Access Dataset on Dryad

Overview

This dataset contains 4 types of data: two-photon imaging (2-photon) fluorescence data, Forster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) data, Western blot data, and immunohistochemistry (IHC) fluorescence data.

2-photon fluorescence data was collected using a custom 2-photon microscope. Green fluorescence was acquired in channel 1 and red was acquired in channel 2. Analysis was performed using custom MATLAB software.

FRET-FLIM data was collected using a custom-built 2-photon microscope with time correlated single photon counting. Lifetime data is stored in channel 1 and the fluorescence intensity from a red cell fill is stored in channel 2. Data was analyzed using custom MATLAB software.

IHC fluorescence data was collected using a Zeiss Apotome 3 structured illumination microscope. Data was captured from blue, green, and red wavelength channels and stored in a single z-stack image. Data was deconvolved in Zeiss ZenPro software.

Western blot data was collected using a LI-COR Odyssey CLx blot imager. Fluorescence data was acquired from 700nm and 800nm wavelength channels and stored separately.

Description of the data and file structure

We have provided our raw data in the “PetshowEtAl_2025_RawData.zip” file and our analysis files in the “PetshowEtAl_2025_Analysis.zip” file. These two folders are sub-divided into files for each individual figure.

Raw data:

Folders for Figures 4 & 5 contain data from 2-photon imaging experiments. These Figure folders are separated into folders containing individual cells folders, under ‘Cells.’ Cell folders are named with the following convention: [initials, cell number]. Each cell folder contains all of the images for that cell with the image name convention: [initials, cell number, dendrite identifier (a or b)]. Z-stacks and maximum projection images are both included and stored in .tif format. A zoomed out image of the whole cell is included in .tif format with the name ‘m’ in place of the dendrite identifier. Cell folders may also contain files with microscope stage position data (.pos files) and screenshots taken during the experiment (.bmp or .jpg files).

Folders for Figures 1, 3, 4, & 5 contain data from 2-photon FRET-FLIM experiments. These Figure folders are separated into folders (‘Cells’) containing individual cells folders with the naming convention: [initials, cell number]. The cell folder contains the raw fluorescence data from channel 2 (red) and a sub-folder named “spc” which contains the raw single photon counting data per pixel. Both are stored in .tif format and use the naming convention [initials, cell number, dendrite identifier (a or b)]. The “spc” folders also include .mat files used to determine whether enough photons were collected. Cell folders may also contain files with microscope stage position data (.pos files) and screenshots taken during the experiment (.bmp or .jpg files).

Folders for Figures 1, 2, & 3 contain data from Western blot experiments. These Figure folders are separated into folders containing individual blots, under ‘Blots.’ Blot folders are named with the following convention: [date of acquisition, experiment/stain used]. Each blot folder contains a single raw image of the blot, stored in .tif format, with the naming convention [arbitrary number string, wavelength of acquisition (700 or 800)]. Blot folders also contain a .txt file with metadata on the instrument settings used to acquire the image, and low-resolution summary image of the blot as a .jpg.

Folders for Figures 3 & 4 contain data from IHC experiments. These Figure folders are separated into folders containing IHC experiments, under ‘IHC.’ IHC folders are named with the following convention: [date of acquisition, experiment/stain used]. Each IHC folder contains a single raw images taken from a single experiment, stored in .czi format, with the naming convention [experiment, stain, cell number].

Analysis:

Folders for Figures 4 & 5 contain analysis files for 2-photon imaging experiments. These Figure folders contain a ‘Cell ANNs’ folder and a ‘Cell Excels’ folder. Cell ANNs folders contain .ann files and .csv files. The .ann files store integrated fluorescence data for green and red fluorescence (channel 1 and channel 2, respectively) and the positions of the analyzed ROIs. These have the naming convention [initials, cell number, dendrite identifier (a or b)]. The .csv files store the integrated background subtracted fluorescence for the analyzed spines with the naming convention [initials, cell number, dendrite identifier (a or b)]. Their data structure is: individual ROIs are separated by row, and for each row/ROI, column entries for the mean background pixel value, integrated pixel density, and pixel area of the ROI (‘Background, ‘Integral’ ‘IntegralPixelCount’) exist in triplets for each timepoint/image in the series. The Cell Excels folders contain .xls files which organize and display the .ann and .csv data for each cell in a user-friendly way. Their data structure is: rows display the normalized fluorescence values for target ROIs or dendrites, columns display the timepoint. These files contain figures not necessary for reanalysis of the data. They have the naming convention [initials, cell number, dendrite identifier (a or b)].

Folders for Figures 1, 3, 4 & 5 contain analysis files for FRET-FLIM experiments. Figure folders contain a ‘Cell MATs’ folder and a ‘Cell Excels’ folder. Cell MATs folders contain .MAT files which store data detailing the ROI position, the average lifetime (tau_m) and the red fluorescence intensity (int_int2). These .MAT files are named with the following convention:[“A_”, initials, cell number, dendrite identifier (a or b), “_ROI2”]. The Cell Excels folders contain .xls files which organize and display the .MAT data for each cell in a user-friendly way. Their data structure is: time points are separated by row, columns indicate values obtained from an ROI at each timepoint, including background subtracted red fluorescence intensity, green fluorescence intensity, calculated bound fraction of the FRET sensor, and fluorescence lifetime (‘red_int2,’ ‘int_int2,’ ‘fraction2,’ ‘tau_m’). These files contain figures not necessary for reanalysis of the data. They have the naming convention [initials, cell number, dendrite identifier (a or b)].

All Figure folders contain summary .xlsx files where normalization and averaging of spine size or average lifetime across cells were performed, or averaging of western blot band intensity, or quantification of IHC data. Their structure varies but is labeled in header information. These files contain figures not necessary for reanalysis of the data.

All figure folders contain .prism files where statistical testing was performed on the datasets contained within the summary .xlsx files

Usage Notes

.tif and .czi images can be opened using most image analysis softwares (e.g. ImageJ)
.MAT and .ann files can be opened using MATLAB
.xls and .xlsx files can be opened using Microsoft Excel
.prism files must be opened with GraphPad Prism