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4.0 METHODOLOGY

4.1 Instrumentation
The instrumentation was used for measurement of diffuse reflectance are light
source, fiber optics probe, standard reflectance, probe holder was positioned at
90 degrees and a computer for data acquisition.

Figure 20: Setup of instrument
The spectrometer (Ocean Optics, HR2000+ High-resolution Spectrometer) in
figure 21 was used for 400-1100 nm wavelength range. The HR2000+
Spectrometer integrates a high-resolution optical bench, a powerful 2-MHz
analog-to-digital (A/D) converter, programmable electronics, a 2048-element
CCD-array detector and a high-speed USB 2.0 port. This innovative
combination produces and extremely fast spectrometer and provides
resolution to 0.035 nm (FWHM). The illumination in the VIS and NIR
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spectral ranges was provided by a mini 7W tungsten-halogen light source
(Ocean Optics,HL-2000) as shown in figure 22. The light delivered and
collection was came from a fiber optic probe(Ocean Optics,R200-7) that
consisting of six 200m core optical fiber for light collection as shown in
figure 23. The delivery fibers were was placed around the central collection
fiber such that the center to center distance of the fiber optical was
approximately 200m. The measurement was calibrated using the reflectance
standard (Ocean Optics,WS-1) as shown in figure 24.

Figure 21: Ocean Optics, HR2000+ High-resolution Spectrometer


Figure 22: Mini 7W tungsten-halogen light source (Ocean Optics,HL-
2000)
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Figure 23: Fiber optic probe(Ocean Optics,R200-7)


Figure 24: Reflectance standard (Ocean Optics,WS-1)


4.2 Method
As shown in figure 25, methods that will be used for this study are calibration
of diffuse reflectance measurement, preparation of sample, diffuse reflectance
measurements on sample and spectra data analysis.

4.2.1 Calibration the diffuse reflectance measurement
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The diffuse reflectance measurement was calibrate by using
reflectance standard (Ocean Optics,WS-1). The reflective area of the
WS-1 has a 32 mm OD. The diffusing material is encased in a small
receptacle with screw-on top. The material is almost uniformly
reflective (>95%) by wavelength across the entire range of 250-2000
nm.

4.2.2 Preparation of samples
The samples of liver, heart and meat from cow were purchased. The
samples were kept frozen in commercial freezer (-4C) until they used
for Vis/NIR spectroscopy. Samples were prepared by cutting slices
10mm thick cross-section

4.2.3 Diffuse reflection measurement
The samples were thawed in the fridge for 24 h and taken out, stored
in the plastic bag prevent water evaporation from the surface and left
at least 1h at ambient temperature. The diffuse reflectance spectra
were collected from 400-1100nm (30 data point per sample) using
spectrometer (Ocean Optics, HR2000+ High-resolution Spectrometer).
The samples were put on ceramic plate and not rotated when spectra
collection was made. Spectral data collected were recorded in the form
of the logarithm of reciprocal of reflectance [log (1/R)].
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Figure 26: The process of measurement

4.2.4 Spectra data analysis
PCA, is a mathematical procedure for resolving sets of data into
orthogonal components whose linear combinations approximate the
original data to any desired degree of accuracy (Martens & Neas,
1996; Martens & Martens, 2000). PCA was used to derive the first 20
principle components form condensed spectral data. These were used
in further analysis to examine the natural grouping of the sample. In
order to visualise the relative position of the different samples were
graphically displayed by mean of the first there PCs. Discriminant
analysis was performed using the dummy regression technique as
described elsewhere by other authors (Osborne et al., 1993; Ding et
al., 1999). Each different assigned a dummy value.

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4.3 Flow Chart



















Figure 25: Flow chart of methodology
Start
Calibration the diffuse reflectance
measurement by reflectance standard

Diffuse reflection measurement on
samples between wavelength range of
400nm to 2400nm every 2nm length
Repeated 30 reading per
samples




reflectance
measurement on in vivo
skin finger provocation
with cutt for arterial
occlusion
Result & discussion
Spectra data analysis using Principle
Component Analysis (PCA)
End
Prepare different samples cut of liver,
heart and beef of cow