Post-stimulus adaptation of the resting position of the bundle exhibits at least two time scales.

<p>The slow component of the bundle movement following a prolonged deflection was fitted with the sum of two exponentials, yielding two time constants and , with >. Good fits were obtained ( & record duration) for 282 recordings (of ). The two time constants are represented in the histogram by different shadings of gray.</p

The time required by the bundle to return to its steady-state position depends on the stimulus duration.

<p>For a series of traces from a single cell, each recording was fitted with a sum of two exponentials. Derivatives of the fitted functions are represented in the form of a contour plot with slopes given in the legend (A). Rows correspond to different stimulus durations, with the recording order from top to bottom. We arbitrarily chose nm/ms as the threshold slope defining the steady-state. Similarity between the different contours indicates that the results do not critically depend on the selected criterion. (B) The time to reach steady-state () as a function of stimulus duration was determined for 17 different cells. These cells each had recordings for at least two different stimulus durations. Cells are represented by different symbols. The red line corresponds to the cell shown in (A). Five cells are displayed in color to guide the eye. The remaining cells are shown in gray with different symbols. (C) Semi-log plot of the data shown in (B).</p

Offset in the position of the hair bundle position does not determine its dynamic state.

<p>For a series of traces obtained from a single cell, the bundle’s position, with respect to that at the end of the recording, is represented in the form of a contour plot (A). Different rows correspond to different stimulus durations, with the recording order displayed from top to bottom. (B) from 17 different cells for which recordings at a minimum of two different stimulus durations were obtained. Cells are represented by different symbols. The red line corresponds to the cell shown in (A). Five cells are displayed in color to guide the eye. The remaining cells are shown in gray with different symbols. (C) Same as (B), but plotted on a logarithmic abscissa.</p

Mechanical Overstimulation of Hair Bundles: Suppression and Recovery of Active Motility

<div><p>We explore the effects of high-amplitude mechanical stimuli on hair bundles of the bullfrog sacculus. Under <i>in vitro</i> conditions, these bundles exhibit spontaneous limit cycle oscillations. Prolonged deflection exerted two effects. First, it induced an offset in the position of the bundle. Recovery to the original position displayed two distinct time scales, suggesting the existence of two adaptive mechanisms. Second, the stimulus suppressed spontaneous oscillations, indicating a change in the hair bundle’s dynamic state. After cessation of the stimulus, active bundle motility recovered with time. Both effects were dependent on the duration of the imposed stimulus. External calcium concentration also affected the recovery to the oscillatory state. Our results indicate that both offset in the bundle position and calcium concentration control the dynamic state of the bundle.</p> </div

The initial offset induced in the position of the bundle depends on stimulus duration.

<p>(A) The bundle’s offset immediately following the cessation of the stimulus () as a function of stimulus duration for 17 different cells. Cell are represented by different color/symbols. Results are shown for all the traces which yielded a good fit ( & record duration). (B) Same as (A), but using a logarithmic abscissa.</p

Different calcium ionic conditions on hair bundle recovery.

<p>Calcium concentration in the artificial endolymph was varied, as indicated to the right of the traces. (A) Concentration of calcium was first brought from 250 to 100 µM, which decreased (<i>asterisk</i>). Subsequent raising the external calcium concentration back to 250 µM partially reversed the effect. The stimulus duration was 5 s. (B) Quiescent interval versus the concentration of calcium in the surrounding endolymph, obtained from 6 cells. Different colors/symbols denote different calcium concentration, and the green triangle showed the lone example that displayed an opposite trend as the other 5 cells.</p

The duration of the quiescent interval was not dependent on stimulus amplitude.

<p>Time-dependent traces of a hair bundle’s position after stimulation at various amplitudes, but at a fixed duration (5 s). The stimulus amplitudes are indicated on the right of the traces. The <i>asterisk</i> over each trace shows the time of first bundle oscillation, which does not vary with stimulus amplitude. On the right, same data are represented as a contour plot, with bundle position color-coded as indicated in the legend. The steady state position is affected only weakly by the imposed deflection.</p

Example of hair bundle motility post-stimulus, with a diagram of the extracted parameters.

<p>The gray/black curve shows the position of the bundle, with motion towards the kinocilium being positive. An automatic routine detected bundle oscillations (<i>gray</i>) from which the onset time of the first oscillation () was determined. A sum of two exponentials (see Eq. 1) was fitted to the bundle’s position in the closed state (<i>black</i>), thus ignoring the oscillations. From the fit (<i>red</i>: ), the time to reach steady state () was calculated as the time for which its derivative first reached nm/ms. It was also used to calculate the bundle’s total offset (), defined as the difference between the position at t = 0 and the end of the recording. The stimulus was a one-second DC offset in positive direction. The curve starts at t = 0, which corresponds to 5 ms after cessation of the stimulus.</p

Label-Free Direct Visual Analysis of Hydrolytic Enzyme Activity Using Aqueous Two-Phase System Droplet Phase Transitions

Dextran hydrolysis-mediated conversion of polyethylene glycol (PEG)-dextran (DEX) aqueous two-phase system droplets to a single phase was used to directly visualize Dextranase activity. DEX droplets were formed either by manual micropipetting or within a continuous PEG phase by computer controlled actuation of an orifice connecting rounded channels formed by backside diffused light lithography. The time required for the two-phase to one-phase transition was dependent on the Dextranase concentration, pH of the medium, and temperature. The apparent Michaelis constants for Dextranase were estimated based on previously reported catalytic constants, the binodal polymer concentration curves for PEG-DEX phase transition for each temperature, and pH condition. The combination of a microfluidic droplet system and phase transition observation provides a new method for label-free direct measurement of enzyme activity

Label-Free Direct Visual Analysis of Hydrolytic Enzyme Activity Using Aqueous Two-Phase System Droplet Phase Transitions

Dextran hydrolysis-mediated conversion of polyethylene glycol (PEG)-dextran (DEX) aqueous two-phase system droplets to a single phase was used to directly visualize Dextranase activity. DEX droplets were formed either by manual micropipetting or within a continuous PEG phase by computer controlled actuation of an orifice connecting rounded channels formed by backside diffused light lithography. The time required for the two-phase to one-phase transition was dependent on the Dextranase concentration, pH of the medium, and temperature. The apparent Michaelis constants for Dextranase were estimated based on previously reported catalytic constants, the binodal polymer concentration curves for PEG-DEX phase transition for each temperature, and pH condition. The combination of a microfluidic droplet system and phase transition observation provides a new method for label-free direct measurement of enzyme activity