Hi,
I consider "Total flux (photons/s)" for data analysis. However, when I compare the BLI data with tumor volume data (measured using digital vernier caliper), it mostly doesn't show correlation. I see "stasis" in treatment group, and a significant difference in tumor volume (mm3) when compared to placebo, but BLI data doesn't show a significant difference, sometimes in treatment group the values go more than placebo (even if I see animals in placebo with more tumor burden).
How effective this method is for quantitative purpose?
Please, share your views and observations for this method.
Thank you very much in advance!
Hi Amisha,
There are couple of reasons why you may not be seeing a tight, positive correlation between measured tumor volumes and BLI signal intensities over time:
(1) Tumor Necrosis: As established tumors get larger and grow outward overtime, their central cores can become necrotic due to an absence of necessary nutrients. This necrosis can be the result of disorganized vascular architecture in the tumors, and their inability to maintain functionality as the tissues and vasculatures of the growing edge are added on top of the older, tumor core. The result of this phenomenon over time is that larger and larger tumors will have larger and larger necrotic cores, and their total photons/sec signals will only increase minimally as a function of the tumor's growing surface area, and not as a function of their change in volume.
(2) BLI signal saturation: The fact that you see tumor "stasis" in the treatment group vs. continued growth in the placebo group obviously suggests that there is some therapeutic efficacy going on. The lack of any BLI detected differences between these two groups could be due to image signal saturation. Have you checked to see that none of your images have saturated pixels? If saturation is occurring, some pixels in your image will have signal intensities that are equal to the max of your sensor's dynamic range. For instance, if your imaging device's sensor consists of 16-bit-depth pixels, the maximum count achievable per pixel will be 2^16 or 65,535 counts. Check out the max signal intensity values in your images. If you do have saturation, you will need to reduce signal intensities by reducing exposure time and/or bin level in your camera settings. Once you have achieved unsaturated conditions, a Region of Interest (ROI) analysis of photon flux (reported as Total photons/sec) should yield values in your test and placebo groups that correlates well with their respective tumor sizes.
Comments and/or additional questions are welcome!
Best,
Andrew Van Praagh
Amisha Parmar - I agree with Andrew Van Praagh 's comments and just want to add that your signal intensity also varies with depth (see image below). When your bioluminescent source is deeper, the photons will be attenuated more (i.e., scattered/absorbed) and the signal the detector picks up will be more diffuse and lower intensity than if that source were located more superficially. It sounds like you might be working with subcutaneous tumors so this might not be that big of an issue.