Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy

Abstract

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.

FIGURE 1

Imaging single-channel Ca²⁺ signals by total internal reflection fluorescence microscopy. (A) Schematic of the TIRFM imaging system. The 488-nm beam from an argon ion laser (50 mW) passes through a 5× beam expander (BE) and is focused by a lens (FL; f = 150 mm) via a dichroic mirror (DM) to a spot at the back focal plane of the microscope objective lens (Olympus TIRFM 60×, oil immersion, NA = 1.45). The focusing lens is mounted on a micrometer-driven translation stage, so that the laser beam can 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 (A) located at a conjugate image plane defines the field of excitation. Fluorescence excited in the specimen by the evanescent wave is collected by the same objective, passes through the dichroic mirror and a barrier filter (BF) blocking the laser wavelength, and is imaged by an intensifier tube coupled through a relay lens to a charge-coupled device camera. An oocyte expressing N-type channels is loaded with fluo-4 dextran, stripped of its vitelline envelope and allowed to adhere (animal hemisphere down) to a cover glass forming the base of the imaging chamber. Its membrane potential is controlled by a two-electrode voltage-clamp. (B) Schematic, illustrating the imaging of near-membrane fluorescent Ca²⁺ signals near an open channel by TIRFM. (C) Single video frame obtained by TIRFM illustrating Ca²⁺ signals from simultaneous opening of three channels within an 80 × 80-μm patch of oocyte membrane in response to depolarization to −15 mV. Increasing [Ca²⁺] is denoted both by “warmer” colors and by height.

FIGURE 2

TIRFM enhances resolution of single-channel Ca²⁺ signals by providing a thin optical section. (A) TIRFM image shows fluorescence at the peak of a sparklet, and traces show two examples of fluorescence signals monitored from a 0.9 × 0.9 μm region centered on this sparklet in response to successive depolarizing pulses from −80 mV to −10 mV. Arrow marks the time at which the image was captured and bars indicate depolarizing pulses. (B) Corresponding image and fluorescence traces obtained from the same channel after adjusting the laser beam for conventional wide-field epifluorescence excitation.

FIGURE 3

Simultaneous optical recording from numerous N-type channels within an imaging field. (A) Sequence showing representative TIRFM images captured before and at different times (indicated by numbered arrows in B) after depolarizing the oocyte to −15 mV. Each image is a single video frame, and shows sparklets arising at several channels throughout the field. (B) Traces show fluorescence signals recorded simultaneously during a single depolarizing pulse from independent regions of interest (∼1 μm²) placed over 40 channels.

FIGURE 4

Voltage-dependence of macroscopic and single-channel TIRFM Ca²⁺ signals. (A) Panels show representative single video frames of TIRFM images from a given patch of oocyte membrane captured 100 ms after depolarizing from −80 mV to the various potentials indicated. (B) Voltage dependence of macroscopic Ca²⁺ signal. Graph plots the mean fluorescence increase (ΔF/F) averaged over the entire image field as a function of membrane potential. Measurements were made during the first 300 ms of each depolarizing pulse, and data are mean ± SEM of measurements from six observations. (C) Voltage dependence of fluorescence signal during individual sparklets. Data are measurements of local fluorescence measured from 1 μm² regions of interest centered on individual channels (n = 11). (D) Voltage dependence of frequency of channel openings. Measurements were made by counting the number of discrete sparklets occurring throughout the imaging field during the first 200 ms after onset of depolarization to various voltages. Points show mean numbers of openings (n = 5 trials), scaled relative to that at 0 mV. Data could not be obtained at positive voltages, as the high frequency of events precluded reliable identification of individual sparklets.

FIGURE 5

Kinetics of sparklets. (A) Histogram shows distribution of durations of sparklets, measured at half maximal amplitude. Fitted curve is a single exponential with time constant of 64 ms. Data are from 243 events, n = 40 channels. (B) Decline in opening frequency during sustained depolarization. Histogram bars show the numbers of sparklets observed during successive 0.25-s time bins at a holding potential of −80 mV before and after depolarization, and throughout a 3-s depolarization to −20 mV as indicated. Data are from 40 channels. Fitted curve is a single exponential with a time constant of 1.36 s.

FIGURE 6

Spatial spread and kinetics of fluorescence signal during sparklets. (A) Radial distribution of fluorescence intensity across the sparklet. Dark trace shows the mean of fluorescence measurements made across a line (width 0.5 μm) passing diametrically through images of nine sparklets, each captured as a single video frame at the time of peak fluorescence. Shaded curve is a fitted Gaussian with width (FWHM) = 0.71 μm. (B) Corresponding fluorescence profile and Gaussian fit (FWHM = 0.45 μm) obtained using a 100-nm diameter fluorescent bead to measure the lateral point-spread function of the microscope. (C) Schematic, illustrating annular regions centered on a channel used to measure the traces in D. Scale corresponds to that in A and B, and numbers indicate mean radial distance (in μm) of each annular ring from the center. (D) Traces show fluorescence signals measured from these different annular rings during a depolarizing pulse that evoked several sparklets at a single channel. (E) Superposition of records from a spot (0.27 μm radius) centered on the channel and an annular ring of radius 1.25 μm around the channel. Traces are normalized to the same peak value to facilitate comparison of kinetic differences.

FIGURE 7

N-type Ca²⁺ channels are anchored in the oocyte membrane. Data were obtained from an oocyte stimulated by two trains of depolarizing pulses (−15 mV, train of five 3-s depolarizations at 0.1 Hz) separated by an interval of ∼4 min. (A) Representative images of sparklets arising at a single channel at various times as indicated. Cross-hairs mark the initial position of the sparklet centroid. (B) Traces show fluorescence measurements throughout corresponding depolarizing pulses obtained from a region of interest (1 μm²) centered on this channel.

FIGURE 8

Spatial density of channels and differences in properties of channel within a small area of membrane. (A) Maps variation in channel density between two regions (60 × 60 μm) of the same oocyte membrane ∼200-μm apart. Sparklets were evoked by a 3-s voltage pulse to 0 mV and dots mark locations where at least one sparklet was observed. (B) Diagram showing locations of all channels identified within a 60 × 60 μm region of membrane. The oocyte was stimulated by 10 successive depolarizing pulses to −10 mV. Colors denote the total number of sparklets observed at each site throughout the 10 pulses (black ≥ 2; blue 3–4; green 4–9; and red > 9). (C) Sample traces showing fluorescence records from selected sites, illustrating channels that showed relatively low frequencies of sparklets (upper two records), and relatively high frequencies (lower two records). (D) Red histogram shows the observed distribution of sparklet frequencies among the 72 channels illustrated in B. The mean number of sparklets per pulse was 0.685, and histogram bars indicate the number of channels that showed a given mean number of sparklets per pulse. Blue histogram shows the corresponding distribution expected from stochastic variability, assuming a Poisson distribution with mean of 0.685 events per pulse.