Multiphoton microscopy for tumor regression grading after neoadjuvant treatment for colorectal carcinoma

Figure 1 Representative multiphoton microscopy images from a muscularis propria specimen, color-coded green represents the second-harmonic generation signal and color-coded red corresponds to the two-photon excited fluorescence signal. After neoadjuvant radiochemotherapy, predominant fibrosis changes can be seen with minimal inflammatory cells replacing tumor cells. A: Multiphoton microscopy of second-harmonic generation from collagen; B: Two-photon excited fluorescence of collagen, elastin, inflammatory cells, and blood vessels; C: Overlay image (scale bar: 100 μm); D: Hematoxylin and eosin staining of an adjacent section (magnification × 40). The tumor underwent complete regression and was replaced by fibrous tissues (blue arrows). Blood vessel hyperplasia is denoted with a white arrow. Muscular tissue is denoted with a pink arrow.

Figure 2 Ratios of second-harmonic generation and two-photon excited fluorescence intensity. Error bars indicate the standard deviation. SHG: Second-harmonic generation; TPEF: Two-photon excited fluorescence.

Figure 3 Representative multiphoton microscopy images from a muscularis propria specimen after neoadjuvant radiochemotherapy, color-coded green represents the second-harmonic generation signal and color-coded red corresponds to the two-photon excited fluorescence signal. Remaining malignant glands are dispersed deep within the rectal muscularis propria. A: Multiphoton microscopy of second-harmonic generation from collagen; B: Two-photon excited fluorescence of carcinomatous cells, inflammatory cells, and elastin; C: Overlay image (scale bar: 100 μm); D: Hematoxylin and eosin staining of an adjacent section (magnification × 40). Tumor cells in post-treatment rectal carcinoma show marked changes, and can become more solid (blue arrows) or retain a glandular growth pattern (white arrow). Fragmented elastic fibers are denoted by pink arrows.

Figure 4 Normalized emission spectrum of residual tumor cells. Excitation wavelength (810 nm) revealed two emission peaks (red arrows).