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INT RODUCTIO N
Recent years have seen an explosion in the use of Raman spectroscopy for biological purposes such as tissue
diagnosis, blood analyte detection, and cellular examination. The greatest bene t of this technique is its high
sensitivity to subtle molecular (biochemical) changes, as
well as its capability for nonintrusive application. However, biological applications of Raman spectroscopy involve turbid, chemically complex, and widely varying
target sites. Thus, the challenge in using Raman spectroscopy for biological purposes is not only the acquisition of viable Raman signatures but also the suppression
of inherent noise sources present in the target media. Perhaps the greatest contributor of noise to biological Raman
spectra is the intrinsic uorescence of many organic molecules in biological materials. This uorescence is often
several orders of m agnitude more intense than the weak
chemical transitions probed by Raman spectroscopy and,
if left untreated, can dominate the Raman spectra and
make analysis of sample biochemistry impractical. Therefore, in order to extract Raman signal from the raw spectrum acquired, it is necessary to process the spectrum to
rem ove this uorescence.
A number of methods, both instrumentational and
mathematical, have been proposed for uorescence subtraction from raw Raman signals. M ethods implemented
in hardware such as wavelength shifting and time gating
have been shown to effectively m inimize uorescence inReceived 29 January 2003; accepted 17 June 2003.
* Author to whom correspondence should be sent.
anita.mahadevan-jansen@ vanderbilt.edu.
E-mail:
0003-7028 / 03 / 5711-1363$2.00 / 0
q 2003 Society for Applied Spectroscop y
APPLIED SPECTROSCOPY
1363
F IG . 4. Phantom spectra of varying uorescence/Ram an ratios processed using the m odi ed poly t technique. (A) Smoothed sponge uorescence
spectrum with pure Raman phantom in a ratio of 10:1 and 100:1, and (B) the corresponding extracted Raman spectra along with the pure Raman
phantom, which have R 2 5 0.994 and 0.726 compared to the pure phantom, respectively. (C ) Using a fth-order polynomial t of the sm oothed
sponge spectrum as the uorescence with the pure Ram an phantom in a ratio of 1 000 000:1 and 10 000 000:1, and (D ) the corresponding extracted
Raman spectra along with the pure Raman phantom, which have R 2 5 0.997 and 0.961 compared to the pure phantom, respectively.
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F IG . 5. Comparison of conventional uorescence subtraction techniques with the modi ed poly t techniques using the sponge phantom
with a 20:1 uorescence-to-Raman ratio. (A) Raw phantom spectrum;
(B) processed spectrum using rst derivative (solid line) along with the
referen ce Raman component; (C ) processed spectrum using the manual
polynomial t m ethod by three independent users (solid lines) along
with the generated pure Raman component; and (D ) processed spectrum
using the modi ed poly t method (solid line) along with the generated
pure Raman component.
spectra of human tissue. A linear t of the baseline offset produced by overlapping peaks was subtracted to ensure a null baseline. A series of phantom spectra were
then created by adding the arti cial Raman spectrum to
the sm oothed sponge uorescence spectrum in varying
ratios.
RESULTS AND DISCUSSION
The phantom spectra with various uorescence-to-Raman ratios were processed using the developed modi ed
poly t technique. Since the phantom spectra contained
known quantities of both uorescence background and
Raman scatter, the perform ance of the processing technique could be evaluated by its ability to return only the
added Raman scatter. Figure 4A shows the raw spectra
of two different phantoms prior to uorescence subtraction where m aximum uorescence intensity was 10 and
100 times more than the maximum Raman intensity (at
1451 rel. cm 2 1), respectively. For illustration purposes,
each phantom spectrum was norm alized to its mean intensity and the spectra were offset for clarity. After m odi ed poly t processing (Fig. 4B), the ltered spectra
Centers (and respective full widths at half-maximum) of peaks used
were 568.75 (70), 792.75 (59.5), 1002.75 (35), 1107.75 (105),
1195.25 (94.5), 1450.75 (28), and 1646.75 (31.5) rel. cm 2 1.
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APPLIED SPECTROSCOPY
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