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, USA2Depertment of Veterinary Clinical Science, Oklahoma State University, Stillwater, OK 74078, USA3Depertment of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078, USA

Overview• Background

• Motivation

• System configuration

• Calibration method

• Results

• Summery

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

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

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

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

150 angle polished fiber

Schematic of Single fiber reflectance spectroscopy setup

single fiber

Bifurcated fiber bundle

15°

System configuration

Angular and vertical-horizontal moveable setup to take measurements from calibration standard

single fiber

Calibration standard

Calibration Method

Rair ( λ ) = ηc· ηfib/airS(λ ) + ηintS ( λ )

Rwater ( λ ) = ηc· ηfib/waterS(λ ) + ηintS ( λ )

Rair = Reflectance spectra of airRwater = Reflectance spectra of waterS = System level native spectral profileηc = collection efficiency of fiberηfib/x = fresnel reflection at fiber/medium

Calibration Method

Rtissue ( λ ) = ηc{ exp[-(λ)⟨L⟩] S ( λ ) +ηfib/tissueS(λ )} + ηintS ( λ )

Rtissue = Reflectance spectra of air = reduced scattering coefficient = absorption coefficientL = path length

= fiber diameter, fitting parameter

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 ) =

Rx%= Reflectance spectra of x% reflectance standard

Calibration Method

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 10 20 30 40 50 60 70 80 90 100

0

5

10

15

20

25

30

Relectance Standard (%)

Nor

mal

ized

Inte

nsity

(a.u

.)

Norm with standardNorm with air & water

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 0 0.2 0.4 0.6 0.8 1 1.2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

µs’ (mm-1)

Spe

ctra

l Int

ensity

R2=0.9970

Normalized intensity reach 0 when is 0 NO

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

0 0.2 0.4 0.6 0.8 1 1.20

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

µs’ (mm-1)

Spe

ctra

l Int

ensity

R2=0.9963

Normalized intensity reach 0 when is 0 YES

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