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.      

Water-protein NOEs: Optimized scheme for selective water excitation

An alternative scheme for selective water excitation is proposed. The pulse sequence saturates the resonances from the solute, allowing the observation of water-solute NOEs with low artifact levels. The water resonance is subsequently excited by a relatively non-selective 90° pulse. The scheme is compared to other selective water excitation schemes. 2D NOE-NOESY and ROE-NOESY pulse sequences are proposed which afford high sensitivity by efficient water excitation and flip-back by radiation damping, yet allow the use of short mixing times for the buildup of water-solute NOEs.     

Sex differences in the electrocommunication signals of Sternarchogiton nattereri (Gymnotiformes: Apteronotidae)

In this study we examined electrocommunication behavior in Sternarchogiton nattereri (Apteronotidae), a weakly electric fish from South America. We focused on variation between females and males lacking external dentition and used playbacks of simulated conspecifics to elicit chirps (modulations of their electric organ discharge, EOD). Chirp responses were not affected by the frequency of the playback stimulus. EOD frequency, chirp rate, and chirp duration were not sexually dimorphic; however, the amount of chirp frequency modulation was significantly greater in toothless males than in females. These results reinforce that sex differences in chirp structure are highly diverse and widespread in the Apteronotidae.     

The Steppengrille (Gryllus spec./assimilis): Selective Filters and Signal Mismatch on Two Time Scales

In Europe, several species of crickets are available commercially as pet food. Here we investigated the calling song and phonotactic selectivity for sound patterns on the short and long time scales for one such a cricket, Gryllus spec., available as "Gryllus assimilis", the Steppengrille, originally from Ecuador. The calling song consisted of short chirps (2-3 pulses, carrier frequency: 5.0 kHz) emitted with a pulse period of 30.2 ms and chirp rate of 0.43 per second. Females exhibited high selectivity on both time scales. The preference for pulse period peaked at 33 ms which was higher then the pulse period produced by males. Two consecutive pulses per chirp at the correct pulse period were already sufficient for positive phonotaxis. The preference for the chirp pattern was limited by selectivity for small chirp duty cycles and for chirp periods between 200 ms and 500 ms. The long chirp period of the songs of males was unattractive to females. On both time scales a mismatch between the song signal of the males and the preference of females was observed. The variability of song parameters as quantified by the coefficient of variation was below 50% for all temporal measures. Hence, there was not a strong indication for directional selection on song parameters by females which could account for the observed mismatch. The divergence of the chirp period and female preference may originate from a founder effect, when the Steppengrille was cultured. Alternatively the mismatch was a result of selection pressures exerted by commercial breeders on low singing activity, to satisfy customers with softly singing crickets. In the latter case the prominent divergence between male song and female preference was the result of domestication and may serve as an example of rapid evolution of song traits in acoustic communication systems.     

Diversity in the structure of electrocommunication signals within a genus of electric fish,<Emphasis Type="Italic"> Apteronotus</Emphasis>

Some gymnotiform electric fish modulate their electric organ discharge for intraspecific communication. In Apteronotus leptorhynchus, chirps are usually rapid (10-30 ms) modulations that are activated through non- N-methyl- d-aspartate (non-NMDA) glutamate receptors in the hindbrain pacemaker nucleus. Males produce longer chirp types than females and chirp at higher rates. In Apteronotus albifrons, chirp rate is sexually monomorphic, but chirp structure (change in frequency and amplitude during a chirp) was unknown. To better understand the neural regulation and evolution of chirping behavior, we compared chirp structure in these two species under identical stimulus regimes. A. albifrons, like A. leptorhynchus, produced distinct types of chirps that varied, in part, by frequency excursion. However, unlike in A. leptorhynchus, chirp types in A. albifrons varied little in duration, and chirps were all longer (70-200 ms) than those of A. leptorhynchus. Chirp type production was not sexually dimorphic in A. albifrons, but within two chirp types males produced longer chirps than females. We suggest that species differences in chirp duration might be attributable to differences in the relative proportions of fast-acting (non-NMDA) and slow-acting (NMDA) glutamate receptors in the pacemaker. Additionally, we map species difference onto a phylogeny and hypothesize an evolutionary sequence for the diversification of chirp structure.     

Sensory processing in the fast electrosensory pathway of pulse gymnotids studied at multiple integrative levels

Pulse gymnotids extract information about the environment using the pulsed discharge of an electric organ. Cutaneous electroreceptor organs transduce and encode the changes that objects imprint on the self-generated transcutaneous electric field. This review deals with the role of a neural circuit, the fast electrosensory path of pulse gymnotids, in the streaming of self generated electrosensory signals. The activation of this path triggers a low-responsiveness window slightly shorter than the interval between electric organ discharges. This phenomenon occurs at the electrosensory lateral line lobe where primary afferent terminals project on the somata of spherical neurons. The main subservient mechanism of the low-responsiveness window rely on the intrinsic properties of spherical neurons (dominated by a voltage dependent, low-threshold, non-inactivating and slowly-deactivating K(+) conductance) determining the cell to respond with a single spike followed by a long refractory period. Externally generated signals that randomly occur within the interval between self-generated discharges are likely blocked by the low responsiveness window. Repetitive signals, as those emitted by conspecifics with a slightly lower rate, occur progressively at longer delays beyond the duration of the low responsiveness window. Transient increases of the discharge rate relocate the interference within the low-responsiveness window. We propose that this combination of sensory filtering and electromotor control favors the self-generated signals in detriment of other, securing the continuity of the electrolocation stream.     

Chirp rate variability in male song ofEphippigerida taeniata (Orthoptera: Ensifera)

Variability in the chirp rate of the male song of the ephippigerine speciesEphippigerida taeniata during intraspecific communication was investigated in the laboratory. Conspecific chirps were used as auditory stimuli. The stimulus rate was controlled by computer. Experiments were carried out at 19, 27, and 35°C. Acoustically isolated males ofE. taeniata sang with a relatively constant chirp rate, which depended on the ambient temperature. Chirp rate significantly increased with rising temperature from 19 to 27°C, whereas at 35°C the chirp rate did not differ significantly from that at 27°C. Male chirp rates were affected by stimulus rates. Males significantly increased their chirp rate in response to increasing stimulus rates at temperatures of 19 and 27°C. At 35°C the increase in the chirp rate was not significant. At 27 and 35°C males sang with a higher chirp rate than the stimulus rate within a certain range. Evaluating stimulus and response chirp pattern when the males increased their chirp rate in response to the stimulus rate showed that an alternation pattern was established. More than 50% of the male chirps occurred at a characteristic time range at around 40% of the interstimulus interval, which was slightly affected by temperature.     

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.     

Self-phase-modulated femtosecond laser source at 1603 nm and its application to deep-brain 3-photon microscopy in vivo

3-photon microscopy (3PM) excited at the 1700 nm window enables deep-tissue imaging in vivo, especially in brain. PC rod soliton source has previously been exclusively used as the excitation source, which is rather costly and difficult to align. Here we demonstrate a novel nonlinear optical technique to build femtosecond laser source at the 1700 nm window, based on self-phase modulation (SPM) in a short span of large-mode-area fiber. The spectral broadening experienced by the pump pulse leads to the generation of a red-shifted sidelobe at 1603 nm. After spectral filtering, this sidelobe corresponds to 170-fs, 167-nJ pulses at 1603 nm. Using this SPM source, we further demonstrate deep-brain 3 PM to a depth of 1500 μm below the mouse brain surface in vivo. Our SPM femtosecond laser source thus provides a cost effective and easy-to-align alternative excitation source to the PC rod soliton source.     

Calling,Courtship, and Condition in the Fall Field Cricket,Gryllus pennsylvanicus

Theoretically, sexual signals should provide honest information about mating benefits and many sexually reproducing species use honest signals when signalling to potential mates. Male crickets produce two types of acoustic mating signals: a long-distance mate attraction call and a short-range courtship call. We tested whether wild-caught fall field cricket (Gryllus pennsylvanicus) males in high condition (high residual mass or large body size) produce higher effort calls (in support of the honest signalling hypothesis). We also tested an alternative hypothesis, whether low condition males produce higher effort calls (in support of the terminal investment hypothesis). Several components of long-distance mate attraction calls honestly reflected male body size, with larger males producing louder mate attraction calls at lower carrier frequencies. Long-distance mate attraction chirp rate dishonestly signalled body size, with small males producing faster chirp rates. Short-range courtship calls dishonestly reflected male residual mass, as chirp rate and pulse rate were best explained by a curvilinear function of residual mass. By producing long-distance mate attraction calls and courtship calls with similar or higher effort compared to high condition males, low condition males (low residual mass or small body size) may increase their effort in current reproductive success at the expense of their future reproductive success, suggesting that not all sexual signals are honest.     

Signal components, acoustic preference functions and sexual selection in a cricket

In many sexually reproducing organisms, females choose mates based on multiple male traits. This study examined how two temporal components of the male mating call – chirp rate and chirp duration – affect female mating preference in five populations of a widely distributed North American cricket, Allonemobius socius (Orthoptera, Gryllidae). Chirp rate and chirp duration of the A. socius mating call were varied independently, and the responses of virgin females to these experimentally manipulated calls were repeatedly measured using a sequential single-stimulus design. Significant among- and within-population variation in chirp-duration preferences of females were found. Contrary to many previous studies, call chirp rate had no effect on female phonotaxis. Also there was no evidence of an interaction between chirp rate and chirp duration on female response to male mating calls. Moreover, female responsiveness to average and above-average chirp duration appeared to decline with female (adult) age. Overall, these results suggest evolved differences among populations in chirp-duration preferences, and that selection can act within populations on female chirp-duration preference.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 461–472.     

Revisiting excitation pattern design for magnetic resonance imaging through optimisation of the signal contrast efficiency

The design of excitation signals for Magnetic Resonance Imaging (MRI) is cast as an optimal control problem. Here, we demonstrate that signals other than pulse excitations, which are ubiquitous in MRI, can provide adequate excitation, thus challenging the optimality and ubiquity of pulsed signals. A class of on-resonance piecewise continuous amplitude modulated signals is introduced. It is shown that despite the bilinear nature of the Bloch equations, the spins system response is largely analytically tractable for this class of signals, using Galerkin approximation methods. To challenge the optimality of the pulse excitation, an appropriate cost criterion, the Signal Contrast Efficiency (SCE), is developed. It is to be optimised subject to dynamics expressed by the Bloch equations. To solve the problem the Bloch equation is transferred to the excitation dependent rotating frame of reference. The numerical solutions to the problem for different tissue types show that for a short period of time, pulse excitations provide the maximum signal contrast. However, the problem should be solved for longer periods of time which may result in a different answer than a pulse. For this purpose, the approximate analytic solution which is derived based on averaging the Bloch equation in the excitation dependent rotating frame of reference will be used to find the optimal excitation pattern. The solution to the optimisation problem is potentially useful for all forms of MRI including structural and functional imaging. The objective of this paper is to show that while classically transient response of pulses have been monitored so far, the optimal excitation pattern may be the steady state response of a non-pulse excitation.     

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source

The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near‐infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two‐photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep‐tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher‐order nonlinearities such as three‐photon absorption that we use to induce spatially localized photodamage in DNA. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multivariate Phenotypic Evolution: Divergent Acoustic Signals and Sexual Selection in <Emphasis Type="Italic">Gryllus</Emphasis> Field Crickets

Predicting the response to selection is at the core of evolutionary biology. Presently, thorough understanding of the effects of selection on the multivariate phenotype is lacking, in particular for behavioral traits. Here, we compared multivariate acoustic mating signals among seven field cricket species contrasting two selection regimes: (1) species producing songs with long trains of pulses for which preference functions for acoustic energy (chirp duty cycle) are linear and likely exert strong directional selection (‘trillers’); (2) species producing songs consisting of short chirps and for which preference functions for chirp duty cycle are concave and directional selection is likely weak or absent (‘chirpers’). We compared the phenotypic variance–covariance matrix (P) among species and uncovered two main patterns: First, surprisingly, pulse rate and chirp rate were positively correlated in six of seven species thus suggesting phenotypic coupling of timescales. Second, chirp rate and chirp duty cycle also covaried, but the direction of covariation differed between chirpers (positive) and trillers (negative). Multi-population Bayesian methods for matrix comparisons, Krzanowski’s subspace comparison and tensor analysis, revealed significant variation in P unrelated to phylogenetic distance, but strongly contrasting chirpers and trillers. We also found differences in the predicted selection response between chirpers and trillers. We thus report that variation in P is higher between than within selection regimes. Although effects from drift and shared ancestry cannot be fully excluded, these findings highlight a role for sexual selection in shaping patterns of phenotypic covariation that can ultimately affect the evolutionary trajectory of a multivariate mating signal.     

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genome-editing efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.     

Effect of stimulus (postsynaptic current) shape on fibre excitation.

Effects of variation of the stimulus pulse shape on the excitation of a nonmyelinated nerve fibre were studied using a mathematical model based on the Hodgkin-Huxley equations. Efficiency of smoothly changing pulses was compared with that of rectangular pulses. For pulses shorter than the time to excitation, the rate of the stimulus rise did not determine the ability of a smoothly changing pulse to excite the fibre. For a given stimulus duration, the main factor was the pulse area or the charge delivered by the pulse. The strength-duration curve for smoothly changing pulses was a nonmonotonic function, in contrast to the curve for rectangular pulses. The dependence of latency on changes in the pulse area was non-linear. It would be nonmonotonic when the pulse area variation were due to the stimulus duration or the stimulus rise duration. More that one propagating intracellular action potential (IAP) could arise upon fibre activation by a long smoothly changing threshold stimulus. Upon activation of relatively short fibres the IAP could arise not at the site of the smoothly changing stimulus injection. The rectangular pulses of long duration were more efficient than the corresponding smoothly changing ones. Irrespective of the shape, the pulses whose duration at the foot is 1-2 ms, are more suitable for a prolonged threshold fibre activation.     

北方两种常见蝈螽鸣声与发声器的比较研究

研究了北方常见的优雅蝈螽Gampsocleis gratiosa和暗褐蝈螽Campsocleis sedakovii雄性鸣声特征和发声器结构.优雅蝈螽鸣声规则,脉冲组序列由2种类型的脉冲组组成,第1种类型的脉冲组持续时间约0.09 s,脉冲持续和间隔时问约0.01 5;第2类型的脉冲组持续时间约0,04 s,脉冲持续和间隔时间均约0.003 s;鸣声的主能峰频率约7 kHz.暗褐蝈螽雄性鸣声包含短促的开翅鸣声和由2种类型的脉冲组组成的脉冲组序列构成的闭翅鸣声,第1种脉冲组持续时间约0.012 s,间隔时间约0.002 s;第2种脉冲组持续时间约0.013 s,间隔时间极短;鸣声主能峰频率约9.1kHz.2种蝈螽镜膜的形状、发声锉的形状和长度、发声齿的形状具显著差异.     

Simulation Studies of Photoacoustic Response from Gold-Silica Core-Shell Nanoparticles

Metal nanoparticles especially of noble metals are used as an exogenous contrast agent for biomedical photoacoustic (PA) imaging in the tissue transmission window extending from visible to near infrared 700–1100 nm band. Different geometrical configurations of gold and silver nanoparticles like spherical core-shell, nanorod, and nanocages are promising candidates for thermoplasmonics, photothermal therapy, photothermal imaging, and photoacoustic imaging. In the current study, we simulated the photoacoustic response of gold and silica core-shell nanoparticle in water medium. Finite element simulations were carried out to study the spectral absorption response and effect of nanosecond laser pulse excitation on the spatial/temporal temperature as well as photoacoustic pressure variations of different core-shell geometry of nanoparticle. We have optimized the dimensions of gold nanosphere, gold-silica, and silica-gold core-shell geometries for optimum photoacoustic conversion efficiency. Further, the effect of shell thickness on the pulse photoacoustic signals for core-shell gold-silica and silica-gold nanoparticle has been studied. We concluded that silica-gold core-shell nanoparticles possess better photoacoustic conversion efficiency in comparison to gold nanosphere and gold-silica core-shell geometries. The prime aim of this study is to design efficient nano-probes for photoacoustic imaging, photoacoustic tomography, photothermal therapy, and drug delivery.     

一种基于chirp编码的高帧速率超声成像方法

在医学超声成像系统中,帧速率由每帧图像的扫查发射次数所决定.同时发射多条波束可以提高图像的帧速率,但是这会带来不同波束间相互干扰的问题,形成伪像.本文基于编码激励的原理,提出了一种新的高帧速率成像方法.该方法通过发射一组线性频率调制编码信号,有效的降低了波束间的互扰.可以在不影响图像质量的情况下,成倍的提高图像的帧速率.