Two weeks after orthotopic implantation of MiaPaCa-2-GFP human pancreatic cancer cells, female athymic mice were anesthetized before undergoing a diagnostic laparoscopy under both white and fluorescence modes. By this time, all the animals had tumors that measured approximately 1 cm in diameter. For fluorescence laparoscopy, insufflation to 2 mm Hg allowed ample distention of the abdominal wall to enable adequate visualization and navigation within the abdomen of the mice.
Because peak excitation for the GFP occurs at 488 nm, a 480-nm short-pass excitation filter was used. Three different emission filters were tested that all achieved excellent and equivalent background suppression in the emission band of GFP at 520 nm, which serves to create the best contrast between fluorescence and background. Additionally, the distinguishing advantage of an ideal emission filter would be preservation of sufficient contrast while enough of the background is allowed to leak through to maintain spatial orientation.
Spectral analysis of combinations of the 480-nm excitation filter with each of the three emission filters tested demonstrated that at wavelengths shorter than 520 nm, the GG495 filter allowed the best transmission of background light while achieving the same degree of contrast between fluorescence and background (Fig. ).
Fig. 3 Spectral analysis of filter combinations of the 480-nm excitation filter and three potential emission filters. At 520 nm within the emission band of green fluorophores (green fluorescent protein [GFP], Alexa-488, fluorescein), all three combinations (more ...)
When either the OG515 or 515LP emission filters were used, the fluorescent tumor became visible. However, the background became so darkened that it was impossible to pinpoint the location of the tumor (Fig. a, b). To visualize the surrounding tissue and locate the tumor, the laparoscope would have to be switched back and forth between white and fluorescence modes. The GG495 filter overcame this problem (Fig. c). The combination of the Xenon lamp, the 480-nm short-pass excitation filter, and the GG495 long-pass emission filter produced a spectrum that not only provided a sufficient background but also resulted in proper color balance. By using a camera with adjustable exposure time and gain, these two parameters can be optimized to compensate for the lack of intensity from the Xenon lamp.
Fig. 4 Fluorescence signal from a green fluorescent protein (GFP)-expressing pancreatic tumor as seen with three different emission filters. A When an interference filter was used, the fluorescence signal was strong, but the background was almost completely (more ...)
For the purpose of our experiment, an exposure time of 110 ms and a gain of 97 afforded clear visualization of both the fluorescent tumor and the surrounding structures. The increasing noise due to increased gain was dynamic and did not interfere with the diagnostic purpose. The exposure time, however, had to be kept shorter than the time frame of typical biologic reactions, such as the interval of heart beats, to remain useful for navigation.
With this proper combination of instruments and light parameters, fluorescence laparoscopy permitted rapid and easy identification of the fluorescent tumor while still permitting visualization of the surrounding tissue. In fact, the GFP-expressing tumor was sufficiently bright to enable its instant localization (Fig. ). Navigation through the abdominal cavity under this fluorescent mode was straightforward because we were able to maintain an adequate view of the surrounding structures.
Fig. 5 Fluorescence laparoscopy at different angles and distances in a mouse with orthotopically implanted green fluorescent protein (GFP)-expressing pancreatic cancer. Due to the fluorescence of the tumor, a green glow (arrow) was quickly detected at entry (more ...)
At termination of laparoscopy, the mice were sacrificed, and the identified lesions were collected for histology. The use of H&E stain confirmed that these fluorescent lesions were indeed pancreatic adenocarcinoma tumors (Fig. ).
Fig. 6 Histologic image of a resected green fluorescent protein (GFP)-expressing, orthotopic tumor implant. Hematoxylin and eosin (H&E)-stained sections demonstrated the expansile tumor formed by orthotopically implanted GFP-expressing human pancreatic (more ...)