6. For Raman imaging carry out the analyses of the selected
marker bands:
(a) Distribution of carotenoids (see Note 27) (Fig. 3c).
(b) Homogeneity of carotenoid composition (see Note 28)
(Fig. 3d).
4 Notes
1. It is recommended to use Ø 90 mm petri dish, however a
smaller size can be used, but then the volume of solutions
should be downscaled proportionally. Protoplast isolation can
also be done in 2 ml Eppendorf tubes using one-eighth volume
of recommended solutions, however in this case protoplast
yield may be too low for carotenoid crystals isolations. Use
glass petri dishes if it is necessary to cut the tissue.
2. Start preparing the ES solution with dissolving the salts, then
pectolyase, and then cellulase by continuous low-speed stirring
as enzymes require long time to dissolve.
3. The pH adjusts very slowly. To avoid changing the volume of
extraction buffer use high concentrated solution of NaOH or
solid NaOH in powder or tablets.
4. Raman measurements of carotenoids can be done using glass
microscopic slides or CaF 2 slides of Raman grade. The Raman
signal coming from microscopic glass is significantly lower than
the signal coming from carotenoids. Glass microscope slides
can be safely used when measurements are focused only on
carotenoids (without measuring biological matrix, e.g., in protoplasts). Nevertheless, it is recommended to perform Raman
measurements with the use of CaF 2 slides. Raman signal coming from CaF 2 substrate (one single Raman band at
ca. 320 cm
À1 ) do not interfere with Raman signal originating
from samples.
5. To localize small objects, a microscope (or at least a camera) is
required. To optimize measurement parameters (e.g., spatial or
spectral resolution vs. signal enhancement) the possibility of
using various lasers is convenient.
6. When using a soft tissue like callus, place small pieces
(0.3 cm  0.3 cm) into an Eppendorf tube with ca. 1 ml 1 N
HCl (to cover the tissue) and incubate on a dry block thermostat at 60
C for 5–7 min to soften the specimen.
7. Apply slight and uniform pressure to avoid breaking the
coverslip.
8. If microscopic observations are to be done later, seal the coverslip with colorless nail polish for longer storage of the slide.
Light Microscopy and Raman Imaging of Carotenoids
255
marker bands:
(a) Distribution of carotenoids (see Note 27) (Fig. 3c).
(b) Homogeneity of carotenoid composition (see Note 28)
(Fig. 3d).
4 Notes
1. It is recommended to use Ø 90 mm petri dish, however a
smaller size can be used, but then the volume of solutions
should be downscaled proportionally. Protoplast isolation can
also be done in 2 ml Eppendorf tubes using one-eighth volume
of recommended solutions, however in this case protoplast
yield may be too low for carotenoid crystals isolations. Use
glass petri dishes if it is necessary to cut the tissue.
2. Start preparing the ES solution with dissolving the salts, then
pectolyase, and then cellulase by continuous low-speed stirring
as enzymes require long time to dissolve.
3. The pH adjusts very slowly. To avoid changing the volume of
extraction buffer use high concentrated solution of NaOH or
solid NaOH in powder or tablets.
4. Raman measurements of carotenoids can be done using glass
microscopic slides or CaF 2 slides of Raman grade. The Raman
signal coming from microscopic glass is significantly lower than
the signal coming from carotenoids. Glass microscope slides
can be safely used when measurements are focused only on
carotenoids (without measuring biological matrix, e.g., in protoplasts). Nevertheless, it is recommended to perform Raman
measurements with the use of CaF 2 slides. Raman signal coming from CaF 2 substrate (one single Raman band at
ca. 320 cm
À1 ) do not interfere with Raman signal originating
from samples.
5. To localize small objects, a microscope (or at least a camera) is
required. To optimize measurement parameters (e.g., spatial or
spectral resolution vs. signal enhancement) the possibility of
using various lasers is convenient.
6. When using a soft tissue like callus, place small pieces
(0.3 cm  0.3 cm) into an Eppendorf tube with ca. 1 ml 1 N
HCl (to cover the tissue) and incubate on a dry block thermostat at 60
C for 5–7 min to soften the specimen.
7. Apply slight and uniform pressure to avoid breaking the
coverslip.
8. If microscopic observations are to be done later, seal the coverslip with colorless nail polish for longer storage of the slide.
Light Microscopy and Raman Imaging of Carotenoids
255