band location strongly depends on the number of the conjugated
double bonds, length of the polyene chain, terminal moieties and
their interaction with the environment. Thus, the band position in
the Raman spectrum and the band shape can be used for identification of different carotenoids present in the sample, including
biological material.
Carotenoids in plant tissues were thoroughly investigated using
Raman spectroscopy. For fresh samples containing mainly
β-carotene the Raman spectra exhibited the symmetric ν 1 band
located at ca. 1520 cm
À1 , while for samples containing a higher
amount of lutein and α-carotene that marker band was asymmetric
and exhibited a shoulder at 1527 cm
À1
. Lycopene rich samples
provided Raman spectra with a band at 1510 cm
À1 [10]. Raman
imaging applied to leaf, fruit and root sections revealed different
content and distribution of carotenoids, depending on the tissue.
Point measurements and high resolution imaging of carotenoid
crystals performed directly in the plant cell as well as after crystal
isolation indicated that the crystals may have different compositions
depending on their morphology, that is, rhomboidal, needle-like,
or ribbon-like shape. Moreover, the distribution of various carotenoids within an individual crystal was visualized [11, 12].
Here we present protocols that enable sample preparation for
both light microscopy and Raman spectroscopy of carotenoids. We
describe the preparation of intact macroscopic biological samples as
well as microscopic slides with a monolayer of cells [11] and
isolated protoplasts [13]. The protocol extends to the procedure
of carotenoid crystals isolation from protoplasts and is a modification of the method reported by Wang et al. [14]. The protocols are
optimized for carrot samples but can be applied to other species as
well, in particular when intact tissue samples or monolayer cell
specimens are desired.
2 Materials
2.1 Sample
Preparation
2.1.1 Plant Material
1. Any of the following carrot material:
(a) Carrot storage root.
(b) Callus tissue maintained in vitro.
2.1.2 Protoplast Isolation
1. Centrifuge capable to achieve at least 150 Â g and equipped
with an angle-tilt rotor for 15 ml tubes.
2. 26
C rotary incubator with a flat table to hold petri dishes.
3. Laminar flow cabinet.
4. Autoclave.
5. pH meter.
Light Microscopy and Raman Imaging of Carotenoids
247
double bonds, length of the polyene chain, terminal moieties and
their interaction with the environment. Thus, the band position in
the Raman spectrum and the band shape can be used for identification of different carotenoids present in the sample, including
biological material.
Carotenoids in plant tissues were thoroughly investigated using
Raman spectroscopy. For fresh samples containing mainly
β-carotene the Raman spectra exhibited the symmetric ν 1 band
located at ca. 1520 cm
À1 , while for samples containing a higher
amount of lutein and α-carotene that marker band was asymmetric
and exhibited a shoulder at 1527 cm
À1
. Lycopene rich samples
provided Raman spectra with a band at 1510 cm
À1 [10]. Raman
imaging applied to leaf, fruit and root sections revealed different
content and distribution of carotenoids, depending on the tissue.
Point measurements and high resolution imaging of carotenoid
crystals performed directly in the plant cell as well as after crystal
isolation indicated that the crystals may have different compositions
depending on their morphology, that is, rhomboidal, needle-like,
or ribbon-like shape. Moreover, the distribution of various carotenoids within an individual crystal was visualized [11, 12].
Here we present protocols that enable sample preparation for
both light microscopy and Raman spectroscopy of carotenoids. We
describe the preparation of intact macroscopic biological samples as
well as microscopic slides with a monolayer of cells [11] and
isolated protoplasts [13]. The protocol extends to the procedure
of carotenoid crystals isolation from protoplasts and is a modification of the method reported by Wang et al. [14]. The protocols are
optimized for carrot samples but can be applied to other species as
well, in particular when intact tissue samples or monolayer cell
specimens are desired.
2 Materials
2.1 Sample
Preparation
2.1.1 Plant Material
1. Any of the following carrot material:
(a) Carrot storage root.
(b) Callus tissue maintained in vitro.
2.1.2 Protoplast Isolation
1. Centrifuge capable to achieve at least 150 Â g and equipped
with an angle-tilt rotor for 15 ml tubes.
2. 26
C rotary incubator with a flat table to hold petri dishes.
3. Laminar flow cabinet.
4. Autoclave.
5. pH meter.
Light Microscopy and Raman Imaging of Carotenoids
247