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Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth

Martin Vanek
Martin Vanek
, 
Filip Mravec
Filip Mravec
, 
Martin Szotkowski
Martin Szotkowski
, 
Dana Byrtusova
Dana Byrtusova
, 
Andrea Haronikova
Andrea Haronikova
, 
Milan Certik
Milan Certik
, 
Volha Shapaval
Volha Shapaval
 e 
Ivana Marova
Ivana Marova
   | 25 apr 2018
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Article Category: Research Article
Pubblicato online: 25 apr 2018
Pagine: 114 - 120
DOI: https://doi.org/10.2478/ebtj-2018-0015
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© 2018 Martin Vanek, Filip Mravec, Martin Szotkowski, Dana Byrtusova, Andrea Haronikova, Milan Certik, Volha Shapaval, Ivana Marova, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Carotenoids I lifetime of β-carotene (±SD) at different trigs-to-lipid ratio in model systems.
Carotenoids I lifetime of β-carotene (±SD) at different trigs-to-lipid ratio in model systems.

Figure 2

Relative abundances of different β-carotene lifetimes in ergosterol:SDS micelles.Note: Relative abundances were derived as relative amplitudes. Abundances of 3.2 ns and 356 ns lifetimes are very low and, thus, they are overlapping in the graph.
Relative abundances of different β-carotene lifetimes in ergosterol:SDS micelles.Note: Relative abundances were derived as relative amplitudes. Abundances of 3.2 ns and 356 ns lifetimes are very low and, thus, they are overlapping in the graph.

Figure 3

Relative abundances of different β-carotene lifetimes in ergosterol:lecithin liposomes. Relative abundances were derived as relative amplitudes.
Relative abundances of different β-carotene lifetimes in ergosterol:lecithin liposomes. Relative abundances were derived as relative amplitudes.

Figure 4

Relative abundance of lifetimes during growth on agar.Note: Lifetimes: Carotenoids I (≈3.5 ns), NADP(H) (≈0.6 ns), carotenoids II (≈0.03 ns), carotenoids III (≈0.3 ns).
Relative abundance of lifetimes during growth on agar.Note: Lifetimes: Carotenoids I (≈3.5 ns), NADP(H) (≈0.6 ns), carotenoids II (≈0.03 ns), carotenoids III (≈0.3 ns).

Figure 5

Carotenoids I lifetime value during growth on agar.Note: Each point is an average from 2-3 groups of cells measured at that time.
Carotenoids I lifetime value during growth on agar.Note: Each point is an average from 2-3 groups of cells measured at that time.

Figure 6

Cystofilobasidium capitatum cell half an hour after inoculation to solid media in flow cell.Note: False-colour representation used: red – Carotenoids I, green – NADP(H), blue – Carotenoids II. Cells were inoculated from late stationary phase when preserved on malt agar media at 4 °C. Cytosolic membrane appeared to be stacked with carotenoids as well as lipid bodies inside the cell.
Cystofilobasidium capitatum cell half an hour after inoculation to solid media in flow cell.Note: False-colour representation used: red – Carotenoids I, green – NADP(H), blue – Carotenoids II. Cells were inoculated from late stationary phase when preserved on malt agar media at 4 °C. Cytosolic membrane appeared to be stacked with carotenoids as well as lipid bodies inside the cell.

Figure 7

Cystofilobasidium capitatum cells during growth on agar.Note: False-colour representation used: red – Carotenoids I, green – NADP(H), blue – Carotenoids II. Top line from the left: approximately 4, 5.5, 7.5 and 11.5 hours after inoculation; bottom line from the left: 24, 33, 49 and 56 hours after inoculation. At 4, 5.5 and 7.5 hours, when in exponential phase, it can be seen that Carotenoids I (membrane form) is decreasing.
Cystofilobasidium capitatum cells during growth on agar.Note: False-colour representation used: red – Carotenoids I, green – NADP(H), blue – Carotenoids II. Top line from the left: approximately 4, 5.5, 7.5 and 11.5 hours after inoculation; bottom line from the left: 24, 33, 49 and 56 hours after inoculation. At 4, 5.5 and 7.5 hours, when in exponential phase, it can be seen that Carotenoids I (membrane form) is decreasing.

Figure 8

Cystofilobasidium capitatum cells during fermentation in 2l laboratory fermentor.Note: In liquid media the cells show different patterns during time. In the upper part row intensity based preliminary lifetime images are illustrated, while row FLIM images with falsecolour representation (as mentioned above) are at the bottom. In exponential phase Carotenoid I lifetime seems to be disappeared, while in early stationary phase this membrane form is concentrated in endoplasmic reticle. In the stationary phase this form is present both in endoplasmic reticle and cytosolic membrane (yellow arrow).
Cystofilobasidium capitatum cells during fermentation in 2l laboratory fermentor.Note: In liquid media the cells show different patterns during time. In the upper part row intensity based preliminary lifetime images are illustrated, while row FLIM images with falsecolour representation (as mentioned above) are at the bottom. In exponential phase Carotenoid I lifetime seems to be disappeared, while in early stationary phase this membrane form is concentrated in endoplasmic reticle. In the stationary phase this form is present both in endoplasmic reticle and cytosolic membrane (yellow arrow).

Figure 9

Relative abundance changes during fermentation.
Relative abundance changes during fermentation.

Carotenoid fluorescence lifetimes in lipid clusters in different structures – micelles, liposomes and cells (lipid bodies and membranes)

Micelles with ergosterolMicelles with CoQLiposomes with ergosterolCultivation on agar
τ (ns)Relative abundance (%)τ (ns)Relative abundance (%)τ (ns)Relative abundance (%)τ (ns)
τ10.01385.80.013790.03373.140.04b
τ20.034a14.10.028a20.950.29710.420.303
τ30.3560.070.4640.021.1127.070.7
τ43.20.043.1450.033.949.363.5
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