May 20, 2025
Pipeline for Image Processing and Quantification of Striatal Dopamine Innervation using Fiji (IF/IHC)
- James Lyman1,
- Natalia Lopez-GonzalezdelRey1
- 1Northwestern University
Protocol Citation: James Lyman, Natalia Lopez-GonzalezdelRey 2025. Pipeline for Image Processing and Quantification of Striatal Dopamine Innervation using Fiji (IF/IHC). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qojpl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 09, 2024
Last Modified: May 20, 2025
Protocol Integer ID: 99543
Keywords: ASAPCRN, striatal innervation, dopaminergic innervation, striatal quantification, quantification of striatal dopamine innervation, striatal dopamine innervation, quantifying striatal innervation, striatal innervation, measuring mean fluorescence intensity, mean fluorescence intensity, th immunofluorescence, fluorescence, immunofluorescence, imagej pipeline, image processing, imagej tool, analysis with imagej tool, th immunofluorescence coronal section, pipeline for image processing, image
Funders Acknowledgements:
Align Science Across Parkinson's
Grant ID: ASAP
Disclaimer
This is a method adapted from ImageJ available tools.
Abstract
Method for quantifying striatal innervation by measuring mean fluorescence intensity using ImageJ. This protocol outlines the image preprocessing steps required prior to analysis with ImageJ tools as well as the ImageJ pipeline to quantify striatal innervation. Here DAT and TH immunofluorescence coronal sections but this method can be adapted for other markers and XYZ planes.
Troubleshooting
Obtaining & Opening Image Files
Obtain single channel TIFF images for desired brain section and desired channels for analysis.
Obtain Allen Atlas TIFF image files that correspond with the Bregma distance of the brain section levels to be analyzed.
Open fully updated version of FIJI ImageJ2 (version 2.14.0 used for this analysis).
Scale the Atlas TIFF image to be just smaller than smallest brain section TIFF to be warped (path: Image>Adjust>Size...). This limits the amount of stretching that occurs from warping later on. (For example: if the smallest brain section image is 12500x13500 pixels, make the atlas TIFF image 12000x13000pixels.
Open Allen Atlas TIFF image and corresponding TIFF image of the fluorescent channel to be warped.
Processing & Warping Image Files
Using the Brightness/Contrast feature (path: Image>Adjust>Brightness/Contrast), adjust the brightness of the section such that key features are clearly visible, including background features. This often requires increasing the brightness.
Once adjusted, do not select “Apply” or “Set” as this will change the raw pixel values. Instead, click off the Brightness/Contrast window.
Navigate to the BigWarp function (path: Plugins>BigDataViewer>Big Warp). This will open up a “Big Warp Setup” window.
Select the previously brightened TIFF image of the brain to be warped as the “moving image.”
Select the scaled TIFF file of the Allen Atlas as the “target image.”
Drag and drop a .csv file into the “Landmarks file” box if a landmarks file has been previously generated. Leave blank to create landmarks as described in step 7.
Uncheck the “apply transform from landmarks” box.
Enable “landmark mode” by pressing the spacebar. Place landmarks on key anatomical structures in the Atlas image window.
Once placed export the landmark files if repeated and consistent warping is intended (path: File>Export Landmarks).
After placing all the landmark points on the Atlas image, open the “Landmarks” window (which opens automatically along with the two target and moving images). Select the first point, this should highlight the first point placed on the target (Atlas) image. Next, place a point in the moving image (the brain section) at the corresponding location. Repeat for all landmark points.
Warp the target image (the brain section TIFF) by pressing “T”.
Export the warped target image as a TIFF file (path: File>Export Moving Image).
Open the Brightness/Contrast feature (path: Image>Adjust>Brightness/Contrast) and select “Reset” to adjust the brightness settings back to the original values. Save the image as a new TIFF file.
Analysis of Warped Images
Open the scaled TIFF file of the Allen Atlas image corresponding
with the brain section to be analyzed
Draw desired Region(s) Of Interest (abbreviated: “ROI”) on the
Atlas image, add them to the ROI Manager (path for manager: Analyze>Tools>ROI
Manager).
Include square ROI(s) of same size for
background sampling and subtraction in data analysis. (For example: 150x150
pixels, total area of 22500 pixels)
Open the warped TIFF file of the warped fluorescent
channel for analysis.
Adjust the ROIs drawn on the Atlas image to fit the exact borders of the brain section (if warped correctly, very minor or no adjustments should be needed).
Regions "I936_R_sox6+" and "I936_R_sox6-" were drawn first on Atlas image corresponding to the Bregma level of this section, then adjusted as needed to fit the proper outline of the warped image. "I936_ACbgr", "I936_CCbgr", and "I936_CXbgr" are 150x150 pixel square ROIs placed in the anterior commissure (AC), corpus callosum (CC), and cortex (CX) respectively in order to measure the background intensity at these locations.
Set the measurements to be taken by accessing
the “Set Measurement…” feature (path: Analyze>Set Measurements…).
For example:
Using the ROI Manager with the warped fluorescent
channel image selected, select the ROIs to be measured and click “measure” in
the ROI Manager. Repeat for all ROIs to be measured. A “Results” window
will be generated containing the resulting measurement data.
Take note of the order in which each ROI is measured as the resulting data will not include the name of the ROI in which it originates from but only a number corresponding to the order in which it was measured. (For example: for the image of mouse brain I936 included above, the ROIs were measured in the following order: Sox6+, Sox6-, ACbgr, CCbgr, CXbgr. This order was maintained throughout the analysis of all images to simplify data analysis in later steps.)
Open the “Results” window, save the file as a .csv
file (path: File>Save As…)
Open the saved .csv file and transfer data to a
Microsoft Excel spreadsheet for data analysis.