Nonlinear optical microscopy for histology of fresh normal and cancerous pancreatic tissues

Abstract

Background: Pancreatic cancer is a lethal disease with a 5-year survival rate of only 1-5%. The acceleration of intraoperative histological examination would be beneficial for better management of pancreatic cancer, suggesting an improved survival. Nonlinear optical methods based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) of intrinsic optical biomarkers show the ability to visualize the morphology of fresh tissues associated with histology, which is promising for real-time intraoperative evaluation of pancreatic cancer.

Methodology/principal findings: In order to investigate whether the nonlinear optical imaging methods have the ability to characterize pancreatic histology at cellular resolution, we studied different types of pancreatic tissues by using label-free TPEF and SHG. Compared with other routine methods for the preparation of specimens, fresh tissues without processing were found to be most suitable for nonlinear optical imaging of pancreatic tissues. The detailed morphology of the normal rat pancreas was observed and related with the standard histological images. Comparatively speaking, the preliminary images of a small number of chemical-induced pancreatic cancer tissues showed visible neoplastic differences in the morphology of cells and extracellular matrix. The subcutaneous pancreatic tumor xenografts were further observed using the nonlinear optical microscopy, showing that most cells are leucocytes at 5 days after implantation, the tumor cells begin to proliferate at 10 days after implantation, and the extracellular collagen fibers become disordered as the xenografts grow.

Conclusions/significance: In this study, nonlinear optical imaging was used to characterize the morphological details of fresh pancreatic tissues for the first time. We demonstrate that it is possible to provide real-time histological evaluation of pancreatic cancer by the nonlinear optical methods, which present an opportunity for the characterization of the progress of spontaneous pancreatic cancer and further application in a non-invasive manner.

Figure 1. Schematic of a nonlinear optical microscopic system.

Figure 2. Visualization of the normal rat pancreas using nonlinear optical microscopy and conventional histology.

(A) The anatomical organization of the normal rat pancreas is composed of exocrine acini and endocrine pancreatic islets. RBC: red blood cell. (B) The nonlinear optical image of the normal rat pancreas at an imaging depth of 6 µm. The red color-coded structure is collagen, and the green color for fluorescent component. Scale bar is 30 µm. (C) The nonlinear optical image of the normal rat pancreas at an imaging depth of 23 µm. The asterisks, arrowheads and arrows indicate the nuclei, acinar cells, the collagen fibers, respectively. (D) The nonlinear optical image of the normal rat pancreas at an imaging depth of 27 µm. The dotted circle indicates the pattern of pancreatic acini. (E) The pancreatic acini can be observed in the hematoxylin and eosin image. (F) The Masson's trichrome image show small amount of collagen fibers are distributed around the acini of normal rat pancreatic samples.

Figure 3. The TPEF images of the pancreatic samples prepared under different conditions.

The intrinsic fluorescence of (A) the fresh tissues, tissues stored in 4°C for (B) 4 hours, (C) 12 hours, and (D) 24 hours, (E) fixed tissues as well as (F) frozen tissues were detected. Scale bar is 30 µm.

Figure 4. Visualization of the cancerous rat pancreatic samples using nonlinear optical microscopy and conventional histology.

(A) The pancreatic cancer cells with various size and shape as well as linear collagen fibers can be identified in the nonlinear optical image. The red color-coded structure is collagen and the green color for fluorescent component. Scale bar is 30 µm. (B) The hematoxylin and eosin image and (C) the Masson's trichrome image show the morphology of cancerous pancreatic tissues in correspondence with (A).

Figure 5. The nonlinear optical images and corresponding histology of subcutaneous pancreatic tumor xenografts.

The pancreatic tumor xenografts harvested at different stages, including (A) 5 days, (B) 10 days, (C) 20 days, and (D) 30 days after implantation, were imaged and related with conventional histology. The SHG images (red color-coded), the TPEF images (green color-coded), and the 3-D superimposed SHG/TPEF images are shown in the first three columns respectively, while the hematoxylin and eosin images and the Masson's trichrome images are displayed in the last two columns. All the 3-D images are 211 µm×211 µm×50 µm. Scale bar is 30 µm.

Figure 6. The nuclear sizes for the subcutaneous pancreatic tumor xenografts harvested at different stages.

The nuclear size at 5 days is significantly lower than those at 20 days and 30 days. *, p<0.001, ANOVA linear contrast. The sample size is 5 for each group (5 mice). +, outliers on the box-and-whisker diagram; bars, total extent of the data.

Figure 7. Quantitative analysis of the collagen fibers in the subcutaneous pancreatic tumor xenografts.

(A) The collagen density decreases as the tumor xenografts grow. The density of the tumors at 5 days is significantly higher in comparison to those at 20 days (*, p = 0.033, ANOVA linear contrast) and 30 days (**, p = 0.005, ANOVA linear contrast). +, outliers on the box-and-whisker diagram; bars, total extent of the data. (B) The organization of collagen fibers changes during the growth of the subcutaneous pancreatic tumor xenografts. An overall comparison of the correlation values shows the greatest difference between the tumor xenografts harvested at 5 days and those harvested after 10 days, as indicated by the Corr₅₀ value, the distance where the correlation crossed 50% of the initial correlation. *, p = 0.035, ANOVA linear contrast. The sample size is 5 for each group (5 mice). Error bars are one standard deviation above and below each data point.