25. The Raman imaging parameters including laser wavelength,
laser power, integration time (for dispersive Raman spectrometers), size of the image and sampling density, are interdependent. For example, by increasing the laser power the
integration time can be reduced. Lower laser power or integration time can be applied when the measurement is performed
using a laser of a shorter wavelength (i.e., from UV-Vis
region). Optimization of spectrometer parameters is crucial
for successful Raman measurements. Measurement parameters
are different for various samples and always must be determined individually taking into account which spectrometer
type is used. Sample parameters for measuring specimens containing carotenoids are presented in Table 1.
26. Generally, Raman spectrum of carotenoids contain three
intense
bands
located
at
1500–1550
cm
À1 ,
1150–1170 cm
À1
, and 1000–1020 cm
À1 assigned to the
C¼C stretching modes, C–C stretching modes, and in-plane
rocking modes of CH 3 groups attached to the polyene chain,
respectively. The exact positions of these bands highly depend
on the number of the conjugated double bonds in the structure of carotenoid molecule (i.e., length of the polyene chain),
terminal moieties as well as on the interaction between carotenoids and other compounds in the sample. The band located in
the 1500–1550 cm
À1 wavenumber range is the most
discriminating one.
27. Distribution of carotenoids can be visualized by plotting intensities of the selected marker band. Generally, the intensity can
be calculated as the band height or band integral intensity (area
under the band). As the result a 3-dimensional matrix is
obtained (Fig. 2c), where the x and y dimensions indicate the
location of sample point within the measured area where
Raman signal was acquired, and the z dimension provides
information about the intensity of the marker band in the
Raman spectrum at individual points. The intensity is proportional to the carotenoid amount occurring in the sample at that
point.
28. Homogeneity of carotenoid compositions can be visualized by
plotting the intensities of the marker bands characteristic for
various carotenoid compounds. Positions of the marker bands
in Raman spectra are shown in colors corresponding to the
chosen range of band wavelengths. Carotenoids occurring in
specific places of the sample can be identified by referring to the
color scale indicating the position of the marker Raman band
(Fig. 2d).
Light Microscopy and Raman Imaging of Carotenoids
259
laser power, integration time (for dispersive Raman spectrometers), size of the image and sampling density, are interdependent. For example, by increasing the laser power the
integration time can be reduced. Lower laser power or integration time can be applied when the measurement is performed
using a laser of a shorter wavelength (i.e., from UV-Vis
region). Optimization of spectrometer parameters is crucial
for successful Raman measurements. Measurement parameters
are different for various samples and always must be determined individually taking into account which spectrometer
type is used. Sample parameters for measuring specimens containing carotenoids are presented in Table 1.
26. Generally, Raman spectrum of carotenoids contain three
intense
bands
located
at
1500–1550
cm
À1 ,
1150–1170 cm
À1
, and 1000–1020 cm
À1 assigned to the
C¼C stretching modes, C–C stretching modes, and in-plane
rocking modes of CH 3 groups attached to the polyene chain,
respectively. The exact positions of these bands highly depend
on the number of the conjugated double bonds in the structure of carotenoid molecule (i.e., length of the polyene chain),
terminal moieties as well as on the interaction between carotenoids and other compounds in the sample. The band located in
the 1500–1550 cm
À1 wavenumber range is the most
discriminating one.
27. Distribution of carotenoids can be visualized by plotting intensities of the selected marker band. Generally, the intensity can
be calculated as the band height or band integral intensity (area
under the band). As the result a 3-dimensional matrix is
obtained (Fig. 2c), where the x and y dimensions indicate the
location of sample point within the measured area where
Raman signal was acquired, and the z dimension provides
information about the intensity of the marker band in the
Raman spectrum at individual points. The intensity is proportional to the carotenoid amount occurring in the sample at that
point.
28. Homogeneity of carotenoid compositions can be visualized by
plotting the intensities of the marker bands characteristic for
various carotenoid compounds. Positions of the marker bands
in Raman spectra are shown in colors corresponding to the
chosen range of band wavelengths. Carotenoids occurring in
specific places of the sample can be identified by referring to the
color scale indicating the position of the marker Raman band
(Fig. 2d).
Light Microscopy and Raman Imaging of Carotenoids
259