Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi
2022
Аутори:
Pajić, TanjaTodorović, Nataša
Živić, Miroslav
Rabasović, Mihailo
Krmpot, Aleksandar
Остала ауторства
Lukić, DraganLekić, Marina
Grujić, Zoran
Тип документа:
Конференцијски прилог (Објављена верзија)
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© 2022 by the Institute of Physics
Метаподаци
Приказ свих података о документуАпстракт:
Lipids in oleaginous filamentous fungi are considered to be a valuable alternative resource for various biotechnological applications. In vivo label-free imaging enables monitoring of fungi lipid droplets (LD) accumulation with the minimal unwanted effects on the metabolism. LDs are the main source of contrast [1] in the Third Harmonic Generation (THG) microscopy method [2] due to their optical properties (high refractive index). The THG phenomenon is utilized in nonlinear laser scanning microscopy that employs ultra-short laser pulses for imaging. We present in vivo and label-free THG imaging of the individual hyphae of Phycomyces blakesleeanus (Figure 1), an oleaginous filamentous fungus with very rapid growth rate. The THG signal was detected in the forward direction (transmission arm) by PMT through Hoya glass UV filter with peak transmission at 340 nm. The Yb KGW laser, wavelength 1040 nm, pulse duration 200 fs, and repetition rate 83 MHz, has been the source of the infrared femtosecond pulses. The LDs from THG images were quantified by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting Particle Size Analysis (PSA). We used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and increase of cellular lipid reserve. The two analysis methods gave similar results. The applicability of the described procedure can be easily extended to other unicellular organisms for the in vivo quantification of LDs since there is no need for sample labeling, fixation or any other specific preparation. In addition, we demonstrate that the ICA is suitable for THG images, although it is primarily developed and have been mostly used for fluorescence signals so far. REFERENCES [1] D. Débarre, W. Supatto et al., Nat. Methods, 3(1), (2006), 47-53. [2] R. W. Boyd, Nonlinear Optics, Academic Press (2008).
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200178 (Универзитет у Београду, Биолошки факултет) (RS-MESTD-inst-2020-200178)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200007 (Универзитет у Београду, Институт за биолошка истраживања 'Синиша Станковић') (RS-MESTD-inst-2020-200007)
- HEMMAGINERO - Hemoglobin-Based Spectroscopy and Nonlinear Imaging of Erythrocytes and Their Membranes As Emerging Diagnostic Tool (RS-ScienceFundRS-Promis-6066079)
У:
- Lukić D, Lekić M, Grujić Z, editors. Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia. Belgrade: Institute of Physics; 2022. p. 52.