(A) In ASAP-type sensors, voltage-induced movement of a positively charged transmembrane helix of a voltage-sensing domain (VSD) is thought to perturb the protonation state of a circularly permuted …
(A) Mean maximal response of ASAP1 variants to 500 ms, 100 mV steps in HEK293A cells from a holding potential of −70 mV. The x-axis labels represent the amino acid substituted in the place of I66 in …
(A) One-photon relative brightness of ASAP1 and ASAP2s transiently expressed in HEK293-Kir2.1 cells. Cells were illuminated with 470/24 nm light, and emitted photons were filtered using a 520/23 nm …
For all panels, HEK293-Kir2.1 cells transiently expressing the voltage indicators or the ASAP1::EGFP control were imaged under widefield one-photon illumination. (A) One-photon photobleaching …
For all panels, HEK293-Kir2.1 cells transiently expressing the voltage indicators (or the ASAP1::EGFP control) were imaged under two-photon illumination using a femtosecond pulsed Ti:sapphire laser. …
(A) Representative human embryonic stem-cell-derived cardiomyocytes (hESC-CMs) expressing ASAP1, ASAP2s, or ArcLight. Scale bar, 10 μm. (B) Representative single-trial responses to spontaneous …
(A) Representative human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) expressing ASAP1, ASAP2s, or ArcLight. Scale bar, 10 μm. (B) Representative single-trial responses to …
Confocal microscopy images of ASAP1 (A), ASAP2f (B), and ASAP2s (C). For each indicator, images of two representative cells are shown, along with magnified views of the boxed regions. Scale bars, 10 …
(A) Representative single-trial responses of ASAP1 (left) and ASAP2s (right) to APs in a cultured hippocampal neuron. These responses are from the same experiment as Figure 2H. (B) One-photon …
(A) Schematic illustration of the imaging setup (top) and the fruit fly visual system around L2 cells (bottom). Inset, example of the region imaged, with six L2 terminals expressing ASAP2s. …
(A) Two-photon photobleaching kinetics. L2 terminals were excited (920 nm, 14 mW) for 30 min at an identical scan rate to the experiments in Figure 3. Illumination was continuous except for 10 s …
MacQ-mCitrine (A) and Ace2N-2AA-mNeon (B) are expressed in L2 axon terminals and visible under two-photon illumination. Imaging conditions were identical to those used in Figure 3B. Scale bar, 5 μm. …
(A) Schematic of our random-access multi-photon (RAMP) imaging system. (B) Representative two-photon single-plane image of ASAP2s-expressing neurons in an organotypic hippocampal slice culture. …
(A–C) Box and whisker plots of the resting membrane potential (A), membrane capacitance (B), and input resistance (C) of neurons expressing ASAP1 and ASAP2s in organotypic hippocampal slice …
Representative single-trial ASAP2s responses to spontaneous APs inorganotypic hippocampal slice cultures imaged at 925 Hz by RAMP microscopy. Twenty voxels were imaged per neuron. Voltage traces …
(A) Representative ASAP1 and ASAP2f responses to a single current-evoked AP (black trace). Optical recordings were acquired at 925 Hz with 20 voxels per neuron. Traces are the average of 10 trials. …
(A) Two-photon images of two representative neurons expressing Ace2N-4AA-mNeon, displayed as maximal intensity Z-projections. Scale bar, 20 μm. (B) Representative two-photon single-plane image of a …
(A–F) ASAP1 and ASAP2s can detect subthreshold potential and hyperpolarization waveforms in single trials in organotypic hippocampal slice cultures. Optical recordings were acquired at 462 Hz. (A) …
(A) ASAP2s and AlexaFluor594 fluorescence from a representative neuron. Overlay shows the five positions (black squares) selected for imaging at 3700 Hz. Scale bar, 20 μm. (B) Representative ASAP1 …
(A) ASAP2s-expressing cells in an organotypic hippocampal slice culture were illuminated with a Ti:sapphire laser tuned to 900 nm and set to a power level of 25 mW at the sample plane. Each pixel …
(A) Representative optical responses of ASAP1 and ASAP2f to a single current-evoked AP. The AP voltage trace corresponds to the ASAP2f trace. For each neuron, 10 trials were conducted, each imaging …
(A) Peak amplitude of current-evoked APs as a function of the scanning frequency for ASAP2s and ASAP1 in single trials. Voxel dwell time was kept constant at 50 μs, and the number of voxels scanned …
(A) Peak amplitude of current-evoked APs as a function of scanning frequency for ASAP1 and ASAP2f in single trials. Both ASAP1 and ASAP2f show a similar pattern with a drop in the amplitude for …
(A–C) Representative responses to trains of five APs were evoked by current injection at 10 Hz (A), 30 Hz (B), or 100 Hz (C) in organotypic hippocampal slice cultures. Optical recordings were …
Trains of current-evoked APs were triggered in bursts of five APs at different frequencies in organotypic hippocampal slice cultures. The 1 Hz condition corresponds to a single AP. GEVI responses …
(A–C) Trains of five APs were evoked by current injection at 10 Hz (A), 30 Hz (B), or 100 Hz (C) in organotypic hippocampal slice cultures. Optical recordings were acquired at 925 Hz with 20 voxels …
(A) Left, overlay image of ASAP2s and AlexaFluor594 fluorescence from a representative neuron in an organotypic hippocampal slice culture. The recording site for this example is shown with an …
(A,B) Representative examples of single-trial, single-voxel responses to a single current-evoked AP in an organotypic hippocampal slice culture using ASAP1 (panel A) or ASAP2f (panel B). (C) …
(A) Optical responses to a backpropagating, current-evoked AP were recorded at multiple sites in the dendritic arbor. Recorded points at different distances from the cell body are shown with their …
(A) Normalized peak amplitude as a function of distance from the cell body shows a large decrease in amplitude with distance (n = 7 neurons, 27 to 41 spatial locations recorded per neuron). Each mark…
A hESC-CM was transfected with ASAP2s at 27 days post-differentiation and was imaged three days later at 100 Hz and with a power density of 11 mW/mm2 at the sample plane. Quantification of the …
ASAP2s fluorescence was captured while its transmembrane voltage was stepped from –70 to 0, 30, or 50 mV as labelled. Frames were acquired at 20 Hz and played back in real time. The movie …
Response kinetics of ASAP indicators and ArcLight Q239 in HEK293A cells at 22°C.
ASAP1 | ASAP2s | ArcLight Q239 | |
---|---|---|---|
Depolarization (–70 to 30 mV) | |||
τfast (ms) | 2.9 ± 0.3 | 5.2 ± 0.4 | 20 ± 2 |
τslow (ms) | 161 ± 33 | 63 ± 11 | 267 ± 13 |
% fast | 74 ± 5 | 56 ± 7 | 37 ± 7 |
Repolarization (30 to –70 mV) | |||
τfast (ms) | 2.3 ± 0.4 | 24 ± 7 | 113 ± 11 |
τslow (ms) | 177 ± 38 | 106 ± 47 | 367 ± 32 |
% fast | 63 ± 6 | 49 ± 17 | 53 ± 8 |
Hyperpolarization (–70 to –100 mV) | |||
τfast (ms) | 11 ± 3 | 8.2 ± 0.6 | 20 ± 5 |
τslow (ms) | 131 ± 16 | 104 ± 10 | 208 ± 23 |
% fast | 59 ± 3 | 53 ± 2 | 49 ± 3 |
Repolarization (–100 to –70 mV) | |||
τfast (ms) | 15 ± 3 | 13 ± 1 | 42 ± 12 |
τslow (ms) | 131 ± 14 | 114 ± 10 | 265 ± 75 |
% fast | 52 ± 2 | 51 ± 1 | 57 ± 3 |
n = 4–5 cells per sensor. Data are presented as mean ± SEM.