Fibrin fiber imaging with OCM is nonspecific. Thus, if collagen was mixed into the fibrin gel, imaging signals from OCM could not be easily processed to differentiate between the two matrix proteins. However, SHG in collagen is readily observable, constituent specific, and may be used to segment it from fibrin. Serial images acquired by integrated NLOM-OCM, in which NLOM and OCM images were simultaneously acquired, are shown in Fig. 2 of NIH 3T3 cell seeded fibrin-collagen mixture matrix at a depth of 100 μm below the surface. Total protein concentration was with ratio of fibrin:collagen, i.e., . NLOM-OCM images from two representative regions are shown at three time points (1, 12, 24 h) containing two 3T3 cells (upper images) and from an acellular region (lower images). TPF from the two regions are shown for the three time points in Fig. 2(a). Cellular autofluorescence indicated two cells within the field of view that extended processes over 24 h [Fig. 2(a), upper images]. No TPF was detected from the acellular region [Fig. 2(a), lower images]. Also, notably absent was collagen autofluorescence, which originates from covalent cross-linking and is generally weak from reconstituted collagen33 [Fig. 2(a)]. Images rendered using SHG are shown in Fig. 2(b), which identified a subset of collagen fibers within the composite matrix. In both cellular (upper images) and acellular regions (lower images), an increase in collagen density was observed, though the increase in collagen density was more pronounced in the cellular region. Nonspecific OCM images simultaneously acquired with NLOM (TPF and SHG) are shown in Fig. 2(c). OCM images rendered from backscattered laser light revealed a predominantly fibrous morphology. NLOM-OCM images were overlaid and are shown in Fig. 2(d) with cellular TPF, collagen SHG, and OCM false colored blue, red, and green, respectively. In this simple three component tissue construct, OCM structures not overlapping with NLOM (TPF and SHG) were assumed to be fibrin and, thus, green in Fig. 2(d). The overlaid images in Fig. 2(d) revealed dynamic relative spatial distributions of 3T3 cells, collagen, and fibrin that occurred in remodeling of the tissue.