DOI: 10.5061/dryad.w6m905qwm
title: Data for: PIEZO1-HaloTag hiPSCs: bridging molecular, cellular and tissue imaging

description: PIEZO1 channels play a critical role in numerous physiological processes
 by transducing diverse mechanical stimuli into electrical and
 chemical signals. Recent studies underscore the importance of endogenous
 PIEZO1 activity and localization in regulating mechanotransduction. To
 enable physiologically and clinically relevant human-based
 studies, we genetically engineered human induced pluripotent
 stem cells (hiPSCs) to express a HaloTag fused to endogenous
 PIEZO1. Combined with super-resolution imaging, our chemogenetic approach
 allows precise visualization of PIEZO1 in various cell types. Further,
 the PIEZO1-HaloTag hiPSC technology allows non-invasive
 monitoring of channel activity via Ca2+-sensitive HaloTag ligands, with
 temporal resolution approaching that of patch clamp electrophysiology.
 Using lightsheet imaging of hiPSC-derived neural organoids, we
 also achieve molecular scale PIEZO1 imaging in three-dimensional
 tissue samples. Our advances offer a novel platform for studying PIEZO1
 mechanotransduction in human cells and tissues, with potential for
 elucidating disease mechanisms and development of targeted therapeutics.
description: Micropatterned neural rosettes (MNRs) of 140 µm in diameter grown
 on 19 mm square Arena A CYTOO chips (Cat. No.10-020-00-18) were imaged on
 a modified adaptive optical lattice light-sheet microscope (AO-LLSM).
 Prior to all imaging, the microscope was calibrated to correct for optical
 aberrations from the system. The Cytoo chip was mounted on a custom
 designed sample holder and immersed in 40 ml of phenol-free MNR culture
 medium. The excitation (Thorlabs 0.6 NA, TL20X-MPL) and detection (Zeiss
 1.0 NA, 421452-9800-000) objectives were also immersed into the imaging
 medium. The MNRs were imaged with a multiBessel square lattice light sheet
 with the NA<sub>sq </sub>of 0.35 and a 0.3/0.4 NA annular
 mask. The 488 nm, 560 nm and 642 nm lasers were used to visualize
 SPY505-DNA (DNA, Spirochrome Cat# CY-SC101), SPY555-actin (actin,
 Spirochrome Cat# CY-SC202), and Janelia Fluor® 635-HaloTag Ligand
 (Non-Bapta) or Janelia Fluor® 646-BAPTA-AM- HaloTag ligand (PIEZO1),
 respectively, with power at the back pupil of the excitation objective of
 45 µW for 488 nm, 50 µW for 560 nm and ranging between 2.1-2.9 mW for 642
 nm. To balance volumetric imaging speed, signal to noise and
 photobleaching, images were acquired using camera exposure between 30–50
 milliseconds. All data from the AO-LLSM were collected on two Hamamatsu
 ORCA-Fusion sCMOS cameras. Emission light from actin, DNA, and JF635 or
 JF646 was separated by a dichroic mirror (Chroma T600DCRB) and passed to
 two cameras equipped with either Semrock FF01-600/37-25 emission filter
 for actin or Semrock FF01-538/685-25 filter for DNA and JF635 or
 JF646-BAPTA . The 3D volumetric imaging of MNRs was
 performed by tiling AO-LLSM across the sample. Each tile (approximately
 200 x 100 x 15 µm<sup>3</sup>) was scanned by moving the
 sample stage at 400 nm step sizes. Each neighboring tile had a 5 µm
 overlap between each adjacent tile. Mismatch between the excitation LLS
 and the detection focal plane caused by the MNR were corrected prior to
 every acquisition and was essential to ensure optimal imaging of PIEZO1,
 DNA and actin structures. The AO-LLSM data was processed using MATLAB
 versions R2022b and R2023a. The large 3D MNR volumes were stitched in
 skewed space, deconvolved, deskewed and rotated on Advanced Bioimaging
 Center’s computing cluster at UC Berkeley using the computational
 pipelines published on GitHub
 (https://github.com/abcucberkeley/LLSM5DTools). The skewed space
 deconvolution was performed with experimentally measured point spread
 functions obtained from 200 nm fluorescent beads (Invitrogen FluoSpheres
 Carboxylate-Modified Microspheres, 505/515 nm, F8811). The nuclei were
 denoised using Content-Aware Image Restoration (CARE). The training data
 for the denoising model was collected using lattice light-sheet microscopy
 by volumetrically scanning LLC-PK1 cells expressing nuclear marker (H2B)
 to record low SNR and corresponding high SNR 3D stacks. The AOLLSM
 instrument was controlled using a custom LabVIEW based image acquisition
 software (National Instruments, Woburn, MA) licensed from Janelia Research
 Campus, HHMI. To observe the dynamics of PIEZO1-HaloTag
 in MNRs, 3-plane stack videos were acquired. For this, the sample stage
 (using the SmarAct MLS-3252 Electromagnetic Direct-Drive) was rapidly
 scanned across a 1 µm sample range (comprised of three image planes spaced
 400 nm along the sample stage scan axis, corresponding to 215 nm along the
 optical z axis) at an interval between 160-210 ms per stack for 30 time
 points. The dynamic time series datasets were deskewed and maximum
 intensity projected prior to analysis. The corresponding DNA and actin
 volumes were recorded at the same location prior to acquiring the
 PIEZO1-HaloTag time series. A new dataset of 30 time points was collected
 every ~10 µm throughout the MNR and was repeated on multiple
 samples.
description: # Data for "PIEZO1-HaloTag hiPSCs: Bridging Molecular, Cellular and
 Tissue Imaging" This data is organized for some of the analysis and
 visualization results shown in the paper "PIEZO1-HaloTag hiPSCs:
 Bridging Molecular, Cellular and Tissue Imaging"
 ([https://doi.org/10.1101/2023.12.22.573117](https://doi.org/10.1101/2023.12.22.573117)): ## Description of the data and file structure ### Schema of data archive <pre> three_plane_stack_data.zip ├── three_plane_stack_data │ ├── BaptaWT │ │ └── 140um │ │ ├── 2022 │ │ │ ├── 0609 │ │ │ │ └── exp_* │ │ │ │ ├── actin_mask │ │ │ │ │ ├── *_lumen_edge.tif │ │ │ │ │ └── *_outer_edge.tif │ │ │ │ ├── Actin_with_DNA │ │ │ │ │ └── *.tif │ │ │ │ ├── HaloLigand │ │ │ │ │ └── *.tif │ │ │ │ ├── results │ │ │ │ │ ├── *_locs.csv │ │ │ │ │ └── *_locs_filtered.csv │ │ │ │ └── settings_info │ │ │ │ ├── *_JSONsettings.json │ │ │ │ ├── *_Settings.txt │ │ │ │ ├── *_slicelist.sqlite3 │ │ │ │ └── *_TargetPositions.csv │ │ │ └── 0612 │ │ │ └── exp_* │ │ │ ├── actin_mask │ │ │ │ ├── *_lumen_edge.tif │ │ │ │ └── *_outer_edge.tif │ │ │ ├── Actin_with_DNA │ │ │ │ └── *.tif │ │ │ ├── HaloLigand │ │ │ │ └── *.tif │ │ │ ├── results │ │ │ │ ├── *_locs.csv │ │ │ │ └── *_locs_filtered.csv │ │ │ └── settings_info │ │ │ ├── *_JSONsettings.json │ │ │ ├── *_Settings.txt │ │ │ ├── *_slicelist.sqlite3 │ │ │ └── *_TargetPositions.csv │ │ └── 2023 │ │ └── 0303 │ │ └── exp_* │ │ ├── Actin │ │ │ └── *.tif │ │ ├── actin_mask │ │ │ ├── *_lumen_edge.tif │ │ │ └── *_outer_edge.tif │ │ ├── DNA │ │ │ └── *.tif │ │ ├── HaloLigand │ │ │ └── *.tif │ │ ├── results │ │ │ ├── *_locs.csv │ │ │ └── *_locs_filtered.csv │ │ └── settings_info │ │ ├── *_JSONsettings.json │ │ ├── *_Settings.txt │ │ ├── *_slicelist.sqlite3 │ │ └── *_TargetPositions.csv │ └── NonBaptaWT │ └──140um │ ├── 2022 │ │ ├── 0609 │ │ │ └── exp_* │ │ │ ├── actin_mask │ │ │ │ ├── *_lumen_edge.tif │ │ │ │ └── *_outer_edge.tif │ │ │ ├── Actin_with_DNA │ │ │ │ └── *.tif │ │ │ ├── HaloLigand │ │ │ │ └── *.tif │ │ │ ├── results │ │ │ │ ├── *_locs.csv │ │ │ │ └── *_FinalTracks_filtered.csv │ │ │ └── settings_info │ │ │ ├── *_JSONsettings.json │ │ │ ├── *_Settings.txt │ │ │ ├── *_slicelist.sqlite3 │ │ │ └── *_TargetPositions.csv │ │ └── 0612 │ │ └── exp_* │ │ ├── actin_mask │ │ │ ├── *_lumen_edge.tif │ │ │ └── *_outer_edge.tif │ │ ├── Actin_with_DNA │ │ │ └── *.tif │ │ ├── HaloLigand │ │ │ └── *.tif │ │ ├── results │ │ │ ├── *_locs.csv │ │ │ └── *_FinalTracks_filtered.csv │ │ └── settings_info │ │ ├── *_JSONsettings.json │ │ ├── *_Settings.txt │ │ ├── *_slicelist.sqlite3 │ │ └── *_TargetPositions.csv │ └── 2023 │ ├── 0228 │ │ └── exp_* │ │ ├── Actin │ │ │ └── *.tif │ │ ├── actin_mask │ │ │ ├── *_lumen_edge.tif │ │ │ └── *_outer_edge.tif │ │ ├── DNA │ │ │ └── *.tif │ │ ├── HaloLigand │ │ │ └── *.tif │ │ ├── results │ │ │ ├── *_locs.csv │ │ │ └── *_FinalTracks_filtered.csv │ │ └── settings_info │ │ ├── *_JSONsettings.json │ │ ├── *_Settings.txt │ │ ├── *_slicelist.sqlite3 │ │ └── *_TargetPositions.csv │ └── 0301 │ └── exp_* │ ├── Actin │ │ └── *.tif │ ├── actin_mask │ │ ├── *_lumen_edge.tif │ │ └── *_outer_edge.tif │ ├── DNA │ │ └── *.tif │ ├── HaloLigand │ │ └── *.tif │ ├── results │ │ ├── *_locs.csv │ │ └── *_FinalTracks_filtered.csv │ └── settings_info │ ├── *_JSONsettings.json │ ├── *_Settings.txt │ ├── *_slicelist.sqlite3 │ └── *_TargetPositions.csv volumetric_data.zip ├── volumetric_data │ ├── KO │ │ └── exp1_140um_1 │ │ ├── Actin │ │ │ ├── DS │ │ │ │ ├── MIPs │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ └── *.tif │ │ │ └── *.tif │ │ ├── DNA │ │ │ ├── DS │ │ │ │ ├── MIPs │ │ │ │ │ └── *.tif │ │ │ │ └── *.tif │ │ │ └── *.tif │ │ ├── HaloLigand │ │ │ ├── DS │ │ │ │ ├── MIPs │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ └── *.tif │ │ │ ├── results │ │ │ │ └── Detection3D.mat │ │ │ └── *.tif │ │ └── settings_info │ │ ├── v_JSONsettings.json │ │ ├── v_Settings.txt │ │ ├── v_slicelist.sqlite3 │ │ └── v_TargetPositions.csv │ ├── WT │ │ └── exp1_140um_2 │ │ ├── Actin │ │ │ ├── DS │ │ │ │ ├── MIPs │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ └── *.tif │ │ │ ├── matlab_decon │ │ │ │ ├── DS │ │ │ │ │ ├── MIPs │ │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ │ └── *.tif │ │ │ │ ├── psfgen │ │ │ │ │ └── 560_CamA_ch0.tif │ │ │ │ ├── MIPs │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ └── *.tif │ │ │ └── *.tif │ │ ├── DNA │ │ │ ├── DS │ │ │ │ ├── MIPs │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ └── *.tif │ │ │ ├── denoised │ │ │ │ └── *.tif │ │ │ └── *.tif │ │ ├── HaloLigand │ │ │ ├── DS │ │ │ │ ├── MIPs │ │ │ │ │ └── *_MIP_z.tif │ │ │ │ └── *.tif │ │ │ ├── results │ │ │ │ └── Detection3D.mat │ │ │ └── *.tif │ │ └── settings_info │ │ ├── v_JSONsettings.json │ │ ├── v_Settings.txt │ │ ├── v_slicelist.sqlite3 │ │ └── v_TargetPositions.csv │ └── PSF │ ├── DS │ │ └── *.tif │ ├── *.tif │ └── settings_info │ ├── *_JSONsettings.json │ ├── *_Settings.txt │ └── *_TargetPositions.csv README.md </pre> ### File Details #### three\_plane\_stack\_data (after decompressing the zip file): BaptaWT: folder containing max intensity projection (MIP) data of three planes of micropatterned neural rosettes (MNRs) in deskewed space for quantifying JF646-BAPTA labeled PIEZO1-HaloTag puncta localizations NonBaptaWT: folder containing max intensity projection data of three planes of micropatterned neural rosettes (MNRs) for quantifying JF635 labeled PIEZO1-HaloTag puncta localizations #### three\_plane\_stack\_data/BaptaWT/140um/2022/\*/exp\_\*: actin_mask: folder containing masks for the lumen and outer edges Actin_with_DNA: folder containing image data of the actin and nuclei channel HaloLigand: folder containing image data of the raw PIEZO1 channel results: folder containing csv files obtained from performing puncta localization using ThunderSTORM settings_info: folder containing information on data acquisition process #### three\_plane\_stack\_data/BaptaWT/140um/2022/\*/exp\_\*/actin\_mask: *_lumen_edge.tif: lumen edge mask *_outer_edge.tif: outer edge mask #### three\_plane\_stack\_data/BaptaWT/140um/2022/\*/exp\_\*/Actin\_with\_DNA: *.tif: image data of the actin and nuclei channel #### three\_plane\_stack\_data/BaptaWT/140um/2022/\*/exp\_\*/HaloLigand: *.tif: image data of the raw PIEZO1 channel #### three\_plane\_stack\_data/BaptaWT/140um/2022/\*/exp\_\*/results: *_locs.csv: csv file containing information on all the localizations obtained using ThunderSTORM *_locs_filtered.csv: csv file containing information on localizations after filtering out autofluorescence blobs #### three\_plane\_stack\_data/BaptaWT/140um/2022/\*/exp\_\*/settings\_info: *_JSONsettings.json: json file containing meta data *_Settings.txt: txt file containing meta data *_slicelist.sqlite3: database file containing the slice list *_TargetPositions.csv: csv file containing target positions #### three\_plane\_stack\_data/BaptaWT/140um/2023/\*/exp\_\*: Actin: folder containing image data of the actin channel actin_mask: folder containing masks for the lumen and outer edges DNA: folder containing image data of the nuclei channel HaloLigand: folder containing image data of the raw PIEZO1 channel results: folder containing csv files obtained from performing puncta localization using ThunderSTORM settings_info: folder containing information on data acquisition process #### three\_plane\_stack\_data/BaptaWT/140um/2023/\*/exp\_\*/Actin: *.tif: image data of the actin channel #### three\_plane\_stack\_data/BaptaWT/140um/2023/\*/exp\_\*/actin\_mask: *_lumen_edge.tif: lumen edge mask *_outer_edge.tif: outer edge mask #### three\_plane\_stack\_data/BaptaWT/140um/2023/\*/exp\_\*/DNA: *.tif: image data of the nuclei channel #### three\_plane\_stack\_data/BaptaWT/140um/2023/\*/exp\_\*/HaloLigand: *.tif: image data of the raw PIEZO1 channel #### three\_plane\_stack\_data/BaptaWT/140um/2023/0303/exp\_\*/results: *_locs.csv: csv file containing information on all the localizations obtained using ThunderSTORM *_locs_filtered.csv: csv file containing information on localizations after filtering out autofluorescence blobs #### three\_plane\_stack\_data/BaptaWT/140um/2023/\*/exp\_\*/settings\_info: *_JSONsettings.json: json file containing meta data *_Settings.txt: txt file containing meta data *_slicelist.sqlite3: database file containing the slice list *_TargetPositions.csv: csv file containing target positions #### three\_plane\_stack\_data/NonBaptaWT/140um/2022/\*/exp\_\*: actin_mask: folder containing masks for the lumen and outer edges Actin_with_DNA: folder containing image data of the actin and nuclei channel HaloLigand: folder containing image data of the raw PIEZO1 channel results: folder containing csv files obtained from performing puncta localization and tracking using ThunderSTORM settings_info: folder containing information on data acquisition process #### three\_plane\_stack\_data/NonBaptaWT/140um/2022/\*/exp\_\*/actin\_mask: *_lumen_edge.tif: lumen edge mask *_outer_edge.tif: outer edge mask #### three\_plane\_stack\_data/NonBaptaWT/140um/2022/\*/exp\_\*/Actin\_with\_DNA: *.tif: image data of the actin and nuclei channel #### three\_plane\_stack\_data/NonBaptaWT/140um/2022/\*/exp\_\*/HaloLigand: *.tif: image data of the raw PIEZO1 channel #### three\_plane\_stack\_data/NonBaptaWT/140um/2022/\*/exp\_\*/results: *_locs.csv: csv file containing information on all the localizations obtained using ThunderSTORM *_FinalTracks_filtered.csv: csv file containing information on the final tracks after filtering out autofluorescence blobs #### three\_plane\_stack\_data/NonBaptaWT/140um/2022/\*/exp\_\*/settings\_info: *_JSONsettings.json: json file containing meta data *_Settings.txt: txt file containing meta data *_slicelist.sqlite3: database file containing the slice list *_TargetPositions.csv: csv file containing target positions #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*: Actin: folder containing image data of the actin channel actin_mask: folder containing masks for the lumen and outer edges DNA: folder containing image data of the nuclei channel HaloLigand: folder containing image data of the raw PIEZO1 channel results: folder containing csv files obtained from performing puncta localization and tracking using ThunderSTORM settings_info: folder containing information on data acquisition process #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*/Actin: *.tif: image data of the actin channel #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*/actin\_mask: *_lumen_edge.tif: lumen edge mask *_outer_edge.tif: outer edge mask #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*/DNA: *.tif: image data of the nuclei channel #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*/HaloLigand: *.tif: image data of the raw PIEZO1 channel #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*/results: *_locs.csv: csv file containing information on all the localizations obtained using ThunderSTORM *_FinalTracks_filtered.csv: csv file containing information on the final tracks after filtering out autofluorescence blobs #### three\_plane\_stack\_data/NonBaptaWT/140um/2023/\*/exp\_\*/settings\_info: *_JSONsettings.json: json file containing meta data *_Settings.txt: txt file containing meta data *_slicelist.sqlite3: database file containing the slice list *_TargetPositions.csv: csv file containing target positions #### volumetric\_data (after decompressing the zip file): KO: folder containing 3d volumetric data for knockout sample WT: folder containing 3d volumetric data for wild-type sample PSF: folder containing point spread function (PSF) images needed for point detection, and deconvolution #### volumetric\_data/KO/exp\_\*: Actin: folder containing image data of the actin channel DNA: folder containing image data of the nuclei channel HaloLigand: folder containing image data of the raw PIEZO1 channel along with puncta detection results settings_info: folder containing information on data acquisition process #### volumetric\_data/KO/exp\_\*/Actin: DS: folder containing deskewed image of the actin channel *.tif: image data of the actin channel in skewed space #### volumetric\_data/KO/exp\_\*/Actin/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed image of the actin channel *.tif: deskewed image data of the actin channel #### volumetric\_data/KO/exp\_\*/Actin/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed image of the actin channel #### volumetric\_data/KO/exp\_\*/DNA: DS: folder containing deskewed image of the nuclei channel *.tif: image data of the nuclei channel in skewed space #### volumetric\_data/KO/exp\_\*/DNA/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed image of the nuclei channel *.tif: deskewed image data of the nuclei channel #### volumetric\_data/KO/exp\_\*/DNA/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed image of the nuclei channel #### volumetric\_data/KO/exp\_\*/HaloLigand: DS: folder containing deskewed image of the raw PIEZO1 channel results: folder containing mat file obtained from performing puncta localization using LLSM5DTools ([https://github.com/abcucberkeley/LLSM5DTools/tree/dev](https://github.com/abcucberkeley/LLSM5DTools/tree/dev)) on the deskewed data *.tif: image data of the raw PIEZO1 channel in skewed space #### volumetric\_data/KO/exp\_\*/HaloLigand/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed image of the raw PIEZO1 channel *.tif: deskewed image data of the raw PIEZO1 channel #### volumetric\_data/KO/exp\_\*/HaloLigand/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed image of the raw PIEZO1 channel #### volumetric\_data/KO/exp\_\*/HaloLigand/results: Detection3D.mat: mat file containing information on all the localizations obtained using LLSM5DTools #### volumetric\_data/KO/exp\_\*/settings\_info: v_JSONsettings.json: json file containing meta data v_Settings.txt: txt file containing meta data v_slicelist.sqlite3: database file containing the slice list v_TargetPositions.csv: csv file containing target positions #### volumetric\_data/WT/exp\_\*: Actin: folder containing image data of the actin channel DNA: folder containing image data of the nuclei channel HaloLigand: folder containing image data of the raw PIEZO1 channel along with puncta detection results settings_info: folder containing information on data acquisition process #### volumetric\_data/WT/exp\_\*/Actin: DS: folder containing deskewed image of the actin channel matlab_decon: folder containing results obtained using RL deconvolution *.tif: image data of the actin channel in skewed space #### volumetric\_data/WT/exp\_\*/Actin/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed image of the actin channel *.tif: deskewed image data of the actin channel #### volumetric\_data/WT/exp\_\*/Actin/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed image of the actin channel #### volumetric\_data/WT/exp\_\*/Actin/matlab\_decon: DS: folder containing deskewed and deconvolved image of the actin channel psfgen: folder containing preprocessed PSF (removing background and empty regions) as input for the deconvolution program MIPs: folder containing maximum intensity projection (MIP) of the deconvolved image of the actin channel in skewed space *.tif: deconvolved image of the actin channel in skewed space #### volumetric\_data/WT/exp\_\*/Actin/matlab\_decon/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed and deconvolved image of the actin channel *.tif: deskewed and deconvolved image of the actin channel #### volumetric\_data/WT/exp\_\*/Actin/matlab\_decon/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed and deconvolved image of the actin channel #### volumetric\_data/WT/exp\_\*/Actin/matlab\_decon/psfgen: 560_CamA_ch0.tif: preprocessed PSF (removing background and empty regions) as input for the deconvolution program #### volumetric\_data/WT/exp\_\*/Actin/matlab\_decon/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deconvolved image of the actin channel in skewed space #### volumetric\_data/WT/exp\_\*/DNA: DS: folder containing deskewed image of the nuclei channel denoised: folder containing deskewed image of the nuclei channel, which has been denoised using a CARE model ([https://github.com/abcucberkeley/piezo1_analysis_pipeline/tree/main/nuclei_denoising_model](https://github.com/abcucberkeley/piezo1_analysis_pipeline/tree/main/nuclei_denoising_model)) *.tif: image data of the nuclei channel in skewed space #### volumetric\_data/WT/exp\_\*/DNA/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed image of the nuclei channel *.tif: deskewed image data of the nuclei channel #### volumetric\_data/WT/exp\_\*/DNA/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed image of the nuclei channel #### volumetric\_data/WT/exp\_\*/DNA/denoised: *.tif: deskewed and denoised image data of the nuclei channel #### volumetric\_data/WT/exp\_\*/HaloLigand: DS: folder containing deskewed image of the raw PIEZO1 channel results: folder containing mat file obtained from performing puncta localization using LLSM5DTools ([https://github.com/abcucberkeley/LLSM5DTools/tree/dev](https://github.com/abcucberkeley/LLSM5DTools/tree/dev)) on the deskewed data *.tif: image data of the raw PIEZO1 channel in skewed space #### volumetric\_data/WT/exp\_\*/HaloLigand/DS: MIPs: folder containing maximum intensity projection (MIP) of the deskewed image of the raw PIEZO1 channel *.tif: deskewed image data of the raw PIEZO1 channel #### volumetric\_data/WT/exp\_\*/HaloLigand/DS/MIPs: *_MIP_z.tif: maximum intensity projection (MIP) of the deskewed image of the raw PIEZO1 channel #### volumetric\_data/WT/exp\_\*/HaloLigand/results: Detection3D.mat: mat file containing information on all the localizations obtained using LLSM5DTools #### volumetric\_data/WT/exp\_\*/settings\_info: v_JSONsettings.json: json file containing meta data v_Settings.txt: txt file containing meta data v_slicelist.sqlite3: database file containing the slice list v_TargetPositions.csv: csv file containing target positions #### volumetric\_data/PSF: DS: folder containing deskewed images of the point spread function (PSF) needed for point detection, and deconvolution *.tif: images of the point spread function (PSF) in skewed space settings_info: folder containing information on data acquisition process #### volumetric\_data/PSF/DS: *.tif: deskewed images of the point spread function (PSF) #### volumetric\_data/PSF/settings\_info: *_JSONsettings.json: json file containing meta data *_Settings.txt: txt file containing meta data *_TargetPositions.csv: csv file containing target positions ## Code/Software The data were processed and analysed with LLSM5DTools ([https://github.com/abcucberkeley/LLSM5DTools/tree/dev](https://github.com/abcucberkeley/LLSM5DTools/tree/dev)) and [https://github.com/abcucberkeley/piezo1_analysis_pipeline.git](https://github.com/abcucberkeley/piezo1_analysis_pipeline.git) to generate the plots in the paper.