Page 191 - Vibrational Spectroscopic Imaging for Biomedical Applications

W idefield Raman Imaging of Cells and T issues 167

known spectral profiles that may be used to correct for instrument
response in Raman experiments.
The SRM provides relative intensity corrections for Raman
spectrometers. The SRM used is dependent on the laser excita-
tion wavelength. For example, NIST SRM 2242 corrects for instru-
ment response in instruments using a 532-nm laser excitation,
while instruments using a laser excitation of 785 nm requires the
NIST SRM 2241 for instrument response correction. The standard
is a manganese-doped borate matrix glass that emits a broad-
band luminescence spectrum. This spectrum is described by a
polynomial expression that relates the relative spectral intensity
to the wavenumber expressed as the Raman shift from the laser
wavelength excitation. This polynomial, when compared to the
measurement of the luminescence spectrum on the specific
Raman instrument used, can determine the spectral intensity-response
correction known as instrument response. When corrected with the
instrument response correction, Raman spectra are instrument
independent. 61
To correct widefield Raman images, an image of the SRM is
acquired immediately after the acquisition of a widefield Raman
image of a tissue sample. All experimental parameters are kept the
same with the exception of the exposure time which may be
decreased to avoid camera overexposure. Upon completion of
acquisition, the widefield Raman image of the sample is divided
by the instrument response image that was derived from the image
collection on the SRM. Evidence of improvement in the spectrum
associated with the widefield Raman image can be seen in Fig. 6.3.
The image in Fig. 6.3a has not yet undergone instrument response
correction, whereas the image in Fig. 6.3b has been corrected. It is
evident that both the spectral features and baseline are smoother
and free from instrument-contributed signal in Fig. 6.3b.

6.4.5 Flatfielding
The SRM may also be used to flatfield the resultant images. In wide-
field Raman imaging, the entire field of view is illuminated. This illu-
mination is frequently not uniform across the field of view, and results
in images where the intensity is greatest at the center. The flatfield
correction improves the image quality and minimizes pixel intensity
variations. The SRM image collected is cosmic filtered, blurred, and
normalized over the image frames to produce an image that repre-
sents the illumination pattern and contains no Raman signal. The
sample image is then divided by this image to produce a more evenly
distributed data across the field of view. Samples other than the SRM
may be used for this procedure, such as silicon; however, it is convenient
to use the SRM since an image is already being collected for instrument
response correction.