We observed reproducible results in
human prostate and lung cancer tissue
studies, as well. In both cases, as with
the breast tissue, two inverse spectral
properties were observed in S3 measurements for cancerous and normal tissues.
To explicitly understand the diagnostic
significance of these two inverse properties, we measured the S3 of a mixed solution of tryptophan, NADH, and flavin with the concentration of ~0.4 mg/
cm3 with various Δλi—from 20 to 140
nm with step increases of 20 nm (see
Fig. 3). The results show that the peak
intensities of three fluorophores ascend
with the increase of Δλi, at first, and then
drop at different intervals (see Figs. 2a
and 3). The FWHM of S3 profiles for
all fluorophores expands at a consistent
rate with the increase of Δλi, and when
Δλi reaches 120 nm, the NADH signal
almost takes over the flavin signal.
How do you choose an optimal Δλi?
To answer this question, we quantitatively studied how S3 changes with Δλi
by plotting the FWHM and the peak
intensities of S3 profiles as a function
of Δλi for three fluorophores in the solution (see Fig. 4). As it turns out, the
two most important properties for determining the quality of signals are resolution and magnitude—and the greater the F WHM, the worse the resolution.
Our studies show that the resolutions for
tryptophan, NADH, and flavin decrease
at a rate consistent with the increase of
Δλi, indicating that the smaller Δλi, the
higher the resolution of the S3 signal (see
Fig. 3a). For instance, choosing Δλi = 20
nm, the three fluorophores have approximately the same peak intensities. And
as Δλi increases, the peak intensities of
the three flurophores ascend at first, but
they descend at different critical values
of Δλi: The curve for tryptophan falls at
Δλi 80 nm, that of flavin drops at Δλi =
60 nm, and the NADH curve decreases
at Δλi = 120 nm (see Fig. 3b).
The results detailed in Figures 2 and
3 show that the optimal Δλi should be
as small as possible if resolution is the
only consideration. However, in or-
der to enhance the signal-to-noise ra-
tio (SNR), the optimal Δλi should be
chosen as close as to Δλss. In particular,
application in breast cancer detection,
the alterations of fluorophores due to
the cancer development direct an opti-
mal Δλi. In the spectral analysis on can-
cer detection, it is very difficult to cal-
culate the absolute concentration of the
fluorophores. One needs to find a sta-
ble reference to observe changes in the
fluorophores of interest.
Evidence of an increase of tryptophan
and a decrease of collagen in cancerous
tissue demonstrate that S3 is superior
to other approaches. Because the Δλss
is 40–50 nm for collagen and ~70–80
nm for tryptophan, Δλi = 40 nm should
be chosen as an optimal scan wavelength interval because it provides balance between resolution and the SNR.
Furthermore, the S3 with Δλi = 40 nm
investigates the signal from tryptophan
and collagen in tissue, which change inversely in cancerous and normal tissues.
Therefore, the S3 with selective Δλi = 40
nm highlights the difference between
cancerous and normal tissues and
causes the two inverse spectral properties exhibited by Figure 1.
It is worth noting that S3 actually acquires the signal of fluorescence. The
S3 profile of each biomolecule is determined by corresponding peak positions
of absorption and emission. The difference between the emission and absorption peaks is known as the Stokes shift
interval, Δλss, and when synchronized
scan wavelength shift interval Δλf approaches the Δλss, the S3 signal magnitudes will access the maximum. Where
Δλi crosses Δλss, the intensity of S3 will
fall. The S3-related parameters of different fluorophores of interest are listed
in the table. As a comparison, the values of Δλdrop for the fluorophores observed in our experiment are also listed.
The S3-related parameters of key fluo-
rophores can explain the changes of the
S3 peak intensities with Δλi in relation
to those fluorophores. When S3 is ac-
quired by Δλi = 20 nm, the excitation
of all three fluorophores is far from the
Δλss, which results the smallest S3 signal
intensity. Because the Δλss of flavin and
tryptophan is 70 nm and ~70–80 nm,
respectively, the magnitude of S3 signals
from these two fluorophores is boosted
with the increase of Δλi within the range
of Δλss at first, but the intensity drops at
Δλi = 60 nm for flavin and 80 nm for
tryptophan after it exceeds their Δλss.
This is the same reason that the peak
intensity of NADH regresses at Δλi =
120 nm after it increases. The observed
Δλdrop is in good agreement with Δλss.
Efficient and effective
This work demonstrates that the S3 measurements can acquire information for
different key fluorophores in one scan.
It also demonstrates that S3 can effectively investigate the changes of the relative contents of the key fluorophores in
breast, prostate, and lung tissues related to cancer development. In summary,
then, the S3 method offers an efficient
way to rapidly measure spectral fingerprints of complex mixtures such as tissue, and adds to the armamentarium by
highlighting differences between normal
tissues and those affected by cancer and
1. R. R. Alfano et al., IEEE J. Quantum Electron.,
20, 1507 (1984).
2. R. R. Alfano and Y. Yang, IEEE J. Quantum
Electron., 9, 148 (2003).
3. Y. Pu, W. B. Wang, G. C. Tang, and R. R.
Alfano, J. Biomed. Opt., 15, 047008 (2010).
4. H. J. G. Bloom and W. W. Richardson, Br. J.
Cancer, 11, 359 (1957).
5. G. Fenhalls, D. M. Dent, and M. I. Parker, Br. J.
Cancer, 81, 1142 (1999).
Editor’s note: This article first appeared in the
November/December 2012 issue of BioOptics
World, and is drawn from the paper published
in Optics Letters, 37, 16, 3360 (2012).
Yang Pu is a postdoctoral fellow, Wuabo
Wang is senior research associate, Yuanlong Yang is a former research fellow, and
Robert R. Alfano is Director of the Institute
for Ultrafast Spectroscopy and Lasers and
Distinguished Professor of Science and Engineering in the Department of Physics at City
College of the City University of New York,
New York, NY; www1.ccny.cuny.edu/ci/iusl.
Contact Prof. Alfano at firstname.lastname@example.org.