The overall goal of this imaging procedure is to standardize bioluminescence acquisition procedures and image analyses to obtain quantitative data for comparison within or among different experiments. This method can help answer key questions in the bioluminescence imaging field, such, how can imaging data be quantified in a reproducible way? The main advantages of this technique are that it is easy to set up in any animal facility that own a bioluminescence imager, and that it does not require complex calculations.
Turn on the imager, and open the Acquisition software. Click Initialize, to initialize the camera, stages, and lenses. After confirming the appropriate level of sedation by lack of response to leg pinch, place a mouse on a warming pad and apply eye ointment.
Then, use a 25 gauge needle to inject 100 microliters of freshly prepared D-Luciferin potassium salt solution intraperitoneally. In this case, tumor cells express firefly luciferase. This protein oxidizes D-Luciferin and produces light that can be detected with the imager.
After 10 minutes, place the mouse, prone, into the imager with its back facing the camera in as flat a position as possible. Select the auto exposure feature and click Acquire to obtain a posterior image of the animal. Then, place the mouse in the supine position, spreading the anterior limbs so they do not block the chest.
Confirm that the auto exposure checkbox is still selected and Acquire an anterior image of the animal. Then, return the mouse to the warming pad until it is fully recovered, at which point, the animal can be returned to its cage. To quantify the bioluminescence, click View on the menu and select the Tool Palette if it is not already displayed.
Next, click on the region of interest tool tab, choose the contour button, and select Free Draw. Using the Free Draw tool, follow the edges of the whole mouse or a specific area in the front view image to mark the region of interest, right clicking to close the contour. Select the Radiance in the Units scrolling menu at the top left of the picture if it is not already selected.
Afterwards, read the value in the Total Flux photons per second box to measure the photon flux. Then, click on the View menu and the region of interest measurements. After repeating the analysis for the back view image, add the photo flux values acquired for both views to obtain the luminoscore.
In this experiment, the tumor was detectable as early as 10 minutes after the injection. The images alone verify that the tumor cells are present in the correct location. However, quantifying the bioluminescence demonstrated a clear difference between the animals that did and did not receive injections.
Interestingly, the signal obtained in the subcutaneous model was 100 times higher than in the primary intraocular lymphoma model, consistent with the number of cells injected. Here, representative tumor growth images in control and CpG treated primary intraocular lymphoma mice demonstrate that after a sufficient period, metastases begin to appear in the control group. Although the primary tumor did not appear sensitive to treatment, fewer metastases were observed in the CpG treated group.
Indeed, quantitative analysis of the tumor burden in each group demonstrates that CpG slows the tumor development, producing a statistically significant difference in the tumor luminescence between the treated and control groups after 28 days. In this subcutaneous model, two tumors were grafted onto each side of the mouse with the treatment administered in situ to a single side on day zero and the contralateral tumor serving as its control. Two regions of interest were then drawn per mouse, and the ratio between the luminoscore on the treated and control sides was measured.
13 days after treatment administration, a significant decrease in the luminoscore ratio was observed revealing that the treated tumor has been resorbed, as further evidenced by the corresponding bioluminescence images. Once administered, this analysis can be completed in two minutes per animal if it's performed properly. Following this procedure, other method like the qualitative location of tumor site can be performed to answer additional question about tumor cell homing or metastasizing mechanisms.
After its development, this technique paved the way for researcher and the field of bioluminescence imaging to explore tumor progression in pre-clinical small animal models. After watching this video, you should have a good understanding of how to perform bioluminescence imaging to quantify the tumor burden in a reproducible manner. Don't forget that working with small animals and surgical instruments can be extremely hazardous, and that precautions such as the proper sterilization of the instruments should always be taken when performing this procedure.
