How to calculate fluorescence intensity using ImageJ (Image Processing Software) for biliary anatomy delineation?

How to Calculate Fluorescence Intensity Using ImageJ for Biliary Anatomy Delineation

For accurate biliary anatomy delineation using fluorescence imaging, semi-quantification of fluorescence signal using ImageJ software is recommended to standardize measurements and improve reproducibility.

Basic Principles of Fluorescence Quantification

• Fluorescence imaging allows real-time visualization of biliary structures during surgery, but quantification is essential for objective assessment and standardization 1
• Semi-quantification (rather than absolute quantification) is currently the most practical approach for clinical applications 1
• ImageJ software provides accessible tools for standardized fluorescence intensity measurement 2

Step-by-Step Process for Fluorescence Intensity Calculation

Image Acquisition and Preparation

• Import fluorescence images into ImageJ software 1
• Include both fluorescence images and corresponding bright field images for reference 1
• Ensure consistent imaging parameters (distance, exposure time) between compared images 1

Region of Interest (ROI) Selection

• Define regions of interest (ROIs) around target structures (bile ducts) 2
• Select additional ROIs for background tissue (liver) and surrounding fat for reference 2
• Use consistent ROI sizes when comparing multiple images 1

Intensity Measurement

• Measure mean fluorescence intensity (MFI) within each ROI 1
• Record raw numeric values for each structure (bile duct, liver, background) 1
• Calculate signal-to-background ratios using the formula: MFI of bile duct/MFI of background tissue 2

Standardized Reporting

• Calculate bile duct-to-liver fluorescence intensity ratio 2
• Calculate bile duct-to-background fat fluorescence intensity ratio 2
• Higher ratios indicate better biliary visualization (optimal ratios: bile duct-to-liver >3.0; bile duct-to-background >7.0) 2

Advanced Analysis Techniques

Surface Area Adjustment

• For more accurate quantification, adjust measurements for surface area using the formula: MFIcorrected = fluorescence intensity of tissue of interest / (injected dose/surface area of the tissue) 1
• This approach is similar to SUV calculations in PET imaging but adapted for fluorescence 1

Heat Map Generation

• Create pseudo-colored heat maps in ImageJ to visualize intensity differences on a color spectrum 1
• Use consistent color scales when comparing multiple images 1

Clinical Applications and Optimization

Timing Considerations

• For optimal bile duct visualization, analyze images taken at least 90 minutes after ICG injection 3
• Longer intervals between ICG injection and imaging (median 90 minutes) significantly improve bile duct detection compared to shorter intervals (median 47 minutes) 3

Dosage Optimization

• Low-dose ICG (0.05 mg) provides significantly higher bile duct-to-liver fluorescence intensity ratios compared to standard-dose (2.5 mg) 2
• Low-dose protocol minimizes background liver fluorescence, improving contrast 2

Common Pitfalls and Solutions

• Variable tissue depth: Fluorescence signal decreases with increasing tissue depth; maintain consistent laparoscope distance from target structures 3
• Inconsistent ROI selection: Use anatomical landmarks to ensure consistent ROI placement across images 1
• Background fluorescence interference: Always subtract background fluorescence from measurements 2
• Equipment variability: Different imaging systems produce different signal contrasts; maintain consistency in equipment when comparing results 3

By following these standardized methods for fluorescence intensity calculation using ImageJ, surgeons can objectively assess biliary anatomy delineation, potentially reducing the risk of bile duct injuries during laparoscopic procedures 4.