Uplansapo 60

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).

The dynamics of the CA1 pyramidal neuron dendritic spines were examined using an Olympus laser-scanning confocal microscope (FV-300; with single-photon optics employing a 60× UPlanSApo60 objective) following the protocol described previously12 (link). The timeline of examination is shown in Fig. 1d. One day before the RISE-producing stimulus (i.e. PS day −1), one of the slices grown on the filter was cut out together with a piece of underlying filter and transferred to an observation chamber fixed on a temperature-regulated stage (Tokai hit; warmed to 34 °C) of the microscope. A segment of the first or second branch of apical dendrite of a pyramidal cell (located in the anteroposteriorly central region of area CA1 and running nearly horizontally to the focal plane for >10 μm) was imaged at Z-steps of 0.75 μm. The culture was returned to the incubator after imaging. The same dendritic segment was imaged at PS day 3, 6, and 10 repeatedly across days. Examinations of control and experimental specimens in a single series of experiment were made using a single lot of culture. The same examination following the same protocol was repeated multiple times using separate lots of culture to confirm reproducibility. The times of repetition of experiment (i.e. the number of lots used) were indicated in figure legends.

Samples were examined by confocal laser scanning microscopy using a confocal FV-1000 station installed on an inverted microscope IX-81 (Olympus, Rungis, France). Multiple fluorescence signals were acquired sequentially to avoid crosstalk between image channels. Fluorophores were excited with a 405 nm diode (for DAPI), the 488 nm line of an argon laser (for AF488), and the 543 nm line of a HeNe laser (for AF594. The emitted fluorescence was detected through spectral detection channels between 425 to 475 nm, 500 to 530 nm, and 555 to 655 nm for blue, green, and red fluorescence, respectively. Objective lens ×10, ×20 (Olympus) or Olympus UplanSapo ×60 oil, 1.4 NA were used to obtain maximal resolution. When necessary, optical sectioning of the specimen (Z series) was driven by a Z-axis stepping motor through the entire thickness of the cell layer and analyzed with Imaris 3D software (Bitplane, Oxford instruments, Oxfordshire, UK).
Videomicroscopy was carried out with the same confocal laser microscope using ×40 oil UAPO NA 1.30 with 488 nm laser illumination for green fluorescence detection (500-600 nm). Green fluorescent images were acquired at the rate of 1 image per second 85 times, at 5 frames per second corresponding to a 17 s movie.