Page 188 - Vibrational Spectroscopic Imaging for Biomedical Applications

164 Cha pte r S i x

6.4 Background and Image Preprocessing Steps for
Widefield Raman Images
Biological samples such as tissues and cells are highly complex and
comprise building blocks such as amino acids, proteins, and DNA
which exhibit very similar Raman spectra. Consequently, the subtle
existing spectral differences between biological samples may be over-
shadowed by signal attributed to instrument components or back-
ground light. In order to eliminate this background signal, a series of
preprocessing steps must be completed prior to any data analysis. 56
This way, any differences found in the analysis will be exclusive to
the biological content of the tissue only.
Any type of mathematical operation applied to the data prior to
57
analysis is considered preprocessing. In effect, preprocessing
separates the Raman signal from the noise, thus removing noninforma-
tive data. Contributing factors to the background include instrument
response, cosmic events, source illumination intensity variations, and
fluorescence. Noise is also present in the data from instrumental
components, software computations, and surrounding light. 56

6.4.1 Fluorescence
Fluorescence is a major source of background in widefield Raman
images of biological samples because of the radiation source used and
the chemical composition of tissue. Raman scattering occurs as a result
of the inelastic scattering of photons. When tissue samples are excited
with a 532-nm laser, the Raman signal is often masked by a broad
fluorescence emission that occurs simultaneously. This fluorescence is
reduced through the process of photobleaching.
Photobleaching is a poorly understood phenomenon that occurs
when a fluorophore permanently loses its ability to fluoresce. This
loss occurs as a result of photon-induced chemical damage and
covalent modification during transitions from excited singlet states to
excited triplet states. The number of transitions a fluorophore undergoes
prior to photobleaching is dependent upon the molecular structure and
the local sample environment. As a result, some fluorophores bleach
quickly while others take much longer to bleach due to thousands of
58
transition cycles. For this reason, photobleaching must be completed
prior to any Raman image collection.
A method to eliminate the majority of fluorescence masking
Raman signal is to monitor the photobleaching process through
collection of Raman dispersive spectra prior to image collection.
This process is illustrated in Fig. 6.2, where Raman dispersive
spectra are collected at 1-second intervals for 30 seconds. The top
spectrum collected at 1 second is absent of any Raman signal. Raman
peaks become more evident with time as the fluorescence burns down.
The last spectrum collected at 30 seconds has peaks evident in both the