dissolved carotenoids in plastoglobuli of various sizes and are more
often found in tissues exposed to light [4, 6, 7]. Both types can be
identified using a light microscopy. Due to their absorbance in the
visible light spectrum, carotenoids can be recognized in bright field
mode without any staining. To avoid destruction of these pigments
(dissolving in washing buffers), microscopic preparations should be
prepared from a nonfixed specimen. A low resolution of light
microscopy allows only for indicating the localization of crystalloid
and globular chromoplasts in the cells, but their high number or
close localization may restrict unambiguous determination of their
shape, size, and number. Thus, chromoplast isolation from cells
may be required for detailed characterizations. The other two types
of chromoplasts—tubular and membranous—are less frequently
observed; they have a fine ultrastructure and can be identified
mainly by using transmission electron microscopy (TEM). However, this technique is destructive to carotenoids which are dissolved during sample fixation and usually only traces of preserved
carotenoid remnants are finally visualized [6].
Carotenoids can be detected and identified nondestructively
using Raman spectroscopy. This technique allows for measurements of carotenoids present in either intact living tissues or single
cells; thus, neither chemical nor structural changes to carotenoids
are expected, as neither exogenous chemicals nor physical forces are
applied during sample preparation. A high resolution Raman spectroscopy enables also the measurements of individual carotene
crystals both when present in the cell or after their extraction.
The Raman spectra of the samples can be collected from a single
point or by measuring several points of the defined area. The latter
approach, Raman mapping or Raman imaging, can be used to assess
the distribution of carotenoids and the homogeneity of the sample.
Such information is retrieved by calculating the integral intensity of
the carotenoid marker band at every point of the mapped sample
and visualized using a color 2D surface plot.
Carotenoid molecules contain a chromophore, which is a polyene chain with alternating double and single bonds. When the
wavenumber of the laser excitation used for Raman measurements
coincides with an electronic transition of the individual carotenoid,
that is, it is in the visible range (or near-infrared range, NIR), the
resonance (or preresonance) Raman effect is observed [8]. Due to
this effect, Raman spectra of carotenoids can be recorded even
when these compounds are present at very low concentrations
(down to 10
À8 M) and when they are embedded in a complex
biological matrix. The most intense and characteristic bands in
the Raman spectra of carotenoids are located at 1500–1550 cm
À1
(ν 1 ) and 1150–1170 cm
À1 (ν 2 ), and are assigned to the C¼C and
C–C stretching modes, respectively. Additionally, in-plane rocking
modes of CH 3 groups attached to the polyene chain can be
observed as a medium intense band at 1000–1020 cm
À1 [9]. The
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Tomasz Oleszkiewicz et al.
often found in tissues exposed to light [4, 6, 7]. Both types can be
identified using a light microscopy. Due to their absorbance in the
visible light spectrum, carotenoids can be recognized in bright field
mode without any staining. To avoid destruction of these pigments
(dissolving in washing buffers), microscopic preparations should be
prepared from a nonfixed specimen. A low resolution of light
microscopy allows only for indicating the localization of crystalloid
and globular chromoplasts in the cells, but their high number or
close localization may restrict unambiguous determination of their
shape, size, and number. Thus, chromoplast isolation from cells
may be required for detailed characterizations. The other two types
of chromoplasts—tubular and membranous—are less frequently
observed; they have a fine ultrastructure and can be identified
mainly by using transmission electron microscopy (TEM). However, this technique is destructive to carotenoids which are dissolved during sample fixation and usually only traces of preserved
carotenoid remnants are finally visualized [6].
Carotenoids can be detected and identified nondestructively
using Raman spectroscopy. This technique allows for measurements of carotenoids present in either intact living tissues or single
cells; thus, neither chemical nor structural changes to carotenoids
are expected, as neither exogenous chemicals nor physical forces are
applied during sample preparation. A high resolution Raman spectroscopy enables also the measurements of individual carotene
crystals both when present in the cell or after their extraction.
The Raman spectra of the samples can be collected from a single
point or by measuring several points of the defined area. The latter
approach, Raman mapping or Raman imaging, can be used to assess
the distribution of carotenoids and the homogeneity of the sample.
Such information is retrieved by calculating the integral intensity of
the carotenoid marker band at every point of the mapped sample
and visualized using a color 2D surface plot.
Carotenoid molecules contain a chromophore, which is a polyene chain with alternating double and single bonds. When the
wavenumber of the laser excitation used for Raman measurements
coincides with an electronic transition of the individual carotenoid,
that is, it is in the visible range (or near-infrared range, NIR), the
resonance (or preresonance) Raman effect is observed [8]. Due to
this effect, Raman spectra of carotenoids can be recorded even
when these compounds are present at very low concentrations
(down to 10
À8 M) and when they are embedded in a complex
biological matrix. The most intense and characteristic bands in
the Raman spectra of carotenoids are located at 1500–1550 cm
À1
(ν 1 ) and 1150–1170 cm
À1 (ν 2 ), and are assigned to the C¼C and
C–C stretching modes, respectively. Additionally, in-plane rocking
modes of CH 3 groups attached to the polyene chain can be
observed as a medium intense band at 1000–1020 cm
À1 [9]. The
246
Tomasz Oleszkiewicz et al.