Nonlinear Optical Microscopy for Histology of Fresh Normal and Cancerous Pancreatic Tissues

Abstract

BackgroundPancreatic 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 FindingsIn 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/SignificanceIn 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.

Simultaneous characterization of pancreatic stellate cells and other pancreatic components within three-dimensional tissue environment during chronic pancreatitis

“…27,28 Our previous work on exocrine pancreatic tissues using nonlinear optical microscopy has been reported, demonstrating that the acinar cells and extracellular matrix in fresh pancreatic tissues can be visualized by intrinsic TPEF signals from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) and SHG signals from collagen fibers, respectively. 29 This work did not manage to recognize sparse PSCs from dense acinar cells in tissue environment, though PSCs show long cytoplasmic processes compared with pyramidal acinar cells. 1 A recent study has reported Ca 2þ signaling events in PSCs stained with fluorescent calcium indicators, and the intrinsic fluorescence from cellular vitamin A in PSCs was also detected to show the morphology of PSCs in pancreatic tissues.…”

“…29 It is reported that the two-photon absorption of the major intrinsic fluorophores such as NADH and FAD decreases as the excitation wavelength increases in the range of 700 to 900 nm. 21 We tuned the excitation wavelength in the 730 to 890 nm range and found that 750 nm was the optimal wavelength to give the best signalto-noise ratio in our microscopic system.…”

“…Because of the fibrous connective tissue capsule distributed in the surface of pancreas, 29 the SHG images of the parenchyma at an imaging depth showing the best visualization of acinar morphology were picked out for the analysis of collagen density for all samples. The SHG images were preprocessed using the MATLAB (The MathWorks) function "imadjust" for gray level adjustment to increase the contrast of the image, so that the intensities of the SHG signal acquired for different samples are comparable.…”

“…1(b)], which has been reported previously. 29 The fluorescence intensity of acinar cells detected in the red channel [ Fig. 1(c)] is much lower than that in the green channel [ Fig.…”

Periductal stromal collagen topology of pancreatic ductal adenocarcinoma differs from that of normal and chronic pancreatitis

“…15 Fibrillar collagens possess a unique non-centrosymmetric structure allowing them to act as a frequency doubler when interacting with multiphoton laser light. This coherent, nonabsorptive light interaction process can be exploited to obtain high-resolution, quantifiable images of discrete collagen fibers in a number of sources including two-dimensional histopathology specimens, 16 three-dimensional intact tissues, 17 and live animal models 18 without the need for exogenous staining. Using second harmonic generation imaging, a number of groups have shown that changes in collagen fiber structure and organization during tumor initiation and progression have biological consequences and correlate with clinical outcomes in a number of other solid tumor types.…”