313
mention that such resolution is theoretically achievable for the conventional Raman
imaging with the applied excitation wavelength (514 nm).
Recently, a new approach eliminating CARS complications related to non-resonant background and phase matching conditions, i. e. multiplex Stimulated Raman
Scattering microscopy (SRS) has been applied to study distribution and concentration of main components of the Botryococcus Braunii microalga grown upon different lighting conditions [35]. the application of SRS resulted in obtaining, in a fast
manner, spatially-resolved images of chlorophyll, carotenoids, lipids and proteins
and showed that pigments accumulate in the periphery of the cell. A significant
increase of carotenoids level (by 95 %) in the conditions of continuous illumination
compared to 12:12 light:dark cycle was noticed, probably due to the increase of the
photosynthetic activity of the algal cells upon full light conditions [35].
Works described above were based purely on the experimental approach. Below
the example of successful match between experiment and quantum-chemical calculations will be presented in the analysis of astaxanthin in a single algal cell. Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is announced a superpotent antioxidant, and it is probably the most frequently studied carotenoid in natural sources
such as algal cells. the most potent natural bioreactor for astaxanthin production
is Haematococcus Pluvialis, a unicellular algae that upon starving conditions may
accumulate up to 3–4 % of its total dry weight [36]. to obtain information about
astaxanthin distribution and structure in a single Haematococcus cell, Raman microimaging was applied to study this pigment in the cyst algal cells (aplanospores)
[29]. the Raman distribution maps demonstrate that with the maturation of cysts,
astaxanthin distribution changes with the pigment shifting from the very center of
cells toward the periphery (Fig. 11.1), although the molecular structure of the pigment is uniform for different compartments as well as maturation stages.
Although the astaxanthin spectra in the cell are nearly identical, they differ
considerably from the Raman signature of the synthetic compound. therefore, the
study was complemented by quantum-chemical calculations [B3LYP/6-31+g(d,p)]
that clarified that these differences were not related to the change of stereoisomeric
form from 3R,3’S stereoisomer in the synthetic compound to predominantly 3S,3’S
form in the case of algal cell [29].
As efficiency of astaxanthin production in the Haematococcus cells is, among
others, related to temperature [37, 38], changes in Raman spectra of a single algal
cell upon temperature treatment have been investigated and analyzed with the aid of
quantum calculations at the B3LYP/6-31g(d,p) level [30]. heating of a single Haematococcus cyst cell form −100°C to 150°C resulted in some spectral alterations
that, based on the computations, were assigned to the conversion of the end-ring
conformations in respect to the hydrocarbon chain. Particularly, for theoretically
predicted Raman spectra of 3S,3’S conformers, (1) a decrease of the Raman shift
of the bands due to the stretching vibration of the C=C bonds, (2) a decrease of the
intensity of bands at ca. 1190 cm
−1
relative to the bands at ca. 1160 cm
−1
, with the
increase of energy (and the end-ring conformations change from gauche to trans)
were predicted. the effects opposite to changes 1 and 2 were observed upon heating
of Haematococcus from −100°C systematically up to 150°C (Fig. 11.2).
11 Structural and Spatial Analysis of Carotenoids in a Single Cell monitored …
mention that such resolution is theoretically achievable for the conventional Raman
imaging with the applied excitation wavelength (514 nm).
Recently, a new approach eliminating CARS complications related to non-resonant background and phase matching conditions, i. e. multiplex Stimulated Raman
Scattering microscopy (SRS) has been applied to study distribution and concentration of main components of the Botryococcus Braunii microalga grown upon different lighting conditions [35]. the application of SRS resulted in obtaining, in a fast
manner, spatially-resolved images of chlorophyll, carotenoids, lipids and proteins
and showed that pigments accumulate in the periphery of the cell. A significant
increase of carotenoids level (by 95 %) in the conditions of continuous illumination
compared to 12:12 light:dark cycle was noticed, probably due to the increase of the
photosynthetic activity of the algal cells upon full light conditions [35].
Works described above were based purely on the experimental approach. Below
the example of successful match between experiment and quantum-chemical calculations will be presented in the analysis of astaxanthin in a single algal cell. Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is announced a superpotent antioxidant, and it is probably the most frequently studied carotenoid in natural sources
such as algal cells. the most potent natural bioreactor for astaxanthin production
is Haematococcus Pluvialis, a unicellular algae that upon starving conditions may
accumulate up to 3–4 % of its total dry weight [36]. to obtain information about
astaxanthin distribution and structure in a single Haematococcus cell, Raman microimaging was applied to study this pigment in the cyst algal cells (aplanospores)
[29]. the Raman distribution maps demonstrate that with the maturation of cysts,
astaxanthin distribution changes with the pigment shifting from the very center of
cells toward the periphery (Fig. 11.1), although the molecular structure of the pigment is uniform for different compartments as well as maturation stages.
Although the astaxanthin spectra in the cell are nearly identical, they differ
considerably from the Raman signature of the synthetic compound. therefore, the
study was complemented by quantum-chemical calculations [B3LYP/6-31+g(d,p)]
that clarified that these differences were not related to the change of stereoisomeric
form from 3R,3’S stereoisomer in the synthetic compound to predominantly 3S,3’S
form in the case of algal cell [29].
As efficiency of astaxanthin production in the Haematococcus cells is, among
others, related to temperature [37, 38], changes in Raman spectra of a single algal
cell upon temperature treatment have been investigated and analyzed with the aid of
quantum calculations at the B3LYP/6-31g(d,p) level [30]. heating of a single Haematococcus cyst cell form −100°C to 150°C resulted in some spectral alterations
that, based on the computations, were assigned to the conversion of the end-ring
conformations in respect to the hydrocarbon chain. Particularly, for theoretically
predicted Raman spectra of 3S,3’S conformers, (1) a decrease of the Raman shift
of the bands due to the stretching vibration of the C=C bonds, (2) a decrease of the
intensity of bands at ca. 1190 cm
−1
relative to the bands at ca. 1160 cm
−1
, with the
increase of energy (and the end-ring conformations change from gauche to trans)
were predicted. the effects opposite to changes 1 and 2 were observed upon heating
of Haematococcus from −100°C systematically up to 150°C (Fig. 11.2).
11 Structural and Spatial Analysis of Carotenoids in a Single Cell monitored …