2) Calibrate scale
Draw over 2 μm bar → Analyze → Set Scale
Known distance = 2 μm; Pixel aspect = 1.0

3) Convert to 8-bit
Image → type → 8-bit

4) Optional preprocessing
Subtract Background (rolling ≈ 50 px)
(+ Gaussian Blur ≈ 1 px if needed)

5) Ridge Detection (bright lines)
Line width = fiber diameter (px) ≈ 20–35
Min line length ≈ 50 px; thresholds default
✓ Add to Manager, ✓ DisplayResults, ✗ Show junction points

6) If only overlay is visible:
Image → Overlay → to ROI Manager

7) Count fibers n
ROI Manager → Select All → Measure
(or count unique Contour ID)

8) Measure field area (μm²)
Edit → Select All → Analyze → Measure

9) Compute density
Fibers/m² = (n / area[μm²]) × 10¹²
Save ROIs & Results; repeat for all images

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