Overview

1. Air and water based normalization of single-fiber reflectance spectroscopy measurements: How is it comparing to normalization based on diffuse reflectance standards? Nigar Sultana1, Kenneth E. Bartels2, G. Reed Holyoak 2 , Daqing Piao1 and Jerry W. Ritchey 3 1School of Electrical and Computer Engineering, Oklahoma State University, Stillwater, OK 74078, USA 2Depertment of Veterinary Clinical Science, Oklahoma State University, Stillwater, OK 74078, USA 3Depertment 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-fiberreflectance 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 beangle 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 Bifurcated fiber bundle • single fiber 150angle polished fiber 15° Schematic of Single fiber reflectance spectroscopy setup

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

9. Calibration Method Rair( λ ) = ηc· ηfib/airS(λ ) + ηintS ( λ ) Rwater( λ ) = ηc· ηfib/waterS(λ ) + ηintS ( λ ) Rair= Reflectance spectra of air Rwater= Reflectance spectra of water S = System level native spectral profile ηc= collection efficiency of fiber ηfib/x = fresnel reflection at fiber/medium

10. Calibration Method Rtissue( λ ) = ηc{ exp[-(λ)〈L〉] S ( λ ) +ηfib/tissueS(λ )} + ηintS ( λ ) Rtissue=Reflectance spectra of air = reduced scattering coefficient = absorption coefficient L = path length = fiber diameter , fitting parameter

11. Calibration Method = Normalized spectrum of tissue with respect to air and water Rtissue( λ ) = Neglecting due to closeness of refractive index, and denoting , Rtissue( λ ) =

12. Calibration Method Rx%=Reflectance spectra of x% reflectance standard

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 • Intralipid test is performed for 0.02% to 1% concentrated intralipid • 50 sets of Single fiber reflectance spectroscopy (SfRS) data was taken by varying intralipid concentration with each time increase in concentration to 0.02% • 50 data points of Normalized scattering intensity with respect to calibration standards of 80% and 2% at 632.8 nm plotted against the known value of • While fitted with test data, it gives a coefficient of determination 0.9970 NO Normalized intensity reach 0 when is 0

15. Results • Same intralipid test is performed by normalizing with respect to air and water • Normalized scattering intensity at 632.8 nm plotted against the known value of • While fitted with simple geometric model, it gives a coefficient of determination 0.9963 YES Normalized intensity reach 0 when is 0

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