High-Speed In Vivo Voltage Imaging Dataset of Hippocampal Pyramidal Neurons and Interneurons

Here we present data used to demonstrate Activity Localization Imaging (ALI), a method that visualizes and extracts neuronal voltage signals from densely labeled population. The detailed principles of ALI and the methods for acquiring these data are described in: Chen et al., Imaging neuronal voltage beyond the scattering limit, Nature Methods (2025). File descriptions: - “MouseCA1-PyramidalCells.tsm”: Contains voltage imaging data of CA1 pyramidal neurons (used in Figs. 2-4 of the paper). - “MouseCA1-PV-Interneurons-Targeted-illumination.tsm”: Contains voltage imaging data of CA1 PV interneurons acquired using targeted illumination (used Fig. 5a, b). This dataset was previously published in Abdelfattah et al. Neuron (2023). Each of these files contains 360,000 images of Voltron 2-labeled neurons (160 x 190 pixels per image) taken at 2000 frames per second. The pixel size is 7.5 x 7.5 micrometers. - “tsm_read.m”: A MATLAB script for reading these images. - “MouseCA1-PyramidalCells_2P_zstack.zip”: Contains a high-resolution two-photon z-stack covering the same pyramidal cells as in “MouseCA1-PyramidalCells.tsm”. --- The raw data file (“2P_verification_rawdata.tif”) contains both green and red channels. The Voltron fluorescence is visible in the red channel. --- To facilitate analysis, the red channel images were extracted, rotated 90 degrees to match the neuron orientation in voltage imaging, and stored in a separate file (“2P_verification_RedChannel_Rotated.tif”). The z-stack contains 100 images taken at 2 micrometer steps.