5. Remove supernatant and carefully resuspend the pellet in 50%
sucrose in 1Â EB.
6. Carefully pipette 30% sucrose in 1Â EB on the inner side of the
tube to create a second layer of the sucrose gradient (see Note
15).
7. Repeat the previous step by pipetting 17% sucrose in 1Â EB to
create the third layer of the sucrose gradient.
8. Centrifuge at 26,000 Â g for 45 min in the centrifuge cooled
down to 4
C.
9. Collect intact chromoplasts localized between the 50% and 30%
sucrose layers using an automatic or Pasteur pipette (see Fig. 1).
10. Resuspend the pellet in 1Â EB and centrifuge at 5000 Â g for
5 min.
11. Remove supernatant and resuspend the pellet in 1 ml dH 2 O.
12. Centrifuge at 20,000 Â g for 3 min.
13. Repeat steps 11 and 12 two times more to wash crystals from
the remaining EB solution.
14. Resuspend the pellet in 0.2–0.5 ml dH 2 O (see Note 16).
15. Transfer one drop of carotenoid crystals onto a microscope
slide and smear very gently (to avoid breaking the crystals)
using a coverslip (see Note 17). Let the slides air dry for
3–5 min in the darkness.
16. Use directly for light microscopy or Raman measurements.
3.2 Light Microscopy
1. Put the slide onto microscopic stage and observe using 10Â
objective at first. 40–60 magnifications are recommended
to observe the details of carotenoid crystals both located in the
cell and extracted from the cell (see Fig. 1b).
2. Adjust light intensity and aperture to visualize crystals, whereas
orange colour of carotenoid crystals should contrast with noncolored cell organelles.
3.3 Raman
Spectroscopy
1. Place the slide with the sample onto the instrument table under
the microscope and focus on the sample.
2. For single point Raman measurements select the following
measurement conditions:
(a) Laser wavelength (see Notes 18 and 19).
(b) Laser power (see Note 20).
(c) Integration time (see Note 21).
(d) Number of accumulations (see Note 22).
(e) Spectral resolution (see Note 23).
The scheme of single point Raman measurement is
presented in Fig. 2a.
Light Microscopy and Raman Imaging of Carotenoids
253
sucrose in 1Â EB.
6. Carefully pipette 30% sucrose in 1Â EB on the inner side of the
tube to create a second layer of the sucrose gradient (see Note
15).
7. Repeat the previous step by pipetting 17% sucrose in 1Â EB to
create the third layer of the sucrose gradient.
8. Centrifuge at 26,000 Â g for 45 min in the centrifuge cooled
down to 4
C.
9. Collect intact chromoplasts localized between the 50% and 30%
sucrose layers using an automatic or Pasteur pipette (see Fig. 1).
10. Resuspend the pellet in 1Â EB and centrifuge at 5000 Â g for
5 min.
11. Remove supernatant and resuspend the pellet in 1 ml dH 2 O.
12. Centrifuge at 20,000 Â g for 3 min.
13. Repeat steps 11 and 12 two times more to wash crystals from
the remaining EB solution.
14. Resuspend the pellet in 0.2–0.5 ml dH 2 O (see Note 16).
15. Transfer one drop of carotenoid crystals onto a microscope
slide and smear very gently (to avoid breaking the crystals)
using a coverslip (see Note 17). Let the slides air dry for
3–5 min in the darkness.
16. Use directly for light microscopy or Raman measurements.
3.2 Light Microscopy
1. Put the slide onto microscopic stage and observe using 10Â
objective at first. 40–60 magnifications are recommended
to observe the details of carotenoid crystals both located in the
cell and extracted from the cell (see Fig. 1b).
2. Adjust light intensity and aperture to visualize crystals, whereas
orange colour of carotenoid crystals should contrast with noncolored cell organelles.
3.3 Raman
Spectroscopy
1. Place the slide with the sample onto the instrument table under
the microscope and focus on the sample.
2. For single point Raman measurements select the following
measurement conditions:
(a) Laser wavelength (see Notes 18 and 19).
(b) Laser power (see Note 20).
(c) Integration time (see Note 21).
(d) Number of accumulations (see Note 22).
(e) Spectral resolution (see Note 23).
The scheme of single point Raman measurement is
presented in Fig. 2a.
Light Microscopy and Raman Imaging of Carotenoids
253