Project plus 5

For the in situ determination of the chemical composition of intracellular structures, a confocal Raman microscope (alpha300 RSA; WITec, Germany) was used as previously described (56 (link)– (link)60 (link)). To immobilize the fast-moving flagellates on the quartz slide, 5 μL of the cell pellet was mixed with 5 μL of 1% (wt/vol) solution of low-melting-point agarose (catalog number 6351.5; Carl Roth, Germany), immediately spread as a single-cell layer between a quartz slide and coverslip, and sealed with CoverGrip sealant (Biotium, USA). Two-dimensional Raman maps were obtained with laser excitation at 532 nm (20 mW power at the focal plane) and oil-immersion objective UPlanFLN 100×, numerical aperture (NA) 1.30, or water-immersion objective UPlanSApo 60×, NA 1.20 (Olympus, Japan). A scanning step size of 200 nm in both directions and an integration time of 100 ms per voxel were used. A minimum of 30 cells were measured for each strain. Raman chemical maps were constructed by multivariate decomposition of the baseline-corrected spectra into the spectra of pure chemical components by using Project Plus 5.1 software (WITec, Germany).

A WITec Alpha 300 RAS system (Ulm, Germany) equipped with a 532 nm excitation laser was used to carry out the Raman spectroscopy measurements. The 50× ZEISS LD EC Epiplan–Neofluar Dic 50×/NA 0.55 objective and an optimized power on the sample of 1 mW to minimize heat induced effects were used in the measurements. All spectra were analyzed using WITec Project Plus 5.1 software. A Lorentzian peak was fitted to obtain D and G band intensities and FWHM. The spectrometer diffraction grating of 600 L mm⁻¹ was implemented in the study. The spectra were also corrected for cosmic rays and the background baseline by subtracted a shape‐based correction.