4. Add 8 ml ES solution and incubate the plant material at 26
C
for 3–5 h in the dark on a rotary shaker with gentle rotation
(20–50 rpm) to release protoplasts (see Note 11).
5. Mount a 80 μm nylon sieve in the funnel placed in the 15 ml
centrifuge tube.
6. Separate undigested tissue by transferring the whole suspension from the petri dish to the sieve using a Pasteur pipette and
collect the released protoplasts in the 15 ml centrifuge tube (see
Note 12). Pipet slowly to avoid damaging (cell wall-free)
protoplasts.
7. Centrifuge the filtered suspension of protoplasts and cell debris
at 100 Â g for 5 min.
8. Remove supernatant and resuspend the pellet in 8 ml of
sucrose/MES solution.
9. Carefully pipette 2 ml of W5 solution onto the top of the
sucrose/MES solution with resuspended protoplasts. Two distinct phases should be visible (see Note 13). Centrifuge at
145 Â g for 10 min.
10. After centrifugation a thin layer of protoplasts between two
phases is visible. Using a Pasteur pipette gently collect the
protoplasts and transfer them to a 2 ml tube with a roundshaped bottom (see Fig. 1).
11. Centrifuge at 100 Â g for 5 min.
12. Remove supernatant and use the pellet for carotenoid crystal
extraction (see Subheading 3.1.4).
13. Alternatively add a few drops of W5 and transfer 1–2 drops of
protoplast suspension to a microscopic slide using a Pasteur
pipette and cover with the coverslip.
14. Prepare slides for light microscopy or Raman spectroscopy as
described in the steps 6 or 7 in the Subheading 3.1.2,
respectively.
3.1.4 Carotenoid Crystals
Specimen
The centrifuge and solutions should be cooled down to 4
C before
crystal isolation. During the whole procedure all solutions and
samples should be kept on ice.
1. Prepare 1Â EB by mixing 2Â EB and dH 2 O in 1:1 ratio. Add
0.35 μl 2-mercaptoethanol for each 1 ml of 1Â EB and for each
1 ml of sucrose gradient solutions (50%, 30%, 17%).
2. Add 1 ml of dH 2 O to the protoplast pellet in 2 ml tube (step
12 in the Subheading 3.1.3).
3. Mix tubes with protoplasts for at least 1 min by inverting (see
Note 14).
4. Centrifuge at 350 Â g for 5 min.
Light Microscopy and Raman Imaging of Carotenoids
251
C
for 3–5 h in the dark on a rotary shaker with gentle rotation
(20–50 rpm) to release protoplasts (see Note 11).
5. Mount a 80 μm nylon sieve in the funnel placed in the 15 ml
centrifuge tube.
6. Separate undigested tissue by transferring the whole suspension from the petri dish to the sieve using a Pasteur pipette and
collect the released protoplasts in the 15 ml centrifuge tube (see
Note 12). Pipet slowly to avoid damaging (cell wall-free)
protoplasts.
7. Centrifuge the filtered suspension of protoplasts and cell debris
at 100 Â g for 5 min.
8. Remove supernatant and resuspend the pellet in 8 ml of
sucrose/MES solution.
9. Carefully pipette 2 ml of W5 solution onto the top of the
sucrose/MES solution with resuspended protoplasts. Two distinct phases should be visible (see Note 13). Centrifuge at
145 Â g for 10 min.
10. After centrifugation a thin layer of protoplasts between two
phases is visible. Using a Pasteur pipette gently collect the
protoplasts and transfer them to a 2 ml tube with a roundshaped bottom (see Fig. 1).
11. Centrifuge at 100 Â g for 5 min.
12. Remove supernatant and use the pellet for carotenoid crystal
extraction (see Subheading 3.1.4).
13. Alternatively add a few drops of W5 and transfer 1–2 drops of
protoplast suspension to a microscopic slide using a Pasteur
pipette and cover with the coverslip.
14. Prepare slides for light microscopy or Raman spectroscopy as
described in the steps 6 or 7 in the Subheading 3.1.2,
respectively.
3.1.4 Carotenoid Crystals
Specimen
The centrifuge and solutions should be cooled down to 4
C before
crystal isolation. During the whole procedure all solutions and
samples should be kept on ice.
1. Prepare 1Â EB by mixing 2Â EB and dH 2 O in 1:1 ratio. Add
0.35 μl 2-mercaptoethanol for each 1 ml of 1Â EB and for each
1 ml of sucrose gradient solutions (50%, 30%, 17%).
2. Add 1 ml of dH 2 O to the protoplast pellet in 2 ml tube (step
12 in the Subheading 3.1.3).
3. Mix tubes with protoplasts for at least 1 min by inverting (see
Note 14).
4. Centrifuge at 350 Â g for 5 min.
Light Microscopy and Raman Imaging of Carotenoids
251