In vivo laser-induced fluorescence imaging of a rat pancreatic cancer with pheophorbide-a.

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.

Photodynamic imaging of a rat pancreatic cancer with pheophorbide a.

Laser-induced fluorescence of pheophorbide a (Ph-a) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg-1 body weight Ph-a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph-a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph-a fluorescence by autofluorescence, called Rt for the tumor and Rp for the pancreas) was used for fluorescence contrast calculation (C = Rt/Rp) between six tumors and their paired pancreas. Among five available laser excitation wave-lengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd:YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph-a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph-a as a PDI dye needs strong improvements.