1. 1 Tri-Modality Imaging of Small Animals Mark B. Williams, PhD University of Virginia DOE Awake Animal Meeting, May 20, 2004

2. 2 Photograph of Tri-Modality Gantry

3. 3 Motivation for Tri-Modality Scanner One structural (x-ray) and two functional (single gamma and optical) modalities Functional modalities have complementary strengths and weaknesses Recent advent of single gamma/optical dual reporter constructs

4. 4 Attenuation of Gamma and Optical Radiation in Biological Tissue  optical  mm 

5. 5 Annexin V Apoptotic Marker Apoptosis is genetically programmed cell death Phosphatidylserine is a lipid that is externalized to the outside of the cell membrane during apoptosis Annexin V is a protein with high affinity for phosphatidylserine Annexin V can be labeled with either 99m Tc or the near-IR fluorophore cyanine 5.5

6. 6 99m Tc vs Cy5.5 Bound to Annexin V Facts 1 molecule of Annexin V = 40 kDa = 6.64 x 10 -20 g 1 molecule of Cy5.5 = 1.5 kDa = 2.49 x 10 -21 g From the literature (Blankenberg, Strauss, et al.) Estimated labeling efficiency for 99m Tc -Annexin V: ~ 95 % Reported specific activity of 99m Tc -Annexin V ranges from 10 – 200 µCi/µg Assumptions 2 Cy5.5 molecules for every Annexin V molecule Approximately 50  g of the Annexin V protein is used in a typical study Conclusions 50 µg of Annexin V corresponds to 7.15 x 10 14 molecules of 99m Tc (12.4 mCi max) Reported labeling efficiency and specific activity implies fraction of undecayed 99mTc molecules from 0.08 – 1.61 % We assume 0.13 % for our test Bottom line 50 µg of Annexin V corresponds to 3.75 µg of Cy5.5 50 µg of Annexin V corresponds to 0.8 mCi of 99m Tc

7. 7 Planar Dual Modality System Mouse table

8. 8 Image Fusion 8-bit digital x-ray image 8-bit gamma image 24-bit fused image Rotate, resample, crop and superimpose

9. 9 IVIS™ imaging system Motor controlled stage, focus, f- stop, filter wheel Heated sample stage Gas anesthesia system Courtesy of Brad Rice, Xenogen

10. 10 Top view Bottom view Dual Modality Images 2.8 cts/sec/pix 2.7 cts/sec/pix

11. 11 Fluorescence Images Top view Bottom view 36,000 ADUs/sec/pix 4,900 ADUs/sec/pix

12. 12 Conclusions scatter of optical photons in tissue makes spatial resolution poor and quantification difficult dedicated small animal gamma cameras are capable of sub- millimeter resolution, and attenuation of gamma rays is low and/or largely correctable the signal from bioluminescent or fluorescent probes is likely to decay rapidly in space, but slowly in time the opposite is true for the signal from gamma ray probes combining single gamma and optical small animal scanners in a single scanner could help overcome the weaknesses of each

13. 13 Plans Improve sensitivity of optical camera Add x-ray detector and source Increase field of view of gamma camera