Facile whole-body imaging of internal fluorescent tumors in mice with an LED flashlight.

The use of green fluorescent protein (GFP) for detection and visualization of cells in vivo has been established by our laboratory. With this fluorescent tool, tumor cells were detected and visualized for the first time at the microscopic level in fresh viable tissue in their normal host organ (1). The use of GFP in whole-body imaging was also established by our laboratory (2). Fluorescent tumors growing and metastasizing in live mice were imaged in real time. Wholebody optical imaging, based on GFP, is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals without the need of substrate injection or anesthesia. Quantitative measurement of tumor growth on internal organs was determined using digitized wholebody images. Imaging was with either a transilluminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color charge-coupled device (CCD) camera (2). Recently, Tyas et al. (3) used a blue LED flashlight to genotype GFP transgenic mice. GFP was expressed at high levels in most tissues in these mice (3). Tyas and coworkers used a blue LED flashlight to excite the GFP fluorescence in living animals with appropriate excitation and emission filters to identify GFPpositive transgenic mouse pups (3). It is reported here that a blue LED flashlight (LDP LLC, Woodcliff Lake, NJ, USA; www.maxmax.com/ OpticalProducts.htm) with an excitation filter (midpoint wavelength peak of 470 nm) and an emission D470/40 filter (Chroma Technology, Brattleboro, VT, USA) for viewing could be used for whole-body imaging of mice with GFP and red fluorescent protein (RFP)expressing tumors growing in or on internal organs (2). Figure 1A shows wholebody imaging of two tumors, one expressing GFP and the other expressing RFP implanted in the brain. The image shows that the GFP and RFP tumors are simultaneously excited with the blue LED flashlight and readily imaged. Figure 1B shows a GFP-expressing tumor implanted in the bone and whole-body imaged with the blue LED flashlight. Figure 2A shows a GFP-expressing tumor implanted on the colon and whole-body imaged with the blue LED flashlight. The animal was also opened and imaged in the same way with the blue LED flashlight (Figure 2B). Comparison of Figure 2A and 2B shows the high accuracy of the wholebody image in Figure 2A compared to the image of the opened animal in Figure 2B. Figure 2C shows a whole-body image of an RFP-expressing tumor implanted on the liver and a GFPexpressing tumor on the pancreas of the same mouse. As with the RFP and GFP tumors implanted in the brain in Figure 1, the RFP tumor implanted on the liver and GFP tumor implanted on the pancreas are simultaneously excited with the blue LED flashlight. Figure 2D shows an image, excited with the blue LED flashlight, of an RFP-expressing tumor implanted on the pancreas that has metastasized. The images were captured with a Hamamatsu C5810 three-chip cooled color CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). However, a much simpler digital camera could be used with acceptable results. The images shown in Figures 1 and 2 were readily seen by the naked eye with no anesthesia, substrate, or restraint of the animal needed. A software program was used to calculate the number and intensity of pixels in each image and then convert the numbers to mm2 for each tumor (Table 1). The fluorescence intensity is an average value of all detectable GFP signals. The fluorescence intensity is measured in relative units defined by the gray scale of the green channel of the CCD camera. A threshold value was used that was detectably above background as visualized by eye. Table 2 compares the size and intensity of images of the tumor implanted on the colon visualized by both wholeA