Detecting and Monitoring of Cancer at the Cellular Level - Vibrational Spectroscopy for Tissue Analysis - page 166

Table 6.1
Important Parameters for Reproduc ibility

Reproducibility Factor Achieving Methods

Laser power Fixed on 100%

Number of spectra collected from each
sample

5 spectra from each sample (intra-/intersample
reproducibility check)

Instrumental reproducibility Using the same instrument for all studies

Objective Doing all spectra collections with 10X

Aperture size 100-µm pinhole

Focus Achieved visually

Exposure number and time 64 × 2 seconds

Spectral resolution Low

Spectral region 96-3430 cm −1

Sample storage conditions Freezer (until a max of 4 hours before experiments)

Thermal stability Air-conditioner at 20-22°C

Number of cells in each sample 3 × 10 6 /5 mL

Sample preparation A unique method for all

Raman spectra of the cell lines (see Figure 6.2) reveal interesting and
important differences that help indistinguishing between R and S spectra.
Details of these differences are summarised as follows:

• The 483 cm −1 peak does not exist in R samples. This peak is attrib-
uted to phosphate vibration in nucleic acids and phospholipids.
• The 862 cm −1 shoulder cannot be seen in R samples. This shoulder
belongs to stretching C - C and bending CCH vibrations in proteins
and polysaccharides.
• The CH region shows signiicant differences. While the shoulders of
the CH region in R samples have almost turned to a separate peak,
they illustrate weaker intensities in S ones. This can demonstrate the
difference existing in CH chains between samples in nucleic acids,
proteins, and lipids.
• The ratio of I880 cm −1 /I937 cm −1 is much greater in R samples in
comparison to S samples. These peaks are due to vibrations in CH 2
(rocking), C - C (stretching), and in CH 3 (rocking) and C-C (stretch-
ing) in proteins, respectively.

Quantitative Analysis of the Spectra of 833k Samples
In this section, quantitative analysis of the spectral differences between the
R and S samples is presented to demonstrate the ability of Raman microspec-
troscopy to precisely and statistically analyse cells.