The document presents research using hyperspectral imaging to visualize biochemical changes in tissue during ischemia and reactive hyperemia. A non-invasive imaging system was developed to measure oxyhemoglobin and carboxyhemoglobin levels with spatial resolution. The study aimed to identify areas of high pressure on the feet by comparing biochemical levels during ischemia and reperfusion in those areas to self-reported pain levels. Results showed oxyhemoglobin increased and carboxyhemoglobin decreased significantly in high pressure areas during reperfusion, supporting the use of this technique to identify areas at risk for foot ulcers in diabetic patients.

1. VISIBLE HYPERSPECTRAL IMAGING : VISUALIZING THE NEUROLOGICAL AND BIOCHEMICAL NATURE OF ISCHEMIC TISSUE Presented By: Ramya Ananthanarayanan November, 2008

2. INTRODUCTION Diabetic Neuropathy Diabetic Patients – foot ulcerations Risk factors for developing foot ulcers Diabetic mediated ulceration: Neuropathy

3. ISCHEMIA & REACTIVE HYPEREMIA

4. HYPOTHESIS Hypothesizing that wearing a tight shoe, one that exerts an uncomfortable pressure on one or more locations of the foot, induces a temporary ischemia, and removing the shoe results in a significantly elevated transient bolus of blood perfusing the microvasculature of the skin, reactive hyperemia.

5. OBJECTIVE Develop a non-invasive, i n–vivo h yperspectral imaging system. Determine spatially distributed percentage of biochemical components. Proof of principle: Oxy and Carboxy Hemoglobin levels.

6. HYPERSPECTRAL IMAGING System primarily used for macroscopic applications in this study. Hyper-spectral Imaging: Many - Spectra Collect Spectral information in each detector pixel. Chemo-metric Methods – Color encode a quantity at each pixel detector

7. HYPERSPECTRAL IMAGE CUBE Wavelength LCTF - CCD assembly Series of Image Planes Y X Spatial Localization of Spectra

8. HYPERSPECTRAL IMAGER UV and IR filters Near Infrared CCD Visible CCD QTH Lamp Housing: 250 watts Lens Lens LCTF Control Boxes Near-Infrared LCTF Visible LCTF Beam Shaping optics Tripod Liquid Light guide

9. SYSTEM BLOCK DIAGRAM Spectral Range: 520nm to 645nm CCD Camera Rail LCTF Lens Source Subjects foot is placed here 0.19 m 1.06 m Front View of the Experimental Setup

10. SYSTEM CHARACTERIZATION Spatial characteristics of this imaging system were determined by a contrast transfer function (CTF) analysis. I max : the maximum intensity reflected by a bar of the resolution target (white bar) I min : the minimum intensity from the nonreflecting area between the bars (dark bar) of the resolution target The percent contrast, C , is experimentally determined from the expression, C = Imax – Imin x 100 Imax + Imin

11. The USAF 1951 Quartz resolution target A : a portion of the target B : the corresponding reflected intensity taken along a row of pixels

12. Spatial Resolving Power: CoolSNAP ES and Varispec LCTF Binning: 4x4 Spatial Resolution : 0.39mm

13. CLINICAL DESIGN Visible Reflectance Hyperspectral imaging: general, noninvasive tool Average percentage of oxy and carboxy hemoglobin from a volume of skin tissue. Penetrates the skin up to 2.5 mm. 20 subjects were studied.

14. PROTOCOL Subject Consent Lights will be turned off while the subject will have his feet imaged. Briefly the imager illuminates the skin tissue with visible light. Increase tissue safety Minimize Motion Artifacts

15. Digital camera captures pictures of the skin tissue. Introduce Occlusion creating Ischemia Foot imaged during Reactive Hyperemia: 8 over a period of 20 minutes Self Reported pressure spots ranked on a scale of 1-10.

16. Spectral and Statistical analysis using computer software LCTF tuning, image acquisition, and data storage are managed by a computer program, written in V++ and PVCAM at National Institutes of Health.

17. EXPERIMENTAL TIMELINE

18. DATA & IMAGE ANALYSIS Carboxy Hemoglobin: Before min 0 min 1 min 2 min 3 min 5 min 7 min 10 min 15 min 20

19. Oxy Hemoglobin: Before min 0 min 1 min 2 min 3 min 5 min 7 min 10 min 15 min 20

20. STATISTICAL ANALYSIS 65 pixel values each for 20 subjects Data analyzed using SAS programming by Dr. Doyle Hawkins MIXED Procedure Test Significant for High Pressure vs Control Significant at every minute starting from the 0 th minute to the 20 th .

21. < 0.0001 >0.05 >0.05

22. NEUROLOGICAL NATURE The subject’s self reported high pressure area matches the area of lowest carboxy and highest oxy hemoglobin concentration in the same subject. A : area of pain B : area that felt normal Carboxy Oxy

23. Established Reactive Hyperemia as a Surrogate Marker of High Pressure Determined the time course of reactive hyperemia and the time for which the effect lasts. Levels of target constituents during the period of reactive hyperemia as opposed to the period of ischemia: Carboxy hemoglobin decreases Oxy hemoglobin increases RESULTS & DISCUSSION

24. CONCLUSION The Microscopic Hyperspectral Imager is capable of depicting spatially distributed percentage of oxy and carboxy hemoglobin in the field of view. The system was successfully used to conclude changes in levels of tissue constituents during reactive hyperemia.

25. APPLICATION Visible-reflectance hyperspectral imaging, can be utilized to visualize areas of lower limb reactive hyperemia . Indicates ischemia and a shoe exerting excessive pressure to avoid formation of foot ulcers in neuropathic patients.

26. REFERENCES Zuzak KJ, Schaeberle MD, Lewis NE and Levin IW. Visible reflectance hyperspectral imaging: Characterization of a noninvasive, in-vivo system for determining tissue perfusion. Anal. Chem., May 2002; 74(9):2021-2028 Priceton Instrument Manual www.cvphysiology.com/Blood%20Flow/BF006.htm