Asking the pikas politely…
Quantities: Individual leaf processing, 5 min hypochlorite, 30 min staining, 1 hour destaining, 30 seconds in ethanol:tolueneDuration: Approximately 2 hours total processing time per leafConditions: Room temperature processing
Equipment: sodium hypochlorite, safranin, ethanol, toluene, Permount medium, glass slides, Fisher Scientific Waltham MA USA materials
Microscopic imaging
Imaged each leaf using a dissecting microscope (SZX-12; Olympus) coupled to digital camera (T2i; Canon, Japan). Slides were back-illuminated using a light box. Images obtained at final magnification of 430 pixels per millimeter over full extent of c. 10 mm × 7 mm. Retained only the green channel of each image and applied contrast-limited adaptive histogram equalization procedure to improve image quality.
Quantities: 430 pixels per millimeter magnification, 10 mm × 7 mm field of view per imageDuration: Not specified per imageConditions: Back-illuminated microscopy setup
Equipment: SZX-12 Olympus dissecting microscope, T2i Canon digital camera, light box
Vein density measurement
Estimated vein density on each image using stochastic line-intersection technique. Distance between veins is known to strongly correlate with the density of veins. Distance can be rapidly estimated by counting number of veins crossed by a line of known length. Used collection of previously traced leaves from 25 morphologically diverse species on which simulated placement of random oriented line segments. For as few as 10 random line segments (c. 7 cm total length) found very strong correlation (r² = 0.89, P < 10⁻¹⁵) between vein density and distance.
Quantities: 10 random line segments of approximately 7 cm total length per image, correlation r² = 0.89Duration: Not specified per measurementConditions: Digital image analysis
Equipment: image analysis software