DOI: 10.5061/dryad.m63xsj4dd
title: Positive-going hybrid indicators for voltage imaging in excitable cells and tissues

description: 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.
description: # Positive-going hybrid indicators for voltage imaging in excitable cells
 and tissues Dataset DOI:
 [10.5061/dryad.m63xsj4dd](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: *
 [https://github.com/PKUCHEMZouLab/HVI](https://github.com/PKUCHEMZouLab/HVI)