Demuro A , Parker I .

GK 65830 PHS HHS , GM 48071 NIGMS NIH HHS , R01 GM048071 NIGMS NIH HHS , R37 GM048071 NIGMS NIH HHS , R01 GM065830 NIGMS NIH HHS

	Figure 1. Schematic of the TIRFM imaging system for single-channel imaging. (A) Overview of the experimental system, constructed around an Olympus IX71 inverted microscope. For clarity, the schematic shows only the imaging light path, with the autofocus system omitted. The beam (488 nm) from a 50-mW argon ion laser passed through a 10× beam expander and was focused by a lens (FL; f = 150 mm) via a dichroic mirror (DM) to a spot at the back focal plane of the objective lens (Olympus TIRFM 60×, oil immersion, NA = 1.45). The focusing lens was mounted on a micrometer-driven translation stage, so that the laser beam could be adjusted to enter the periphery of the objective aperture so as to achieve total internal reflection at the interface between the cover glass and the aqueous bathing medium. An adjustable rectangular knife-blade aperture located at a conjugate image plane restricted excitation to the field imaged by the camera. Fluorescence excited in the specimen by the evanescent wave was collected through the objective lens, passed through the dichroic mirror and a barrier filter and was imaged by a Photometrics Cascade 128+ camera (Roper Scientific). The camera utilizes a back-illuminated frame transfer ccd with on-chip electron multiplication, and images (128 × 128 pixels; 0.33 μm per pixel) were captured at a frame rate of 500 s−1 with minimal dark and readout noise. An oocyte expressing nicotinic AChR channels was loaded with fluo-4 dextran, stripped of its vitelline envelope, and allowed to adhere (animal hemisphere down) to a coverglass forming the base of the imaging chamber. Its membrane potential was controlled by a two-electrode voltage clamp, and stepped to strongly negative potentials to enhance Ca2+ influx. (B) Enlarged view (not to scale) illustrating the imaging of near-membrane fluorescent signals from the Ca2+ microdomain (red) around a single open channel by the evanescent wave (green) formed by the TIRF objective lens. (C) Single image frame (2 ms exposure time) obtained by TIRFM illustrating Ca2+ signals from simultaneous opening of nAChR channels within a 40 × 40 μm patch of oocyte membrane. Increasing [Ca2+] is denoted both by “warmer” colors and by height. (D) Schematic of autofocus system. Light paths show: blue, 488 nm excitation beam; red, 670 nm guide beam; green, emitted fluorescence. Components include: 670 laser diode (4 mW; Edmund Optics); LLF, 670 nm laser line filter; DM2, dichroic mirror, 600 nm, to combine 670 and 488 nm laser beams; FL, focus lens; DM1, dichroic mirror transmitting between 500 and 600 nm; BF, barrier filter, sharp band pass between 510 and 600 nm to block 488- and 670-nm laser light from the imaging camera; quadrant photodetector module (Phresh Photonics). See text for further details.
	Figure 2. Image processing. (A–E) Illustration of processing steps. (A) Example of a “raw,” unprocessed frame acquired during polarization to −150 mV in the presence of ACh, showing several SCCaFTs. (B) Averaged image of background fluorescence, formed by averaging 100 frames at a holding potential of 0 mV before the hyperpolarizing step was applied to induce SCCaFTs. (C) Ratio image formed by dividing A by B. (D) Heavily smoothed (10 × 10 pixel) image formed from C. (E) Final, corrected image formed by subtracting D from C. For clarity of presentation, contrast settings are individually adjusted for each panel. (F) Representative example of SCCaFTs at a single channel site, derived from a sequence of ratio images like that in C. The trace represents mean fluorescence ratio within a region of interest (0.6 × 0.6 μm) centered on a channel. (G) Corresponding record from the same channel, derived after frame-by-frame subtraction of a heavily smoothed copy of each image as illustrated in E.
	Figure 3. Recording and depicting the simultaneous activity of hundreds of individual nAChR channels. (A) Functional map showing the locations of 191 channels within a 40 × 40 μm membrane patch. The map was constructed by visually identifying the coordinates of SCCaFTs observed throughout an 8-s imaging period during which the oocyte was polarized to −150 mV in the presence of 100 nM suberyldicholine. (B) Examples of single-channel recordings obtained by monitoring fluorescence from regions of interest (1 μm square) centered on 28 of the channels mapped in A. Traces were acquired simultaneously and show SCCaFTs resulting from Ca2+ influx during channel openings when the membrane was hyperpolarized. (C) Channel-chip depiction of the activity of all channels mapped in A. Channel openings (SCCaFTs) are represented as white streaks, time runs from left to right, and different channels (n = 191) are depicted vertically in random order. (D) Enlarged view of the region of the channel-chip boxed in white in C, showing the activities of 51 channels (numbered along the ordinate) during a shorter time period.
	Figure 4. Exchange between extracellular fluid “trapped” beneath the plasma membrane and the bulk bathing fluid. (A) Reversible blocking of nAChR channel activity by bath-applied curare. Each panel shows a single TIRFM image frame, together with representative traces of SCCaFT activity. Control records (left) were obtained in the presence of 30 nM ACh. The bathing solution was then exchanged for one containing 3 μM curare together with 30 nM ACh, resulting in cessation of SCCaFT activity after ∼60 s (middle). Finally (right), SCCaFT activity resumed ∼10 s after washing with solution containing only 30 nM ACh. (B) Washout of extracellular dye from the fluid film trapped between the cell membrane and coverglass. An oocyte was bathed in extracellular solution containing 1 μM Calcium Green for several minutes, and the dye was then quickly washed out from the bulk bathing solution by bath superfusion. Superimposed traces show the resulting decline of normalized fluorescence measured from regions of interest (1 μm square) spaced at 10-μm increments inward from the edge of the contact patch between the oocyte and coverglass. (C) Time to 90% fall of extracellular fluorescence as a function of distance from the contact zone boundary. (D) The amplitude of SCCaFTs remained constant throughout the duration (7 s) of hyperpolarizing pulses, indicating minimal depletion of extracellular Ca2+. Scatter plot shows measurements of SCCaFT amplitudes from 37 channel records (representative example shown in inset) as a function of time during the hyperpolarizing pulse. Data points are measurements of individual SCCaFT amplitudes. A regression line is fitted to the data.
	Figure 5. SCCaFTs track channel gating with a temporal resolution of 2 ms. Upper trace shows local fluorescence monitored from a single nAChR (30 nM ACh, −150 mV) that displayed four SCCaFTs during a roughly 3-s period. Lower panels show selected SCCaFTs on an expanded time scale. Images were acquired at 500 frames s−1, and dots indicate successive 2-ms time points.
	Figure 6. Optical measurement of nAChR channel open and closed time distributions during activation by two different agonists. (A–C) Data obtained in the presence of 30 nM ACh. (A) Representative records of SCCaFTs. (B) Distribution of SCCaFT durations evoked by ACh. Curve is a single exponential fit, with time constant = 7.9 ms. Data were derived from 1,700 SCCaFTs (280 channels; 3 oocytes). (C) Corresponding distribution of intervals between SCCaFTs (channel closed times). Data are fit by double exponentials, with time constants of 70 and 474 ms. (D–F) Corresponding measurements obtained in the presence of 1 μM suberyldicholine.
	Figure 7. (A and B) Voltage dependence of Ca2+ flux through nAChR. (A) Superimposed traces show SCCaFTs recorded simultaneously from 10 channels in the presence of 30 nM ACh while the membrane potential was pulsed to the voltages indicated. (B) Mean SCCaFT amplitude as a function of membrane potential. Data are from 29 channels in 2 oocytes. Regression line extrapolates to zero at a potential of about +60 mV. (C and D) Concentration dependence of nAChR opening derived from SCCaFT kinetics. (C) Representative traces show SCCaFTs evoked at 10 channel sites while an oocyte was polarized to −150 mV while successively superfusing 3, 10, and 30 nM ACh. (D) Relationship between [ACh] in the superfusate and channel open probability, as measured by the fraction of time individual channel sites displayed SCCaFTs. Data are from 43 channels (2 oocytes).
	Figure 8. Lack of motility of nAChR expressed in the oocyte membrane. (A) Sequence of single image frames shows SCCaFTs evoked at a single site during successive recording periods at ∼10-s intervals. Crosshairs mark the centroid position of the first SCCaFT imaged. (B) Fluorescence traces measured from a 0.6 × 0.6 μm square region of interest centered on the crosshairs in A. The oocyte was repeatedly hyperpolarized (3-s pulses) to −150 mV in the continued presence of 300 nM ACh, and rest intervals of ∼5 s were allowed between each record. (C) Scatter plot shows the centroid positions of SCCaFTs evoked during these recordings. (D) Plot shows the displacement of SCCaFT centroid positions relative to their mean for all events as a function of time through the roughly 60-s total recording.
	Figure 9. Muscle nicotinic AChR are randomly distributed in the oocyte membrane. (A) Representative map of nAChR channels distributed throughout a 60 × 60 μm imaging field, formed by manually plotting the locations of all (370) discrete SCCaFT sites observed during three 7-s recording periods in the presence of 30 nM ACh. (B) Example of a simulated random distribution, created by generating 370 random x-y coordinates within the same image field. (C) Histogram (red bars) shows the distribution of measured interchannel spacings. Data were obtained by calculating the separations between each channel and every other channel within the imaging field. Measurements were taken from three other membrane regions (550 × 400 pixel; 72 × 52 μm) like that illustrated in A, each of which contained 308–380 channels. The blue curve shows the distribution expected if channels with the same overall density as the experimental data were randomly arrayed. It was formed by averaging the interchannel distributions derived from 10 sets of randomly generated coordinates corresponding to each of the three experimental maps. (D) The same data as in C, replotted on an expanded scale to show the distribution of observed (red bars) and random (blue curve) interchannel spacings for spacings up to 6 μm.
	Figure 10. Spatial mapping of channel open probability. (A) Channel-chip representation, showing the simultaneous activity of 236 channels within a 40-μm square imaging field. The oocyte was continually exposed to 1 μM ACh, and the membrane potential was stepped from 0 to −150 mV for 7 s as indicated by the bar. The channels are arranged (from top to bottom) in order of decreasing open probability as calculated from the proportion of time during the hyperpolarizing pulse for which the SCCaFT amplitude exceeded one half of the mean maximal value for that channel. (B) Representative traces showing local fluorescence ratio measurements from the six channels marked by arrows in A. (C) Channel open probabilities (Popen, plotted on logarithmic scale) corresponding to the 236 channels depicted in A. Data points represent the proportion of time for which the fluorescence at that channel exceeded one half of the mean maximum during a single, 7-s hyperpolarizing step. (D) Histogram shows the distribution of open probabilities among the 236 channels. (E) Three-dimensional color contour plot showing the distribution of channel open probabilities throughout the imaging field. Same experiment as A–D. Contours depict mean Popen on a 30 × 30 grid matrix, with different colors corresponding to Popen values as indicated by the scale. Seven channels had Popen values >0.1 and were omitted from this analysis to avoid compressing the color scale. Their positions are marked by red squares, and black dots mark the positions of all other channels. (F) The same color contour analysis of Popen as shown in E, but after randomizing the x-y coordinates of the channels (indicated by black dots). Data in this figure are representative of three similar experiments.

Anderson, Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction. 1973, Pubmed

Anderson, Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction. 1973, Pubmed
Axelrod, Total internal reflection fluorescence microscopy in cell biology. 2003, Pubmed
Borges, Dual role for calcium in agrin signaling and acetylcholine receptor clustering. 2002, Pubmed
Borisenko, Simultaneous optical and electrical recording of single gramicidin channels. 2003, Pubmed
Burnashev, Calcium permeability of ligand-gated channels. 1998, Pubmed
Colquhoun, Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. 1985, Pubmed
Decker, Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant. 1990, Pubmed
Demuro, Optical single-channel recording: imaging Ca2+ flux through individual N-type voltage-gated channels expressed in Xenopus oocytes. 2003, Pubmed , Xenbase
Demuro, Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy. 2004, Pubmed , Xenbase
Demuro, Inhibition of nicotinic acetylcholine receptors by bicuculline. 2001, Pubmed , Xenbase
Demuro, Optical single-channel recording: imaging Ca2+ flux through individual ion channels with high temporal and spatial resolution. 2005, Pubmed
Dumont, Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. 1972, Pubmed , Xenbase
Fucile, Ca2+ permeability of nicotinic acetylcholine receptors. 2004, Pubmed
Hamill, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. 1981, Pubmed
Harms, Single-molecule imaging of l-type Ca(2+) channels in live cells. 2001, Pubmed
Harms, Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy. 2003, Pubmed
Methfessel, Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. 1986, Pubmed , Xenbase
Neher, Single-channel currents recorded from membrane of denervated frog muscle fibres. 1976, Pubmed
Peng, Diffusion of single cardiac ryanodine receptors in lipid bilayers is decreased by annexin 12. 2004, Pubmed
Peng, Imaging single cardiac ryanodine receptor Ca2+ fluxes in lipid bilayers. 2004, Pubmed
Ragozzino, Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells. 1998, Pubmed
Shuai, Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution. 2005, Pubmed , Xenbase
Sigworth, Patch clamp on a chip. 2002, Pubmed
Sonnleitner, Single ion channel imaging. 2003, Pubmed
Sonnleitner, Structural rearrangements in single ion channels detected optically in living cells. 2002, Pubmed , Xenbase
Ventura, A model-independent algorithm to derive Ca2+ fluxes underlying local cytosolic Ca2+ transients. 2005, Pubmed , Xenbase
Wang, Imaging microdomain Ca2+ in muscle cells. 2004, Pubmed
Wang, Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells. 2001, Pubmed
Willmann, Neuromuscular synaptogenesis: clustering of acetylcholine receptors revisited. 2002, Pubmed
Xu, Ion-channel assay technologies: quo vadis? 2001, Pubmed
Zou, Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events. 2004, Pubmed
Zou, Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs--fundamental calcium events. 2004, Pubmed
Zou, Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel. 1999, Pubmed
Zou, Visualization of Ca2+ entry through single stretch-activated cation channels. 2002, Pubmed