CMM Lab - Collagen Fibril Orientation Analyser for SHG images

CMM Lab website Homepage

CMM Lab GitHub Homepage

---

ImageJ / FIJI script designed to automate the quantification of pixel orientation of two-photon second harmonic generation images of collagen fibres.

Requires ImageJ/FIJI 1.38m or above.
Tested working on Mac OSX 10.14 and above.
Tested working on Windows 10.

Last updated: 2nd Oct 2020.

---

Description:
This script wraps the B.I.G. OrientationJ plugin into a batch process that applies user-defined pre-processing and iterates through entire directories (including all sub-directories) of images outputting the colour survey overlay and text file of histogram values for each image.

If using multiple sub-directories of images, please ensure that filenames are unique.

### This scripts requires the OrientationJ plugin to run ###
See https://github.com/Biomedical-Imaging-Group/OrientationJ

Briefly, the orientation is evaluated for every pixel of the image based on the structure tensor. A histogram of orientations is built taking into account pixels that have a coherency larger than 'min-coherency' and an energy larger than 'min-energy'.

Citation for original OrientationJ plugin:
Rezakhaniha et al. Biomech. Model Mechanobiol. (2012)
Experimental Investigation of Collagen Waviness and Orientation in the Arterial Adventitia
doi: 10.1007/s10237-011-0325-z
PubMed link

Citation for script:
Mayorca-Guiliani AE et al. Nature Medicine (2017)
ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix
doi: 10.1038/s41467-019-10968-6
Pubmed link

All processed images should be taken using identical acquisition parameters

---

Installation

Ensure you have ImageJ or FIJI (preferred) installed.

• ImageJ is available from here.
• FIJI is available from here.

The B.I.G. OrientationJ tools are also required.
These are available from https://github.com/Biomedical-Imaging-Group/OrientationJ

Copy the CMM_Fibril_Orientation_Analyser.ijm to the ImageJ/FIJI plugins directory.

Restart ImageJ/FIJI.

The script should now appear in the Plugins Dropdown menu.

---

Basic variables specified by the user include:

Upon launching, the script will ask for the following inputs:

• File Type - Specify input image file type (.tif .jpg etc.)*
• Tensor - Minimum structure tensor - Passed to OrientationJ
• Image pre-processing (Enable/Disable) - Select image pre-processing options.
• Advanced Options (Enable/Disable) - Opens Advanced Options control panel for user input
• Enable Batch Mode (Enable/Disable) - Runs the script silently (faster)
• Launch Memory Monitor (Enable/Disable) - Mainly for debugging

*Output colour overlay files are saved in .png format.
Avoid using input files in the .png format where possible.

---

Image pre-processing steps (if enabled) include:

• 8-bit Conversion - Converts each image to 8-bit
• Brightness Threshold - Minimum brightness threshold
• Sharpen (Enable/Disable) - Runs the ImageJ/FIJI sharpen command prior to analysis
• Despeckle (Enable/Disable) - Runs the ImageJ/FIJI despeckle command prior to analysis
• Auto Enhance Output Image (Enable/Disable) - Enhance Brightness/Contrast of Output images only (does NOT affect analysis - aesthetics only)

---

Advanced variables specified by the user (if enabled) include:

If enabled in the Basic Parameter window, the script will also launch an input window allowing for customisation of analysis parameters which are passed directly to the OrientationJ plugin:

• Gradient - Gradient used (0: Cubic Spline, 1: Finite difference, 2: Fourier; 3: Riesz, 4: Gaussian)
• Min Coherency - Minimum Coherency - The ratio between the difference and the sum of the tensor eigenvalues
• Min Energy - Minimum Energy value - The energy parameter is the trace of the tensor matrix

---

Once basic and advanced options have been chosen, you will be asked to specify the top level input directory containing the image files to be analysed.

---

Output Image files

1. The script will output colour survey overlays (in .png format) of each of the analysed images, in the originating directory.

2. It will also output a saved version of the pre-processed image file in .png format in the originating directory.

(The original image always remains unchanged)

---

Output text files

The analysis will output two text files in the top level directory:

1. Parameters.txt - Contains a list of all the parameters used in the analysis, along with a list of successfully analysed image files.

2. Orientation_Results.txt - Results file containing all of the collated orientations for each file analysed.

---