Advanced Spectroscopy Technique for Biomedicine - Biomedical Optical Imaging Technologies: Design and Applications - page 20

normalization procedures only keep the spectral shape information, while the
absolute intensity information is ignored. The spectral shape provides the informa-
tion of fluorophore species/Raman molecules and their relative concentration, while
the absolute spectral intensity contains the information of the fluorophore/Raman
molecule quantities. Intensity calibration is paramount in many applications, such
as flow cytometry and photodynamic therapy. Quantitative fluorescence or Raman
measurement enables the capability of interlaboratory comparison and evaluation.
Even in the same laboratory, intensity-calibrated fluorescence or Raman measure-
ment is still favorable because the components of the spectroscopy system such
as filters, detectors, fibers, or light source may be failed or aged in the course of
the study, making it necessary to calibrate the intensity before and after the system
configuration change or with time.
Many researchers have proposed instrument-independent fluorescence or Raman
measurement [ 25 ]. The simplest way is to divide the excitation intensity from the
wavelength and spectral calibrated spectra. However, this is invalid for noncontact
measurement where the collection efficiency is not taken into account. Recently, we
proposed an intensity calibration procedure for fluorescence measurements, which
can be written as [ 26 ]

I 0 ./
I R . m /

E L ./
E L . m /

I L . m /
I L ./

I./ D I R . m /; (1.8)

where E L ./ and I L ./ are the emission spectra and measured spectra of current
system of the standard lamp used for spectral response calibration. I R ./ is the
properties of the reference standard. I R ./ and I 0 ./ are the measured spectra of
the reference standard and the sample. m is the wavelength at the maximum of the
spectra of the reference standard.
By inspection of Eq. 1.8 , the physical meaning of the calibration procedure
is straightforward. Intensity-calibrated spectrum is obtained by normalizing the
wavelength and spectral response-calibrated sample spectra to the maximum of
the wavelength and spectral calibrated spectrum of the reference standard. Although
this calibration procedure is obtained from fluorescence spectrum calibration, it is
also valid for Raman spectral calibration, provided the reference standard is a
Raman standard. Note that this calibration procedure is valid for both contact and
noncontact measurement.

1.3 Advanced Spectroscopy Systems

Advanced spectroscopic systems for biomedical applications include diffuse
reflectance spectroscopy, fluorescence spectroscopy, and Raman spectroscopy.
Of all the spectroscopic systems, the probes vary significantly according to the
specific applications. For diffuse reflectance and fluorescence spectroscopy probes,
the readers can refer to the review article of Utzinger et al. [ 27 ]. Here, we are