9. The critical point of this step is to prevent thawing the coverslip, otherwise the specimen will be removed along with the
coverslip.
10. For more efficient enzyme maceration it is recommended to
cut the tissue in PS solution into fine pieces using, for example,
a scalpel. For carotenoid crystals isolation more tissue (ca. 3 g)
is needed.
11. The duration of the enzyme treatment depends on tissue type
and on the desired protoplast yield. For carrot callus tissue
small number of protoplasts can be produced already after
1 h incubation time. For root tissues overnight digestion is
recommended.
12. To isolate protoplasts from undigested tissue, prepare a prefilter column by placing nylon sieve in the plastic funnel
inserted into a centrifuge tube. The nylon sieve will retain
undigested tissue.
13. To achieve distinct phases between two solutions, add the W5
solution drop by drop along the tube wall over sucrose/MES
solution to avoid mixing of the solutions.
14. After water addition the cell membranes break due to osmotic
pressure and carotenoid crystals are released from the chromoplasts into the solution.
15. It is very important to avoid mixing of solutions. This step
should be done carefully, with smoothly working automatic
pipette.
16. Pellet of crystals can be stored at 4
C in the dark until slide
preparation.
17. Insert the coverslip edge into the drop with crystals and drag it
along the slide. Alternatively, use an inoculating needle instead
of the coverslip.
18. The intensity of Raman signal is proportional to the fourth
power of the frequency of the incident light. This means that
the strongest Raman spectra are obtained when a measurement
is performed with a laser emitting in the UV region while the
weakest spectra are obtained in the infrared region. However,
the risk of side effect (e.g., fluorescence, photodamage of a
sample) is higher when using the UV-Vis lasers than the NIR
lasers. Spectrometers may be equipped with several lasers of
different wavelengths. Dispersive Raman spectrometers are
usually equipped with lasers emitting UV, visible and infrared
(IR) light while Fourier transformed (FT, also called interferometric) spectrometers are coupled with a Nd:YAG laser
(yttrium aluminum garnet doped with neodymium Nd
3+
ions) operating in NIR region. So, the shorter the laser wavelength, the stronger Raman signal can be obtained, and
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Tomasz Oleszkiewicz et al.
coverslip.
10. For more efficient enzyme maceration it is recommended to
cut the tissue in PS solution into fine pieces using, for example,
a scalpel. For carotenoid crystals isolation more tissue (ca. 3 g)
is needed.
11. The duration of the enzyme treatment depends on tissue type
and on the desired protoplast yield. For carrot callus tissue
small number of protoplasts can be produced already after
1 h incubation time. For root tissues overnight digestion is
recommended.
12. To isolate protoplasts from undigested tissue, prepare a prefilter column by placing nylon sieve in the plastic funnel
inserted into a centrifuge tube. The nylon sieve will retain
undigested tissue.
13. To achieve distinct phases between two solutions, add the W5
solution drop by drop along the tube wall over sucrose/MES
solution to avoid mixing of the solutions.
14. After water addition the cell membranes break due to osmotic
pressure and carotenoid crystals are released from the chromoplasts into the solution.
15. It is very important to avoid mixing of solutions. This step
should be done carefully, with smoothly working automatic
pipette.
16. Pellet of crystals can be stored at 4
C in the dark until slide
preparation.
17. Insert the coverslip edge into the drop with crystals and drag it
along the slide. Alternatively, use an inoculating needle instead
of the coverslip.
18. The intensity of Raman signal is proportional to the fourth
power of the frequency of the incident light. This means that
the strongest Raman spectra are obtained when a measurement
is performed with a laser emitting in the UV region while the
weakest spectra are obtained in the infrared region. However,
the risk of side effect (e.g., fluorescence, photodamage of a
sample) is higher when using the UV-Vis lasers than the NIR
lasers. Spectrometers may be equipped with several lasers of
different wavelengths. Dispersive Raman spectrometers are
usually equipped with lasers emitting UV, visible and infrared
(IR) light while Fourier transformed (FT, also called interferometric) spectrometers are coupled with a Nd:YAG laser
(yttrium aluminum garnet doped with neodymium Nd
3+
ions) operating in NIR region. So, the shorter the laser wavelength, the stronger Raman signal can be obtained, and
256
Tomasz Oleszkiewicz et al.