1. Air and water based normalization of single-fiber reflectance spectroscopy measurements: How is it comparing to normalization based on diffuse reflectance standards? Nigar Sultana 1, Kenneth E. Bartels 2, G. Reed Holyoak 2, Daqing Piao 1 and Jerry W. Ritchey 3 1 School of Electrical and Computer Engineering, Oklahoma State University, Stillwater, OK 74078, USA 2 Depertment of Veterinary Clinical Science, Oklahoma State University, Stillwater, OK 74078, USA 3 Depertment of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078, USA

2. Overview Background Motivation System configuration Calibration method Results Summery

3. Background Reflectance spectroscopy using single fiber has great implication on in vivo studies to determine tissue optical properties Single-fiber reflectance spectroscopy (SfRS) generally requires a normalization of the spectral measurements in order to isolate the medium-related light scattering and absorption For normalization procedure to conduct, two samples of known optical properties are required In recent times, diffuse reflectance standards are used for normalization process and 2%, 5%, 10%, 20%, 40%, 60%, 80% and 99% reflactance standards are available from spectralon

4. Motivation When measuring optical properties of tisse in vivo, fiber tip becomes contaminated by tissue contact which affects the measurement For several set of normalized data collection from each sample tissue, fiber tip must have to be very cleaned before every measurement Due to damping of source, the source condition vary with time and data taken long before is no longer usable to conduct normalization with in time taken data It is exteremly hard to maintain same fiber condition for in time measurement with reflectance standard

5. Motivation To reduce backscattering, fiber tip to be angle polished, which makes it even more complicated to make fiber tip in contact with the flat surface of reflectance standards Without proper allignment and dark condition maitained, the measurement cannot be accurate A convenint, available and repeatitively used samples for normalization process is needed to overcome the challenges of reflactance standard

6. Objective The objective of this study is to determine samples (air and water) other than reflectance standard that is compatible to normalization process If normalization using air and water could provide better performance than normalized with reflection standards

7. System configuration Computer Light Source Spectrometer The experimental setup includes:  Halogen -Deuterium Source  Spectrometer  Bifurcated fiber bundle  320μm single fiber with 15° angle polished tip  Computer 7 15 0 angle polished fiber Schematic of Single fiber reflectance spectroscopy setup single fiber Bifurcated fiber bundle 15°

8. System configuration Angular and vertical-horizontal moveable setup to take measurements from calibration standard single fiber Calibration standard

9. Calibration Method R air ( λ ) = η c · η fib/air S(λ ) + η int S ( λ ) R water ( λ ) = η c · η fib/water S(λ ) + η int S ( λ ) R air = Reflectance spectra of air R water = Reflectance spectra of water S = System level native spectral profile η c = collection efficiency of fiber η fib/x = fresnel reflection at fiber/medium

10. Calibration Method

13. Results Reflectance standards of 10%, 20%, 40%, 60%, 80% and 99% are normalized with respect to 5% and 2% reflectance and again with respect to air and water Normalized result for 650nm wavelength is presented against percentage of reflectance Normalization result for calibration standards are the same for normalized with air and water, with only difference in scaling factor

14. Results NO

15. Results YES

16. Summery To identify other samples rather than calibration standard, this study was performed Normalization related to Single fiber reflectance spectroscopy with respect to air and water is compared with normalization with respect to reflectance standard At a certain wavelength of 650 nm, the normalized intensity using air and water reflects the same result as with reflectance standards Spectral intensity of intralipid normalized with respect to air and water shows a correct trend to reach zero, while normalized with respect to reflectance standard not reachable to zero