Positive-going hybrid indicators for voltage imaging in excitable cells and tissues
Data files
Jul 09, 2025 version files 23.65 MB
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Code_for_voltage_imaging_with_HVI_and_HVIplus.zip
23.64 MB
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README.md
4.54 KB
Abstract
Membrane potential dynamics are crucial to the physiology of electrically excitable cells, such as neurons, cardiomyocytes, and pancreatic islet cells. Fluorescent voltage indicators have enabled the visualization of electrical activities at the single-cell level across large cell populations. However, most existing indicators have a negative fluorescence-voltage response, resulting in a high background signal when cells are at their resting state. To reduce background interference during voltage imaging, we developed a positive-going hybrid voltage indicator (HVI+), which has a lower intensity at the resting state and becomes brighter upon membrane depolarization. HVI+-Cy3b exhibits a remarkable voltage sensitivity of 55% ΔF/F0 per 100 mV, enabling accurate reporting of action potential waveforms in beating cardiomyocytes and neurons. Additionally, by combining HVI+-Cy3b with HVI-Cy3b, we demonstrate the capability to simultaneously assess the effects of glucose on the spike activities of two cell types in pancreatic islets.
Dataset DOI: 10.5061/dryad.m63xsj4dd
Description of the data and file structure
We have submitted the codes for voltage imaging (LabVIEW code folder) and data analysis (MATLAB code folder) used in the paper “Positive-going hybrid indicators for voltage imaging in excitable cells and tissues”.
Files and variables
File: Code_for_voltage_imaging_with_HVI_and_HVIplus.zip
Description: This file contains LabVIEW code for voltage imaging and MATLAB code for data analysis.
The analysis codes include two parts: analysis in HEK293T and analysis in excitable cells or tissues.
Analysis in HEK293T cells:
1) Kinetics and sensitivity analysis for negative-going indicator: for HVI and other negative-going voltage indicators with an overshoot depolarizing (Table S2).
2) Kinetics and sensitivity analysis for positive-going indicator: for HVIplus and other positive-going voltage indicators with an overshoot depolarizing (Table S2).
3) Ladder waveform analysis for F-V curve: fluorescence responses of voltage indicators to voltage steps from -100 mV to 100 mV. Fluorescence responses were normalized to baseline fluorescence at -70 mV (Figure 2D).
ncol = 176; % x of ROI
nrow = 96; % y of ROI
camera_bias = 400; % background due to camera bias (100 for bin 1x1)
dt_mov = 0.9452; % exposure time in milliseconds (1058 Hz)
4) Photobleaching_analysis: photostability characterization of voltage indicators (Figure S7).
ncol = 324; % x of ROI
nrow = 324; % y of ROI
bkg = 1600; % background due to camera bias (100 for bin 1x1)
dt_mov = 0.1; % exposure time in second
Analysis in excitable cells or tissues:
1) Analysis of stimulated single AP or subthreshold potential: refine the parameters in generation of averaged voltage signals for sensitivity calculation for various frame rates (Figure 2C & S6).
dire = ;% dire = 1 means positive-going GEVI; dire = -1 means negative-going GEVI
dire_for_stim = ;% 1 means positive stimulation (depolarization); -1 means negative stimulation (hyperpolarization)
mode = ; % input the sampling rate of the camera
(if mode == 484; ncol = 312; nrow = 208; dt_mov = 2.0658;
if mode == 1058; ncol = 176; nrow = 96; dt_mov = 0.9452;
if mode == 2343; ncol = 324; nrow = 40; dt_mov = 0.4268; )
2) Analysis of spontaneous voltage imaging: sensitivity analysis of spontaneous voltage activity in pancreatic islets (dF/F per AP, Figures 6 & 7).
dt_mov = 5; % exposure time in milliseconds (200 Hz)
ncol = 256; % x of ROI
nrow = 256; % y of ROI
camera_bias = 1600; % background due to camera bias (100 for bin 1x1)
3) Analysis of dual color and spontaneous voltage imaging: for generating the traces of spontaneous voltage activity in cardiomyocytes (dF/F per AP, Figure 4 & 5).
dt_mov = 10; % exposure time in milliseconds (100 Hz)
4) Analysis of PB removed ratiometric signals based on existing traces: for generating the trace of the ratio of signals from two recording channels with photobleaching correction, based on the traces from “3 Analysis of dual color and spontaneous voltage imaging” (Figure 4 & 5).
5) Analysis of AP frequency and duration for cardiomyocytes: for calculating the averaged AP, AP frequency, and AP duration of cardiomyocytes, based on the traces from “4 analysis of PB removed ratiometric signals based on existing traces” (Figure 5).
6) Analysis of averaged AP AP frequency and width of islet cells: for calculating the averaged AP, AP frequency, and FWHM of islet cells, based on the traces from “2 analysis of spontaneous voltage imaging” (Figures 6 & 7).
7) Analysis of photocurrent_voltageclamp: analysis of steady-state and peak photocurrent of voltage indicators in voltage clamp (Figure S4).
WL = ; % wavelength of the excitation laser
Code/software
The fluorescence voltage images were captured using LabVIEW software (version 2015, Run maincontrol v2.13 to start).
The electrical data and fluorescence images were analyzed using custom MATLAB software (MathWorks, version R2019b).
Access information
Other publicly accessible locations of the data: