DOI: 10.5061/dryad.b5mkkwhp5
title: Synergistic label-free fluorescence imaging and miRNA studies reveal dynamic human neuron-glial metabolic interactions following injury

description: Neuron-glial cell interactions following traumatic brain injury (TBI)
 determine the propagation of damage and long-term neurodegeneration.
 Spatiotemporally heterogeneous cytosolic and mitochondrial metabolic
 pathways are involved, leading to challenges in developing effective
 diagnostics and treatments. An engineered 3D brain tissue model comprising
 human neurons, astrocytes, and microglia is used in combination with
 label-free, two-photon imaging and microRNA studies to characterize
 metabolic interactions between glial and neuronal cells over 72 hours
 following impact injury. We interpret multi-parametric, quantitative,
 optical metabolic assessments in the context of microRNA gene set analysis
 and identify distinct metabolic changes in neurons and glial cells.
 Glycolysis, NADPH and glutathione synthesis, fatty acid synthesis, and
 oxidation are mobilized within glial cells to mitigate the impacts of
 initial enhancements in oxidative phosphorylation and fatty acid oxidation
 within neurons, which lack robust anti-oxidant defenses. This platform
 enables enhanced understanding of mechanisms that may be targeted to
 improve TBI diagnosis and treatment.
description: # Synergistic label-free fluorescence imaging and miRNA studies reveal
 dynamic human neuron-glial metabolic interactions following injury
 [https://doi.org/10.5061/dryad.b5mkkwhp5](https://doi.org/10.5061/dryad.b5mkkwhp5) ## Description of the data and file structure This dataset accompanies the paper titled *"Synergistic label-free fluorescence imaging and miRNA studies reveal dynamic human neuron-glial metabolic interactions following injury."* The dataset includes the following files: * **Code**: The key scripts used for data analysis and figure generation. * **Raw and Calibrated Two Photon Images**: The original two-photon fluorescence image TIFF files, as well as calibrated images saved in .mat format, which are used to generate all the figures. * **Raw miRNA Data**: The unprocessed miRNA results used to generate the results in the provided .xls file. These files have been uploaded to the Dryad/Zenodo databases. ### Files and variables #### File: Round1\_6h.zip **Description:** This ZIP file contains the raw TIFF images from the **Round 1, 6-hour experiment**, including both intensity and FLIM images. These are original images used for analysis. #### File: FLIM\_ExclusionMask\_R1\_6h.zip **Description:** This ZIP file contains manual exclusion masks for each FLIM ROI from the **Round 1, 6-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: FLIM\_ExclusionMask\_R1\_24h.zip **Description:** This ZIP file contains manual exclusion masks for each FLIM ROI from the **Round 1, 24-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: FLIM\_ExclusionMask\_R2\_6h.zip **Description:** This ZIP file contains manual exclusion masks for each FLIM ROI from the **Round 2, 6-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: FLIM\_ExclusionMask\_R2\_24h.zip **Description:** This ZIP file contains manual exclusion masks for each FLIM ROI from the **Round 2, 24-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: FLIM\_ExclusionMask\_R1\_72h.zip **Description:** This ZIP file contains manual exclusion masks for each FLIM ROI from the **Round 1, 72-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: ExclusionMask\_R2\_6h.zip **Description:** This ZIP file contains manual exclusion masks for each Intensity ROI from the **Round 2, 6-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: ExclusionMask\_R1\_6h.zip **Description:** This ZIP file contains manual exclusion masks for each Intensity ROI from the **Round 1, 6-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: ExclusionMask\_R1\_72h.zip **Description:** This ZIP file contains manual exclusion masks for each Intensity ROI from the **Round 1, 72-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: ExclusionMask\_R2\_24h.zip **Description:** This ZIP file contains manual exclusion masks for each Intensity ROI from the **Round 2, 24-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: ExclusionMask\_R2\_72h.zip **Description:** This ZIP file contains manual exclusion masks for each Intensity ROI from the **Round 2, 72-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: ExclusionMask\_R1\_24h.zip **Description:** This ZIP file contains manual exclusion masks for each Intensity ROI from the **Round 1, 24-hour experiment**. These masks confine the cellular regions to ensure no silk signal interference. #### File: FLIM\_Calibrated\_R2\_24h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated FLIM intensity images for each Intensity ROI from the **Round 2, 24-hour experiment**. #### File: FLIM\_Calibrated\_R1\_24h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated FLIM intensity images for each Intensity ROI from the **Round 1, 24-hour experiment**. #### File: FLIM\_Calibrated\_R1\_6h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated FLIM intensity images for each Intensity ROI from the **Round 1, 6-hour experiment**. #### File: FLIM\_Calibrated\_R1\_72h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated FLIM intensity images for each Intensity ROI from the **Round 1, 72-hour experiment**. #### File: FLIM\_Calibrated\_R2\_6h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated FLIM intensity images for each Intensity ROI from the **Round 2, 6-hour experiment**. #### File: Calibrated\_R1\_24h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated intensity images for each Intensity ROI from the **Round 1, 24-hour experiment**. #### File: Round2\_72h.zip **Description:** This ZIP file contains the raw TIFF images from the **Round 2, 72-hour experiment**, including both intensity and FLIM images. These are original images used for analysis. #### File: Round2\_24h.zip **Description:** This ZIP file contains the raw TIFF images from the **Round 2, 24-hour experiment**, including both intensity and FLIM images. These are original images used for analysis. #### File: Round2\_6h.zip **Description:** This ZIP file contains the raw TIFF images from the **Round 2, 6-hour experiment**, including both intensity and FLIM images. These are original images used for analysis. #### File: Round1\_72h.zip **Description:** This ZIP file contains the raw TIFF images from the **Round 1, 72-hour experiment**, including both intensity and FLIM images. These are original images used for analysis. #### File: Round1\_24h.zip **Description:** This ZIP file contains the raw TIFF images from the **Round 1, 24-hour experiment**, including both intensity and FLIM images. These are original images used for analysis. #### File: Calibrated\_R1\_6h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated intensity images for each Intensity ROI from the **Round 1, 6-hour experiment**. #### File: Calibrated\_R1\_72h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated intensity images for each Intensity ROI from the **Round 1, 72-hour experiment**. #### File: Calibrated\_R2\_6h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated intensity images for each Intensity ROI from the **Round 2, 6-hour experiment**. #### File: Calibrated\_R2\_72h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated intensity images for each Intensity ROI from the **Round 2, 72-hour experiment**. #### File: Calibrated\_R2\_24h.zip **Description:** This ZIP file contains illumination power/detector gain calibrated intensity images for each Intensity ROI from the **Round 2, 24-hour experiment**. ### Notes on Naming Conventions In the "ExclusionMask_xxx.zip" files, you will find various `.mat` files named according to the specific treatment, experiment, sample, and peri-injury location. For example, consider two files from the `ExclusionMask_R1_6h.zip`archive: * `ExcludeSilk_Ctrl6_NAM1_Peri_1.mat` * `ExcludeSilk_Inj6_NAM1_Peri_1.mat` Here’s how the naming convention works: * **Ctrl**: Represents the control condition. * **Inj**: Represents the injured condition. * **6**: Indicates that the sample comes from the 6-hour experiment. * **NAM**: Refers to the triculture tissue seeded with neurons (N), astrocytes (A), and microglia (M). * **1**: Refers to the first NAM tissue used in that experiment. * **Peri**: Indicates that the data corresponds to the peri-injury site (as shown in Fig. 1Ab). * **1**: Denotes the first peri-injury location of that NAM tissue. ### **Contents of .mat Files** Inside each `.mat` file, you will find multiple map containers. The key map containers include: * **ExcludeSilkDraw_container**: This container is used as input by the script `1_Int_Cell_Separation.m` to generate cell masks. * **Cell_anno_ima**: Contains the logical masks for the non-silk regions of the sample. * **Bright755_container**: Contains logical masks for apoptotic-body-like features, isolated from the "Cell_anno_ima." * **LipoMask_Container**: Contains logical masks for lipofuscin pixels. * **Cell_No_Lipo**: Provides cell masks excluding lipofuscin pixels. * **SHG_Masks**: Logical masks for collagen fibers that produce strong second harmonic generation signals. * **Overlapped8o7**: Logical masks of cellular pixels that overlap at 860 nm and 755 nm excitation wavelengths from the same volume. * **PixelRatio8o7**: The ratio of cellular pixel amounts between the two wavelengths from the same volume. The "ExclusionMask_xxx.zip" files are for intensity-based data, whereas the "FLIM_ExclusionMask_xxx.zip" files correspond to lifetime-based data. The difference is that the variables in the FLIM files have the "FLIM" prefix attached to each map. ### **Naming Conventions for Roundx\_xh.zip Folders** In the `Roundx_xh.zip` folders, you will find various `.tif` files representing different regions of interest (ROIs) captured at multiple time points and wavelengths. #### Example of File Naming for 1 ROI: For a specific region of interest (ROI), you may find files such as: * `Sequence 001_Int_ROI_Ctrl6_NAM1_Peri_1_EX_755_022_z00_RAW_ch00.tif` * `Sequence 002_Int_ROI_Ctrl6_NAM1_Peri_1_EX_755_086_z00_RAW_ch00.tif` * `Sequence 003_Int_ROI_Ctrl6_NAM1_Peri_1_EX_755_147_z00_RAW_ch00.tif` * `Sequence 001_Int_ROI_Ctrl6_NAM1_Peri_1_EX_860_023_z00_RAW_ch00.tif` * `Sequence 002_Int_ROI_Ctrl6_NAM1_Peri_1_EX_860_087_z00_RAW_ch00.tif` * `Sequence 003_Int_ROI_Ctrl6_NAM1_Peri_1_EX_860_148_z00_RAW_ch00.tif` #### Naming Breakdown: * **Sequence 001, 002, 003**: These numbers represent different time points in sequential order, with `001` being the initial time point and `003` representing the terminal time point (6 hours in this case). * **Int_ROI_**: Prefix indicating intensity images for a specific region of interest (ROI). * **Ctrl6_NAM1_Peri_1**: * **Ctrl**: Control condition. * **6**: 6-hour experiment. * **NAM**: Triculture tissue containing neurons (N), astrocytes (A), and microglia (M). * **1**: First NAM tissue used in the experiment. * **Peri**: Peri-injury site, referring to the sampling region. * **1**: First peri-injury location for that NAM tissue. * **EX_755** / **EX_860**: Represents the excitation wavelengths used, with `755 nm` or `860 nm`. * **022, 086, 147** / **023, 087, 148**: These are system-generated identifiers that increase sequentially for each time point. * **z00**: Represents the depth, starting from `z00` for the first optical section, with increasing values for subsequent sections. * **RAW**: Indicates raw, unprocessed data. * **ch00**: Channel number, ranging from 00 to 04: * **ch00**: Transmittance channel. * **ch01**: 525 nm emission channel. * **ch02**: 460 nm emission channel. * **ch03**: 560–700 nm emission channel. * **ch04**: 430 nm emission channel. * **Calibrated Images**: Power and gain-calibrated images are processed using the above mentioned RAW images and stored in `Calibrated_xxx.zip` via MATLAB for easy access.  ## Code/software To view and process the data, you will need the MATLAB software: * **MATLAB (Version R2023a or later)** – Used to run the image analysis pipeline. Key scripts include: **Intensity Data Pipeline (Starting after power/gain pre-processing):** 1. **1_Int_Cell_Separation.m** – Processes raw calibrated intensity images to generate neuron and glial cell masks. 2. **2_Int_Lipo_Apoptotic_Removal.m** – Removes lipofuscin and apoptotic signals from intensity data. 3. **3_Int_Redox_Beta_Lipo_Overall.m** – Extracts intensity-based parameters from all cell populations, including redox ratio, mitochondrial clustering, lipofuscin, and redox ratio distribution per region of interest (ROI). 4. **3_Int_Redox_Beta_Lipo_Neuron.m** – Extracts intensity-based parameters from neuron cells, including redox ratio, mitochondrial clustering, lipofuscin, and redox ratio distribution per ROI. 5. **3_Int_Redox_Beta_Lipo_Glial.m** – Extracts intensity-based parameters from glial cells, including redox ratio, mitochondrial clustering, lipofuscin, and redox ratio distribution per ROI. 6. **4_Int_RR_DistributionAnalysis.m** – Conducts distribution analysis of redox ratios to generate Low RR, Mid RR, and High RR component shapes and concentrations per ROI. **FLIM Data Pipeline (Starting after power/gain pre-processing):** 1. **1_FLIM_Cell_Separation.m** – Processes calibrated FLIM images to generate neuron and glial cells. 2. **2_FLIM_Lipo_Apoptotic_Removal.m** – Removes lipofuscin and apoptotic signals from FLIM data. 3. **3_FLIM_LifetimeAnalyses_Neuron.m** – Analyzes lifetime data specifically for neurons, including long lifetime, short lifetime, mean lifetime, and bound fraction. 4. **3_FLIM_LifetimeAnalyses_Glial.m** – Analyzes lifetime data specifically for glial cells, including long lifetime, short lifetime, mean lifetime, and bound fraction. 5. **3_FLIM_LifetimeAnalyses_Overall.m** – Analyzes overall lifetime data for all cell populations, including long lifetime, short lifetime, mean lifetime, and bound fraction. 6. **4_FLIM_AUCratioAnalysis.m** – Conducts area-under-the-curve ratio analysis for lifetime data, including Low G, Mid G, and High G shapes and concentrations. **Helper Functions:** * **HelperFunctions.zip** – Contains auxiliary functions used across the pipelines. ## Access information Other publicly accessible locations of the data: * n/a Data was derived from the following sources: * n/a