Jonathan A. N. Fisher

Near infrared two-photon excitation cross-sections of voltage-sensitive dyes

Microscopy based on voltage-sensitive dyes has proven effective for revealing spatio-temporal patterns of neuronal activity in vivo and in vitro. Two-photon microscopy using voltage-sensitive dyes offers the possibility of wide-field visualization of membrane potential on sub-cellular length scales, hundreds of microns below the tissue surface. Very little information is available, however, about the utility of voltage-sensitive dyes for two-photon imaging purposes. Here we report on measurements of two-photon fluorescence excitation cross-sections for nine voltage-sensitive dyes in a solvent, octanol, intended to simulate the membrane environment. Ultrashort light pulses from a Ti:sapphire laser were used for excitation from 790 to 960 nm, and fluorescein dye was used as a calibration standard. Overall, dyes RH795, RH421, RH414, di-8-ANEPPS, and di-8-ANEPPDHQ had the largest two-photon excitation cross-sections ( approximately 15 x 10(-50)cm4 s photon(-1)) in this wavelength region and are therefore potentially useful for two-photon microscopy. Interestingly, di-8-ANEPPDHQ, a chimera constructed from the potentiometric dyes RH795 and di-8-ANEPPS, exhibited larger cross-sections than either of its constituents.

The Spatial Pattern of Cochlear Amplification

Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces.

In vivo fluorescence microscopy of neuronal activity in three dimensions by use of voltage-sensitive dyes.

We report in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The measurements have approximately 16-microm spatial and 3-ms temporal resolution and reach depths of 150 microm below the cortical surface. The depth-dependent differential-fluorescence optical sections of activity are consistent with known cortical architecture and represent an important step toward in vivo measurement of functioning complex neural networks. Our observations employ a custom gradient-index lens probe and voltage-sensitive dye fluorescence; the use of epi-illumination rather than dark-field illumination provides the dramatic signal-to-noise improvement necessary for fast three-dimensional imaging.

Contribution of active hair-bundle motility to nonlinear amplification in the mammalian cochlea

The cochlea’s high sensitivity stems from the active process of outer hair cells, which possess two force-generating mechanisms: active hair-bundle motility elicited by Ca2+ influx and somatic motility mediated by the voltage-sensitive protein prestin. Although interference with prestin has demonstrated a role for somatic motility in the active process, it remains unclear whether hair-bundle motility contributes in vivo. We selectively perturbed the two mechanisms by infusing substances into the endolymph or perilymph of the chinchilla’s cochlea and then used scanning laser interferometry to measure vibrations of the basilar membrane. Blocking somatic motility, damaging the tip links of hair bundles, or depolarizing hair cells eliminated amplification. While reducing amplification to a lesser degree, pharmacological perturbation of active hair-bundle motility diminished or eliminated the nonlinear compression underlying the broad dynamic range associated with normal hearing. The results suggest that active hair-bundle motility plays a significant role in the amplification and compressive nonlinearity of the cochlea.

Real-Time Detection and Monitoring of Acute Brain Injury Utilizing Evoked Electroencephalographic Potentials.

Rapid detection and diagnosis of a traumatic brain injury (TBI) can significantly improve the prognosis for recovery. Helmet-mounted sensors that detect impact severity based on measurements of acceleration or pressure show promise for aiding triage and transport decisions in active, field environments such as professional sports or military combat. The detected signals, however, report on the mechanics of an impact rather than directly indicating the presence and severity of an injury. We explored the use of cortical somatosensory evoked electroencephalographic potentials (SSEPs) to detect and track, in real-time, neural electrophysiological abnormalities within the first hour following head injury in an animal model. To study the immediate electrophysiological effects of injury in vivo, we developed an experimental paradigm involving focused ultrasound that permits continuous, real-time measurements and minimizes mechanical artifact. Injury was associated with a dramatic reduction of amplitude over the damaged hemisphere directly after the injury. The amplitude systematically improved over time but remained significantly decreased at one hour, compared with baseline. In contrast, at one hour there was a concomitant enhancement of the cortical SSEP amplitude evoked from the uninjured hemisphere. Analysis of the inter-trial electroencephalogram (EEG) also revealed significant changes in low-frequency components and an increase in EEG entropy up to 30 minutes after injury, likely reflecting altered EEG reactivity to somatosensory stimuli. Injury-induced alterations in SSEPs were also observed using noninvasive epidermal electrodes, demonstrating viability of practical implementation. These results suggest cortical SSEPs recorded at just a few locations by head-mounted sensors and associated multiparametric analyses could potentially be used to rapidly detect and monitor brain injury in settings that normally present significant levels of mechanical and electrical noise.

Imaging electrical resonance in hair cells

The mechanosensory hair cells of many auditory receptor organs are tuned by an electrical resonance that increases their responses to stimulation over a narrow band of frequencies. The small oscillations of membrane potential characteristic of this phenomenon have previously been detectable only through intracellular electrode measurements, which are laborious and preclude analysis at the level of an entire sensory organ. We used a voltage-sensitive dye to image hair-cell electrical resonance in an intact preparation of the bullfrogs sacculus, a receptor organ sensitive to low-frequency seismic and auditory stimuli. Imaging revealed distinct populations of hair cells whose resonant response varied with the frequency of transepithelial electrical stimulation. Most of the hair cells in the saccular epithelium in vitro were electrically tuned to stimulation at 25–50 Hz. The frequency dependence of the fluorescence signal was sensitive to pharmacological blockade of large-conductance Ca2+-sensitive K+ channels and to enzymatic digestion. At an elevated concentration of Ca2+, we observed transient fluorescence signals that probably represented action potentials. The stroboscopic imaging and analysis techniques described here present a general approach for studying subthreshold oscillations in electrically excitable cells.

Epidermal Electrode Technology for Detecting Ultrasonic Perturbation of Sensory Brain Activity

Objective: We aim to demonstrate the in vivo capability of a wearable sensor technology to detect localized perturbations of sensory-evoked brain activity. Methods: Cortical somatosensory evoked potentials (SSEPs) were recorded in mice via wearable, flexible epidermal electrode arrays. We then utilized the sensors to explore the effects of transcranial focused ultrasound, which noninvasively induced neural perturbation. SSEPs recorded with flexible epidermal sensors were quantified and benchmarked against those recorded with invasive epidural electrodes. Results: We found that cortical SSEPs recorded by flexible epidermal sensors were stimulus frequency dependent. Immediately following controlled, focal ultrasound perturbation, the sensors detected significant SSEP modulation, which consisted of dynamic amplitude decreases and altered stimulus-frequency dependence. These modifications were also dependent on the ultrasound perturbation dosage. The effects were consistent with those recorded with invasive electrodes, albeit with roughly one order of magnitude lower signal-to-noise ratio. Conclusion: We found that flexible epidermal sensors reported multiple SSEP parameters that were sensitive to focused ultrasound. This work therefore 1) establishes that epidermal electrodes are appropriate for monitoring the integrity of major CNS functionalities through SSEP; and 2) leveraged this technology to explore ultrasound-induced neuromodulation. The sensor technology is well suited for this application because the sensor electrical properties are uninfluenced by direct exposure to ultrasound irradiation. Significance: The sensors and experimental paradigm we present involve standard, safe clinical neurological assessment methods and are thus applicable to a wide range of future translational studies in humans with any manner of health condition.

In vivo fluorescence microscopy of neuronal activity in three dimensions using voltage-sensitive dyes

Abstract: We report three-dimensional in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The depth-dependent differential fluorescence optical sections of activity were consistent with known cortical architecture.