The mammary lobule (white arrows), which comprises groups of acini separated by collagen fiber, is visualized distinctively via their strong TPEF signals (Figure 2(a)).
The compressed mammary duct (white arrows) generates strong TPEF signals because of epithelial cells lining the outline of duct (Figure 3(a)).
The lumen is filled with cystic fluid rather than cancerous or benign lump of cells, which can be distinctly observed based on strong TPEF signals.
As illustrated by Table 2, MPM has the ability to reveal morphological changes in both cellular feature and extracellular matrix (ECM) architecture, by combining TPEF signals from epithelial cells and SHG signals from collagen-rich ECM.
Experimental results demonstrate that MPM has the capability to provide structural information comparable to histopathology, allowing for identifying the microstructure of lobule and stroma in normal breast tissue, the interaction of compressed ducts with surrounding collagen fiber in fibroadenoma, and the architecture of cyst filled with cystic fluid in fibrocystic disease, based on endogenous TPEF and SHG signals.
(a) TPEF image (color-coded red) of epithelial cells in lobule, collagen bundles, and blood vessel in stroma; (b) SHG image (color-coded green) of collagen bundles in stroma and collagen fibers inside lobule; (c) the overlaid image of TPEF and SHG; (d) the corresponding H&E stained image; white arrows, pink arrows, and blue arrows indicate mammary lobule, stroma, and the blood vessel, respectively.
The mucin of mucosal cells exhibited no TPEF fluorescence and, therefore, showed a dark pattern in MPM images.
In some biopsies (n = 3; 9%) the collagen bundles in the lamina propria of BE showed the mixture of green and blue color instead of single blue color, coded because of its comparable SHG and TPEF signal yields at the same order of magnitude (Figure 3).
Based on effective TPEF of cellular nicotinamide adenine dinucleotide phosphate and flavin adenine dinucleotide and the lack of TPEF of mucin and cellular nuclei, MPM can easily identify different types of cells, such as squamous epithelial cells, goblet cells, foveolar gastric mucous neck cells, parietal cells, and chief cells.
Based on effective TPEF of elastin and SHG of collagen, connective tissue in the lamina propria and the position of the basement membrane could be visualized in MPM images.