Standard Operating Procedure (SOP) for Quantifying Collagen Fibers after Masson's Trichrome Staining

The quantification of collagen fibers following Masson's trichrome staining is a critical procedure in histopathological analysis, especially for the study of fibrotic conditions. Fibrosis is a pathological feature of numerous chronic diseases, marked by the excessive production and deposition of extracellular matrix components, such as collagen, in various organs including the lungs. The progression of fibrosis can severely impair organ function, making it crucial to quantify the extent of fibrosis for evaluating potential treatments, understanding disease mechanisms, and predicting outcomes. For instance, in pulmonary fibrosis, the quantification of collagen in lung tissue can help assess the severity of the disease and monitor the response to antifibrotic therapies. Similarly, in liver cirrhosis, measuring collagen deposition can aid in evaluating the progression of liver damage. Thus, accurate quantification of collagen fibers is invaluable across a range of clinical and research settings.
 

Preparation and Image Acquisition:

Open ImageJ Software:

• Launch the ImageJ software on your computer.

Open Acquired Image:

• Navigate to File -> Open or use the shortcut Ctrl+O to open the image acquired using a confocal microscope at a magnification of 10x.

Set Image Scale:

• Draw a line on the scale bar present in the image.
• Go to Analyze -> Set Scale.
  
  
  • In the "Known Distance" field, enter the real distance as indicated on the scale bar.
  • Enter the unit of length (e.g., µm).
    
    

Color Deconvolution and Threshold Setting:

Download Color Deconvolution Plugin:

• Install the color deconvolution plugin from the provided link. 

Apply Color Deconvolution:

• Upon applying the color deconvolution plugin, three different images corresponding to three different channels will be generated. Use the green channel for collagen quantification. Plugins -> Scripts -> Color Deconvolution.
  
  
  
  

Set Threshold:

• Select a threshold for the green channel by using the shortcut Ctrl+Shift+T.
• Apply the same threshold settings to all images to maintain consistency.
  
  

Region of Interest (ROI) Selection and Area Measurement:

Create and Save ROI:

• Use the rectangle or circle tool to create a Region of Interest (ROI) where you want to measure the collagen fibers.
• Save the created ROI using Analyze -> Tools -> ROI Manager.
  
  

Measure Area:

• With the ROI selected, go to Analyze -> Measure or use the shortcut Ctrl+M to measure the area of the collagen fibers within the ROI.
  
  

Repeat Measurements:

• Perform measurements in different spots within each image to ensure a more accurate analysis of the collagen fibers.

Documentation and Analysis:

• Record Measurements:
  • Document the measured areas from each ROI and calculate the average to estimate the collagen density in the tissue sample.
• Comparative Analysis:
  • Compare the quantified collagen fiber areas across different samples to assess fibrosis severity or treatment efficacy.

Research and Publication Reference:

This SOP is designed based on the methods outlined in specific research papers. When utilizing this protocol, it's important to cite the original research that has employed this method for collagen quantification. Additionally, incorporating a flowchart from a published paper, that summarizes the steps can enhance the understanding and reproducibility of the protocol.



Note: It is crucial to always ensure that the same scale (e.g., 1.3 pixels/µm) and threshold values (e.g., min 235 and max 255) are used consistently across all images to maintain accuracy in quantification.