400
300
200
100
0
Wavelength (nm)
Intensity (a.u.)
600 500 400 300
40 nm
Flavin NADH
Collagen
Tryptophan
Collagen
0.6
0.5
0.8
0.7
0.4
0.3
0.2
Tryptophan
Collagen
a) b)
0.80.9 0.7 0.6 0.5
Cancer
Normal
800
1000
600
400
200
0
Wavelength (nm)
Intensity (a.u.)
600 500 400 300
20 nm
60 nm
× 10 80 nm
a)
800
1000
600
400
200
0
Wavelength (nm)
Intensity (a.u.)
600 500 400 300
100 nm
120 nm
140 nm
b)
SPECTROSCOPY continued
vide greater resolution and more information than fluorescence, combining
absorption and their spectral shifts.
They can also be used to acquire suf-
ficient information about multiple key
fluorophores in a much lower concen-
tration and relatively higher resolu-
tion by employing a single scan than
can EEM. Thus, S3 can dramatically
reduce data acquisition time and keep
the classification accuracy reasonably
high. Let’s see how it works.
Measuring fluorophores
Tryptophan, collagen, NADH, and flavin are key molecules in cancer diagnosis using spectroscopy. 3 For breast cancer, the most common grading system
used in the U.S. is the Scarff–Bloom–
Richardson (SBR) system, which is a
FIGURE 2. (a) S3 of mixed solution of tryptophan, NADH, and flavin obtained by Δλi = 40
nm and aqueous suspension collagen. This shows that in Fig. 1, the main peak at ~290
nm for the S3 of the breast tissues is from tryptophan, the secondary peak at ~340 nm
corresponds to collagen, and the tiny peak at ~380 nm correlates with NADH. No obvious
peak of flavin was observed. (b) The relative content of tryptophan vs. collagen in cancerous
and normal breast tissues, as revealed by analyzing their S3 obtained with the selective Δλi
= 40 nm. The concentration of data points for normal tissue at the upper left compared to
the concentration of data for the cancerous tissue at the lower right is telling: It indicates that
the relative contribution of collagen to the S3 signal in normal tissue is higher than that of
cancerous tissues, while the relative contribution of tryptophan in normal tissue is lower than
that in the cancerous tissue. The separating line was calculated using linear discriminant
analysis (LDA) for the diagnostic significance of tryptophan vs. collagen.
FIGURE 3. S3 spectra of mixed solution of tryptophan, NADH, and flavin obtained by (a)
Δλi = 20, 60, and 80 nm; for visualization purposes, the S3 obtained by Δλi = 20 nm was
magnified by 10 times. (b) S3 of the same mixture for Δλi = 100, 120, and 140 nm. Note that
Fig. 2a showed the spectrum of the same mixture acquired by Δλi = 40 nm.
