DOI: 10.5061/dryad.tqjq2bw90
title: Data from: Three-photon in vivo imaging of neurons and glia in the medial prefrontal cortex with sub-cellular resolution

description: The medial prefrontal cortex (mPFC) is important for higher cognitive
 functions, including working memory, decision making, and emotional
 control. In vivo recordings of neuronal activity in the mPFC have been
 achieved via invasive electrical and optical approaches. Here we apply low
 invasive three-photon in vivo imaging in the mPFC of the mouse at
 unprecedented depth. Specifically, we measure neuronal and astrocytic
 Ca2+-transient parameters in awake head-fixed mice up to a depth of 1700
 µm. Furthermore, we longitudinally record dendritic spine density (0.41
 ±0.07 µm-1) deeper than 1 mm for a week. Using 1650 nm wavelength to
 excite red fluorescent microglia, we quantify their processes’ motility
 (58.9 ±2% turnover rate) at previously unreachable depths (1100 µm). We
 establish three-photon imaging of the mPFC enabling neuronal and glial
 recordings with subcellular resolution that will pave the way for novel
 discoveries in this brain region. 
description: Deep overview <em>in vivo</em> z-stacks were recorded
 with depth increments of 1-10 µm, 0.2-0.65 µm/pixel resolution and 2-3 µs
 pixel dwell time. Z-stacks (spanning 10-30 µm) of dendritic spines on
 basal dendrites of mPFC LV/VI neurons were imaged in 900-1100 µm depth
 with 1 µm depth increments, 0.15 µm/pixel resolution and 2 µs pixel dwell
 time. For the measurement of microglial fine process motility, z-stacks of
 individual microglia were imaged with 2-3 µm depth increments, 0.16
 µm/pixel resolution, 2 µs pixel dwell time and with 5-10 min
 time-intervals for a period of 30 minutes. Timelapse imaging of astrocytic
 Ca<sup>2+</sup>-activity in mPFC in vivo was performed at 3 Hz
 frame rates with 1 µm/pixel resolution and 2 µs pixel dwell time. Imaging
 the Ca<sup>2+</sup>-activity of layer 5 excitatory neurons in
 mPFC of awake mice was performed at ≥10 Hz frame rates with 1-2 µm/pixel
 resolution and 2 µs pixel dwell time. Recordings of the
 Ca<sup>2+</sup>-activity of dentate gyrus granule cells in the
 hippocampus of awake mice was performed at 5-10 Hz frame rates with
 0.5-1.5 µm/pixel resolution and 2 µs pixel dwell time. Spinal cord in vivo
 z-stacks were recorded with depth increments of 3 µm, 0.25 µm/pixel
 resolution and 2-3 µs pixel dwell time.
description: # Data from: Three-photon in vivo imaging of neurons and glia in the
 medial prefrontal cortex with sub-cellular resolution
 [https://doi.org/10.5061/dryad.tqjq2bw90](https://doi.org/10.5061/dryad.tqjq2bw90) ## Description of the data and file structure This dataset contains the raw (tif-files for each individual channel) and processed imaging files (mp4-file or avi-file) presented in the manuscript COMMSBIO-24-5589 and was collected using 3P and 2P microscopy. The MP4/AVI files are compressed video files of the TIFF files. In addition, the MP4/AVI files contain a scale bar and a marker for the imaging depth [µm] within the 3D volume of the image stack. Time-lapse videos contain a scale bar and a marker for the elapsed time [sec] or [min]. The MP4 file of video11 contains the green fluorescent channel (GCaMP) and the red fluorescent channel (tdTomato), and the overlay channel next to each other. The corresponding TIF files contain the separate channels. The avi-file of video 9 contains the same microglia imaged on two consecutive days (d0 and d1). The two recordings are placed next to each other in the AVI file. The corresponding TIF files contain the two separate recordings. The data are a sequence of individual 2D (x,y-dimension) images along the z-axis of the microscope (3D-imaging volume) or a 2D time-lapse (2D-images at the same position recorded over time. The number of images within one TIF file corresponds to the number of steps along the z-axis or the number of time-lapse images. The spatial spacing along the z-axis and the temporal spacing in time are described below for each file. In addition, the pixel size in 1/µm is described for each tif-file enabling exact 2D size measurements. Deep overview *in vivo* z-stacks were recorded with depth increments of 1-10 µm, 0.2-0.65 µm/pixel resolution, and 2-3 µs pixel dwell time. Z-stacks (spanning 10-30 µm) of dendritic spines on basal dendrites of mPFC LV/VI neurons were imaged in 900-1100 µm depth with 1 µm depth increments, 0.15 µm/pixel resolution, and 2 µs pixel dwell time. For the measurement of microglial fine process motility, z-stacks of individual microglia were imaged with 2-3 µm depth increments, 0.16 µm/pixel resolution, 2 µs pixel dwell time, and with 5-10 min time intervals for a period of 30 minutes. Timelapse imaging of astrocytic Ca2+-activity in mPFC in vivo was performed at 3 Hz frame rates with 1 µm/pixel resolution and 2 µs pixel dwell time. Imaging the Ca2+-activity of layer 5 excitatory neurons in mPFC of awake mice was performed at ≥10 Hz frame rates with 1-2 µm/pixel resolution and 2 µs pixel dwell time. Recordings of the Ca2+-activity of dentate gyrus granule cells in the hippocampus of awake mice were performed at 5-10 Hz frame rates with 0.5-1.5 µm/pixel resolution and 2 µs pixel dwell time. Spinal cord in vivo z-stacks were recorded with depth increments of 3 µm, 0.25 µm/pixel resolution, and 2-3 µs pixel dwell time. ### Files and variables **Supplementary Video 1.** In-vivo 3P z-scan of 320 x-y frames from brain surface to 1600µm below, taken at a depth increment of 5µm in the mPFC of YFP-H transgenic mouse. **Supplementary Video 2.** In-vivo 2P mPFC z-scan with 920nm excitation in a YFP-H transgenic mouse.  **Supplementary Video 3.** In-vivo 3P mPFC z-scan with 1300nm excitation in a YFP-H transgenic mouse. **Supplementary Video 4.** In-vivo 3P Cortex to Hippocampus z-scan of 265 x-y frames from surface to 1325 µm below taken at a depth increment of 5 µm with 1300nm excitation in a GFP.M::Cx3cr1-CreER::Rosa25_tdTomato transgenic mouse. **Supplementary Video 5.** In-vivo 3P z-scan of 132 x-y frames from spinal cord surface to 390 µm below taken at a depth increment of 3 µm with 1300 nm excitation in a Thy1-GFP-M transgenic mouse. **Supplementary Video 6.** In-vivo 3P mPFC z-scan with 1300nm excitation in a Thy1-GFP-M transgenic mouse. **Supplementary Video 7.** In-vivo 3P mPFC z-scan with 1650nm excitation in a Cx3Cr1-creER2 Rosa tdTomato mouse on day 0. **Supplementary Video 8.** In-vivo 3P mPFC z-scan with 1650nm excitation in a Cx3Cr1-creER2 Rosa tdTomato mouse on day 1. **Supplementary Video 9.** Microglial fine process motility in the mPFC at d0 and d1. **Supplementary Video 10.** In-vivo 3P z-scan of 406 x-y frames from brain surface to 1200 µm below, acquired at a depth increment of 3 µm in the mPFC of a GLAST-CreERT2::GCaMP5g::tdTomato transgenic mouse. **Supplementary Video 11.** In vivo 3P recording of GCaMP5g-positive astrocytes at 1000 µm below the surface. GCaMP (green channel), tdTomato (magenta channel), merge. **Supplementary Video 12.** In-vivo 3P functional imaging of the *Drosophila* Mushroom body with intact cuticle. **Supplementary Video 13.** In-vivo 3P z-scan from brain surface to 1420µm below in a vGlut2-Cre mouse expressing GCaMP6s in glutamatergic neurons in the mPFC. **Supplementary Video 14.** In vivo 3P recording of GCaMP6s-positive glutamatergic neurons in the mPFC at a depth of 1100 µm. **Supplementary Video 15.** In-vivo 3P z-scan from SO to SG up to 700 µm deep into the dorsal hippocampus through a hippocampal window in a Thy1-GCaMP6f transgenic mouse.