3 Methods
3.1 Sample
Preparation
3.1.1 Intact Organ
Specimen
1. Cut a thin (0.1–2.0 mm) slice of carrot root or other tissue and
place it on a microscope slide. Cover with another microscopic
slide and press gently to flatten the specimen. Protect the
movement of slides by joining their both ends using an adhesive tape that will protect also any specimen movement.
2. Use directly for Raman measurements.
3.1.2 Cell Monolayer
Specimen
1. Using a knife make manual cross-sections of the storage root of
ca. 1 mm thickness or less. Then cut into 2 Â 5 mm fragments
containing the secondary phloem. Place into an Eppendorf
tube and add 1 N HCl to completely cover the tissue. Incubate
on dry block thermostat at 60
C for 40 min (see Note 6).
2. Using forceps gently transfer small pieces of softened tissue to
the microscope slide. Absorb the excess of HCl with filter paper
and add 1–2 drops of dH 2 O.
3. Attach the edge of the coverslip to the side of the water drop
and slowly lower the coverslip over the specimen to prevent
forming air bubbles.
4. Gently apply pressure to the coverslip using the wooden pencil
with round-shaped end to flatten the specimen and to finally
spread individual cells (see Note 7).
5. Check the quality of the preparation under the light microscope. If a multilayer specimen is visible apply additional pressure to the coverslip with the pencil until cells are dispersed in
monolayers.
6. Start observation under the bright-field microscope immediately as water used to close the preparations evaporates quickly
und samples might shrink due to drought (see Note 8).
7. For Raman spectroscopy remove the coverslip. For this purpose put the water-closed slides to the deep freezer (À80
C)
for 48 h. Then lever the coverslip with the scalpel blade and tear
it out vigorously (see Note 9) and let the slides air dry for
3–5 min.
8. Use the open slide directly for Raman measurements.
3.1.3 Protoplast
Specimen
1. Place 1–3 g of callus or root pieces in a petri dish (see Note 10)
and add 8 ml of PS solution to cover completely the plant
tissue.
2. Incubate plant material at 26
C for 1 h in the dark on a rotary
shaker with gentle rotation (20–50 rpm).
3. Carefully remove as much as possible PS solution by pipetting
while avoiding moving the plant tissue.
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Tomasz Oleszkiewicz et al.
3.1 Sample
Preparation
3.1.1 Intact Organ
Specimen
1. Cut a thin (0.1–2.0 mm) slice of carrot root or other tissue and
place it on a microscope slide. Cover with another microscopic
slide and press gently to flatten the specimen. Protect the
movement of slides by joining their both ends using an adhesive tape that will protect also any specimen movement.
2. Use directly for Raman measurements.
3.1.2 Cell Monolayer
Specimen
1. Using a knife make manual cross-sections of the storage root of
ca. 1 mm thickness or less. Then cut into 2 Â 5 mm fragments
containing the secondary phloem. Place into an Eppendorf
tube and add 1 N HCl to completely cover the tissue. Incubate
on dry block thermostat at 60
C for 40 min (see Note 6).
2. Using forceps gently transfer small pieces of softened tissue to
the microscope slide. Absorb the excess of HCl with filter paper
and add 1–2 drops of dH 2 O.
3. Attach the edge of the coverslip to the side of the water drop
and slowly lower the coverslip over the specimen to prevent
forming air bubbles.
4. Gently apply pressure to the coverslip using the wooden pencil
with round-shaped end to flatten the specimen and to finally
spread individual cells (see Note 7).
5. Check the quality of the preparation under the light microscope. If a multilayer specimen is visible apply additional pressure to the coverslip with the pencil until cells are dispersed in
monolayers.
6. Start observation under the bright-field microscope immediately as water used to close the preparations evaporates quickly
und samples might shrink due to drought (see Note 8).
7. For Raman spectroscopy remove the coverslip. For this purpose put the water-closed slides to the deep freezer (À80
C)
for 48 h. Then lever the coverslip with the scalpel blade and tear
it out vigorously (see Note 9) and let the slides air dry for
3–5 min.
8. Use the open slide directly for Raman measurements.
3.1.3 Protoplast
Specimen
1. Place 1–3 g of callus or root pieces in a petri dish (see Note 10)
and add 8 ml of PS solution to cover completely the plant
tissue.
2. Incubate plant material at 26
C for 1 h in the dark on a rotary
shaker with gentle rotation (20–50 rpm).
3. Carefully remove as much as possible PS solution by pipetting
while avoiding moving the plant tissue.
250
Tomasz Oleszkiewicz et al.