Figure 7. Local alignment vector analysis of in vitro H157 tumor spheroids in a collagen gel.

(a) LKB1^WT + siRNA^ctrl, (b) LKB1^WT + siRNA^MARK1 cases at 0 and 24 hour after seeding a tumor spheroid into a 2 mg/ml collagen gel. The first row in (a and b) is multiphoton microscopy images to visualize tumor spheroids, dyed using CellTracker Red; the second row is multiphoton microscopy images to visualize collagen fibers using second harmonic generation signals; the third row is quantized fiber segments with manually selected circular tumor boundary after extracting collagen fibers using CT-FIRE; the fourth row is local alignment vectors. These 2D fiber images are the first z stack images (the bottom of collagen gel). The fifth row is local alignment vectors of all z stacks in 3D, where we plot only high alignment vectors (alignment > 0.8). For the clear visualization, we magnify 4 times in Z, e.g. X:Y:Z = 1:1:4. (c) Normalized local alignment distribution using all z stack data. The local alignment (d) and the high alignment ratio over the whole z stacks (e) were calculated. (f) LKB1 signaling pathway for LKB1^WT + siRNA^ctrl and LKB1^WT + siRNA^MARK1 case.