Tcs sp8 sted 3

For coincubation and imaging experiments, phenol red was removed by flushing the channels three times with phenol red-free DMEM supplemented with 100 U/ml penicillin–streptomycin. Subsequently, channels were filled with medium containing corresponding Aβ species. Cells were incubated for 24 h. Channels were flushed with fresh medium and supplemented with 50 nM Yellow HCK-123 LysoTracker. Imaging was performed either on a Leica Infinity TIRF microscope or on a confocal microscope using the Leica LAS AF software. Confocal measurements were performed using a TCS SP8 STED 3× (Leica Microsystems) equipped with an HC PL APO CS2 ×100 objective (NA 1.4) at a scan speed of 600 Hz and a line accumulation of 6. A 488 nm of a pulsed white light laser was chosen as excitation for Yellow HCK-123 LysoTracker and AbberiorSTAR520XPS. The emitted fluorescent signal was detected by counting-mode hybrid detectors in the spectral range of 500–531 nm for Yellow HCK-123 LysoTracker and 650–765 nm for AbberiorStar520SXP. Additionally, a time-gating of 0.1 ns was used to avoid laser reflection.

The fungal before or after bacterial-exposure were pretreated based on methods reported by Ding et al. [22 (link)]. Then, 200 μL of the cell suspension was mixed with 300 μL of fluoresce in fluorescein isothiocyanate labeled Concanavalin A (FITC-ConA) for 30 min at 4 °C in darkness. Excess cells and dye were removed by PBS (0.1 M, pH 7.4). Then, propidium iodide (PI) was added to the stained samples for 15 min at 4 °C in darkness. A drop (10 μL) of the sample was placed on a glass slide. The excitation wavelength and emission wavelength of FITC-ConA were 480 and 505/525 nm, respectively. PI was excited at 540 nm, while its emission wavelength was 560/660 nm. The detection parameters were adjusted to capture images. The areas associated with dead, compromised, and viable cells were visualized by LSCM (TCS SP8 STED 3×, Leica Microsystems Inc., Wetzlar, Germany; Ultra VIEW VoX, PerkinElmer, USA).

The produced W/O/W emulsions and centrifuged W/O/W were added onto glass slides (VWR Microscope Slides with cut edges, 1.0 mm thickness, 76 × 26 mm). W/O/W emulsions and centrifuged W/O/W emulsions were imaged using confocal microscopy (Leica TCS SP8, Leica Microsystems Inc. and Leica TCS SP8 STED 3×, Leica Microsystems Inc.). The crowding sensor crGE2.3 was excited at 488 nm and the emission was split into a 510–525 nm channel for the donor detection and a 600–700 nm channel for the FRET detection. For the acceptor excitation a wavelength of 561 nm was used and a 600–700 nm channel was used for emission detection. DiD was excited at a wavelength of 633 nm and for emission a 650–720 nm channel was used. All data were analyzed using ImageJ.

STED imaging was performed on a Leica TCS SP8 STED 3× (Leica Microsystems) equipped with an HC PL APO 100×/1.40 Oil STED WHITE objective. In brief, the fluorophores Alexa Fluor 594 and Abberior Star 635P were excited at 598 and 653 nm, respectively, and STED was performed at 775 nm for both color channels. The dye Alexa Fluor 488 was excited at 499 nm and recorded confocally. The channels were recorded sequentially. All images except the movies are raw data. No image processing, except for contrast stretching, was applied. The 3D STED data were deconvolved using Huygens deconvolution software (Scientific Volume Imaging) and rendered and animated using Imaris (Bitplane).

Single-plane STED images were acquired on a Leica TCS SP8 STED 3× (Leica Microsystems) on a DMI8 stand using a 100×/1.4NA HCS2 PL APO objective. A pulsed supercontinuum light source set at 644 nm was used for excitation and a pulsed depletion laser at 775 nm was added with no pulse delay at 20% intensity. Detection was performed with a hybrid detector (HyD) between 652 and 750 nm and gating between 0.3 and 6 ns. Scanner speed was set at 600 Hz and images were taken with 8× frame accumulation and 2× frame average. Pixel size was set according to the depletion power to 23 nm.