is based on a set of master equations describing the time evolution of the orientational distribution function of the various species resulting from excitation, reorientational dynamics, and chemical kinetics. For intense but short excitation pulses, the changes in absorbance (for arbitrary polarization directions of the excitation and probe pulses) and the absorption anisotropy are expressed in terms of reorientational correlation functions. The influence of the internal motions of the chromophore as well as the overall motions of the molecules is considered. When the duration of the excitation pulse is long compared to the time-scale of internal motions but comparable to the overall correlation time of the molecule that is reorienting isotropically, the problem of calculating the changes in absorbance is reduced to the solution of a set of first-order coupled differential equations. Emphasis is placed on obtaining explicit results for quantities that are measured in photolysis and fluorescence experiments so as to facilitate the analysis of experimental data.     

Chemical excitation of Limulus photoreceptors. I. Phosphatase inhibitors induce discrete-wave production in the dark

Molybdate, tungstate, fluoride, vanadate, and GTP-gamma-S [guanosine-5'- 0-(3-thiotriphosphate)] were injected into Limulus ventral photoreceptors by ionophoresis from microelectrodes. All of these drugs induce discrete waves of depolarization similar in waveform to, but smaller in amplitude than, those normally elicited by dim light. As for light-evoked waves, the amplitude of drug-induced waves decreases with light adaptation. For the compounds examined so far (fluoride, vanadate, GTP-gamma-S), the drug-induced waves share a reversal potential with light-induced discrete waves at about +15 mV. The induction of discrete waves by fluoride, vanadate, and molybdate was found to be reversible, whereas the induction of waves by GTP-gamma-S was not. Unlike fluoride and vanadate, which induce waves when added to the bath, molybdate appears to be ineffective when applied extracellularly. Because of the similarity of the drug-induced waves to light-induced discrete waves, we conclude that the drug-induced waves arise from a process similar or perhaps identical to visual excitation of the photoreceptor. However, the smaller size of drug-induced waves suggests that they arise at a stage of phototransduction subsequent to the isomerization of rhodopsin. On the basis of the chemical properties and action of the drugs, we suggest that discrete waves may arise through the activation of a GTP-binding protein.     

Photoresponses of Halobacterium salinarum to repetitive pulse stimuli.

Halobacterium salinarum cells from 3-day-old cultures have been stimulated with different patterns of repetitive pulse stimuli. A short train of 0.6-s orange light pulses with a 4-s period resulted in reversal peaks of increasing intensity. The reverse occurred when blue light pulses were delivered as a finite train: with a 3-s period, the response declined in sequence from the first to the last pulse. To evaluate the response of the system under steady-state conditions of stimulation, continuous trains of pulses were also applied; whereas blue light always produced a sharply peaked response immediately after each pulse, orange pulses resulted in a declining peak of reversals that lasted until the subsequent pulse. An attempt to account for these results in terms of current excitation/adaptation models shows that additional mechanisms appear to be at work in this transduction chain.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Mechanisms regulating variability of the single photon responses of mammalian rod photoreceptors

Variability in the single photon responses of rod photoreceptors limits the accuracy with which the number and timing of photon absorptions are encoded. We investigated how much single photon responses of mammalian rods fluctuate and what mechanisms control these fluctuations. Mammalian rods, like those of toads, generated responses to single photons with trial-to-trial fluctuations 3-4 times smaller than other familiar signals produced by single molecules. We used the properties of the measured fluctuations to constrain models for how the single photon responses are regulated. Neither feedback control of rhodopsin's activity nor saturation within the transduction cascade were consistent with experiment. The measured responses, however, could be explained by multistep shutoff of rhodopsin or a combination of multistep shutoff and saturation.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10¹³ photons · cm^?2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10¹³–10¹⁶ photons · cm^?2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Photo-bleaching and photon saturation in flow cytometry.

In flow cytometry, small particles travel at a high speed through a bright light spot. The high light intensity at the point of measurement causes measurable photon saturation. This observation indicates that the rate at which individual dye molecules emit photons is close to the maximum emission rate. Despite the short exposure time, individual molecules may go through a few hundred excitation cycles while they are in the light beam. The absorbed light dose causes significant dye destruction. This article presents experimental procedures to determine the extent of photon saturation and photo-bleaching of dyes bound to cell nuclei in a flow cytometer. Measurements of Hoechst and propidium iodide bound to chromatin show that the amount of dye bleached per emitted photon is the same at low and high illumination intensities. This finding indicates that photon emission and dye destruction are both the result of the absorption of single excitation photons. The experimental observations allow rough estimates of the lifetime of the excited state and the lifetime of the molecule. The lifetime of the Hoechst 33258 bound to DNA is estimated to be 100 excitation-relaxation cycles. The average propidium iodide molecule lasts approximately 200 excitation-relaxation cycles. The theoretical considerations show that the optimal illumination conditions are different for bleaching and nonbleaching dyes. An optical arrangement for high precision measurements of bleaching dyes is presented.     

Refractive index and pulse broadening characterization using oil immersion and its influence on three‐photon microscopy excited at the 1700‐nm window

Three‐photon microscopy excited at the 1700‐nm window enables deep‐tissue penetration. However, the refractive indices of commonly used immersion oils, and the resultant pulse broadening are not known, preventing imaging optimization. Here, we demonstrate detailed characterization of the refractive index, pulse broadening and distortion for excitation pulses at this window for commonly used immersion oils. On the physical side, we uncover that absorption, rather than material dispersion, is the main cause of pulse broadening and distortion. On the application side, comparative three‐photon imaging results indicate that 1600‐nm excitation yields 5 times higher three‐photon signal than 1690‐nm excitation.      

Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale.

Bovine rhodopsin and isorhodopsin were excited with a single 530-nm, 7-ps light pulse emitted by a mode-locked Nd 3+ glass laser at room temperature. Within 3 ps of excitation, absorbance changes due to formation of bathorhodopsin were observed. The difference spectra generated during and 100 ps after pulse excitation are presented. The data show that bathorhodopsin formation is completed within 3 ps for both the primary pigments and suggest that a single common bathorhodopsin is photochemically formed from both primary pigments. Our findings provide additional support for the cis-trans isomerization model of the primary event in vision. Additional absorption transients that were observed near 670 and 460 nm are discussed.     

Stochastic Properties of Discrete Waves of the Limulus Photoreceptor

In the dark-adapted photoreceptor of the horseshoe crab, Limulus, transient discrete depolarizations of the cell membrane, discrete waves, occur in total darkness and their rate of occurrence is increased by illumination. The individual latencies of the discrete waves evoked by a light stimulus often cannot be resolved because the discrete waves overlap in time. The latency of the first discrete wave that follows a stimulus can be determined with reasonable accuracy. We propose a model which allows us to make an estimate of the distribution of the latencies of the individual light-evoked discrete waves, and to predict the latency distribution of the first discrete wave that follows a stimulus of arbitrary intensity-time course from the latency distribution of the first discrete wave that follows a brief flash of light. For low intensity stimuli, the predictions agree well with the observations. We define a response as the occurrence of one or more discrete waves following a stimulus. The distribution of the peak amplitudes of responses suggests that the peak amplitude of individual discrete waves sometimes has a bimodal distribution. The latencies of the two types of discrete waves, however, follow similar distributions. The area under the voltage-time curve of responses that follow equal energy long (1.25 sec) and short (10 msec) light stimuli follows similar distributions, and this suggests that discrete waves summate linearly.     

Effects of removing extracellular Ca2+ on excitation and adaptation in Limulus ventral photoreceptors.

In Limulus ventral photoreceptors, removing extracellular calcium (Ca2+o) increases the median latency of light-evoked discrete waves. Removal greatly lengthens the time-to-peak of responses in the dark-adapted cell, but not in the light-adapted cell. Removal does not block light-adaptation or the light-induced rise in intracellular calcium (Ca2+i). These results are interpreted in terms of the hypothesis that both sensitivity and the kinetics of excitation are dependent on Ca2+i, and that Ca2+i is dependent on Ca2+o in the dark-adapted cell, but in the light is dependent largely on Ca2+ released from intracellular compartments.     

Photic sensitivity for circadian response to light varies with photoperiod

The response of the circadian system to light varies markedly depending on photic history. Under short day lengths, hamsters exhibit larger maximal light-induced phase shifts as compared with those under longer photoperiods. However, effects of photoperiod length on sensitivity to subsaturating light remain unknown. Here, Syrian hamsters were entrained to long or short photoperiods and subsequently exposed to a 15-min light pulse across a range of irradiances (0-68.03 μW/cm(2)) to phase shift activity rhythms. Phase advances exhibited a dose response, with increasing irradiances eliciting greater phase resetting in both conditions. Photic sensitivity, as measured by the half-saturation constant, was increased 40-fold in the short photoperiod condition. In addition, irradiances that generated similar phase advances under short and long days produced equivalent phase delays, and equal photon doses produced larger delays in the short photoperiod condition. Mechanistically, equivalent light exposure induced greater pERK, PER1, and cFOS immunoreactivity in the suprachiasmatic nuclei of animals under shorter days. Patterns of immunoreactivity in all 3 proteins were related to the size of the phase shift rather than the intensity of the photic stimulus, suggesting that photoperiod modulation of light sensitivity lies upstream of these events within the signal transduction cascade. This modulation of light sensitivity by photoperiod means that considerably less light is necessary to elicit a circadian response under the relatively shorter days of winter, extending upon the known seasonal changes in sensitivity of sensory systems. Further characterizing the mechanisms by which photoperiod alters photic response may provide a potent tool for optimizing light treatment for circadian and affective disorders in humans.     

Neuronal activity of the pulvinar of the thalamus in the cat

Spontaneous and induced activity in the neurons in the pulvinar of the thalamus (PT) was studied extracellularly in unnarcotized cats immobilized with Remiolan. The PT neurons had a relatively low level of spontaneous activity, and either responded only to light flashes (61%) or did not respond to any of the stimuli applied. The latent periods of the responses varied within wide limits (from 15 to 60 msec), which indicates the possibility of direct peripheral activation of the PT neurons and also of the existence of multisynaptic afferent connections. Acoustic and electrical cutaneous stimuli, although they did not themselves evoke response-discharges, modulated both spontaneous activity and activity evoked by light flashes in the PT neurons. The same effect on the activity of the PT neurons was produced by electrical excitation of the mesencephalic reticular formation. That gives us grounds for surmising that the modulating effects of acoustic and electrical stimuli are produced through activation of reticular structures in the midbrain.Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 3, No. 5, pp. 491–499, September–October, 1971.     

Simulation of the excitation of quasi-plane wake waves in a plasma by a resonant sequence of laser pulses with a variable envelope

Results are presented from full-scale numerical simulations of the excitation of wake waves by a sequence of weakly relativistic laser pulses in a subcritical plasma. Computations were carried out with a 2D3V version of the SUR-CA code that is based on the local-recursive nonlocal-asynchronous algorithm of the particle-in-cell method. The parameters of a train of laser pulses were chosen to correspond to the resonant excitation of the wake field. The curvature of the envelope of the pulses was chosen to depend on the number of the pulse in the train. Numerical simulations showed that there are plane waves during the first period of the plasma wave behind the pulse train.     

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser.

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.     

Mechanisms for ultrafast nonradiative relaxation in electronically excited eumelanin constituents

We investigate the relaxation dynamics of melanin model constituents including monomers, dimers, and tetramers, upon excitation, using state-of-the-art, time-dependent, density functional theory calculations. The results explain the ability of these molecules to transform photon energy into thermal energy in a remarkably short timescale of ∼100 fs. We find that after electronic excitation by light absorption, ultrafast energy conversion takes place through two novel mechanisms: proton transfer on a timescale of 110 fs and state mixing upon oligomerization on a timescale of <50 fs. These results are in good agreement with available experiments and help elucidate melanin's role in photoprotection against ultraviolet radiation.