A hierarchical model of the organization of fibrillar collagen is developed and its implications on polarization-resolved
second harmonic generation (SHG) microscopy are investigated. A “ground-up” approach is employed to develop the
theory for understanding of the origin of SHG from fibrillar collagen. The effects of fibril ultrastructure and fibril
macroscopic organization on the second-order polarization properties of fibrillar collagen are presented in conjunction
with recent ab initio results performed on a collagen triple-helix model (-GLY-PRO-HYP-)n. Various tissues containing
fibrillar collagen are quantified using a polarization-resolved SHG technique, termed polarization-in, polarization-out
(PIPO) and interpreted in light of the aforementioned theory. The method involves varying the incident laser
polarization, while monitoring the SHG intensity through an analyzer. From the SHG polarization data the orientation of
the fibers, in biological tissue, can be deduced. Unique PIPO signatures are observed for different rat tissues and
interpreted in terms of the collagen composition, fibril ultrastructure, and macroscopic organization. Similarities and
discrepancies in the second-order polarization properties of different collagen types and ultrastructures will be presented.
PIPO SHG microscopy shows promise in its ability to quantify the organization of collagen in various tissues. The
ability to characterize the structure of collagen in various tissue microenvironments will aid in the study of numerous
collagen related biological process, including tissue diseases, wound repair, and tumor development and progression.