6. 25 and 50 ml glass or plastic beakers
7. Crushed ice.
8. 2Â Extraction Buffer (2Â EB): dissolve 1.19 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (100 mM
final conc.), 0.06 g ethylenediaminetetraacetic acid disodium
salt (EDTA) (4 mM final conc.), 6.01 g sorbitol (660 mM final
conc.) in dH 2 O. Adjust volume to 50 ml with dH 2 O and pH to
7.5 using NaOH (see Note 3). Store at 4
C.
9. Sucrose gradient solutions: dissolve 5.0 g, 3.0 g, and 1.7 g
sucrose in 5 ml of 2Â EB each, and adjust volume with dH 2 O
to 10 ml each to acquire 50%, 30%, and 17% sucrose in 1Â EB,
respectively. Store at 4
C.
10. 2-Mercaptoethanol.
2.1.4 Microscopic Slide
Preparation
1. Dry block thermostat up to 60
C.
2. 2 ml Eppendorf tubes.
3. Glass petri dish or microscopic slide.
4. Automatic pipette for 100–1000 μl.
5. 100–1000 μl pipette tips.
6. SuperFrost glass microscope slides (75 Â 25 Â 1 mm) or
Raman grade calcium fluoride (CaF 2 ) slides (see Note 4).
7. Coverslips (22 Â 22 mm or 22 Â 32 mm).
8. Small pieces of filter paper (3 Â 2 cm).
9. Wooden pencil with round-shaped end.
10. Adhesive tape.
11. Preparation needle.
12. Tweezers.
13. Knife.
14. Deep freezer for À80
C (Optional).
15. 1 N HCl: take 0.1 mol of HCl and add to dH 2 O, make up to
100 ml.
2.2 Light Microscopy
and Raman
Spectroscopy
1. Bright-field microscope for routine tasks (10–60 objective
magnification).
2. Raman spectrometer equipped with a mapping stage and suitable software (see Note 5).
Light Microscopy and Raman Imaging of Carotenoids
249
7. Crushed ice.
8. 2Â Extraction Buffer (2Â EB): dissolve 1.19 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (100 mM
final conc.), 0.06 g ethylenediaminetetraacetic acid disodium
salt (EDTA) (4 mM final conc.), 6.01 g sorbitol (660 mM final
conc.) in dH 2 O. Adjust volume to 50 ml with dH 2 O and pH to
7.5 using NaOH (see Note 3). Store at 4
C.
9. Sucrose gradient solutions: dissolve 5.0 g, 3.0 g, and 1.7 g
sucrose in 5 ml of 2Â EB each, and adjust volume with dH 2 O
to 10 ml each to acquire 50%, 30%, and 17% sucrose in 1Â EB,
respectively. Store at 4
C.
10. 2-Mercaptoethanol.
2.1.4 Microscopic Slide
Preparation
1. Dry block thermostat up to 60
C.
2. 2 ml Eppendorf tubes.
3. Glass petri dish or microscopic slide.
4. Automatic pipette for 100–1000 μl.
5. 100–1000 μl pipette tips.
6. SuperFrost glass microscope slides (75 Â 25 Â 1 mm) or
Raman grade calcium fluoride (CaF 2 ) slides (see Note 4).
7. Coverslips (22 Â 22 mm or 22 Â 32 mm).
8. Small pieces of filter paper (3 Â 2 cm).
9. Wooden pencil with round-shaped end.
10. Adhesive tape.
11. Preparation needle.
12. Tweezers.
13. Knife.
14. Deep freezer for À80
C (Optional).
15. 1 N HCl: take 0.1 mol of HCl and add to dH 2 O, make up to
100 ml.
2.2 Light Microscopy
and Raman
Spectroscopy
1. Bright-field microscope for routine tasks (10–60 objective
magnification).
2. Raman spectrometer equipped with a mapping stage and suitable software (see Note 5).
Light Microscopy and Raman Imaging of Carotenoids
249