Uplansapo

Fluorescent images of brain slices were obtained using an IX83 inverted-imaging system with a DP80 camera and 10x, 20x, or 40x UPlanSApo (0.75 NA) and 60x UPlanSApo oil-immersion (1.35 NA) objectives (Olympus, Shinjuku, Japan).
For confocal-image acquisition, either an upright TCS SP8 Laser microscope equipped with a 63x objective (oil-immersion, 1.2 NA) using sequential scanning with the 488 nm line of an argon-ion laser and the 543 nm line from helium–neon lasers (for Alexa 488 and Alexa 568, respectively) (Leica Microsystems) or an inverted confocal laser scanning FV3000 microscope equipped with 10x (0.75 NA), 20x (0.8 NA), and 40x (oil-immersed, 1.4 NA) objectives using sequential scanning with 405 nm, 488 nm, 561 nm cw diode lasers (for DAPI, Alexa488, Cy3 or Alexa 568, respectively) (Olympus) was used. Background correction and adjustment of brightness and contrast were performed using either LasX confocal software version 3.5.7 (Leica Microsystems, RRID:SCR_013673), cellSens Dimension version 1.18 (Olympus) or Fiji (NIH, Bethesda, MD, RRID:SCR_002285). Image analysis of protrusions was performed using cellSens Software (Olympus).

For assessment of dendrites and dendritic spine morphology, a Z-stack of the optical section was captured with a confocal microscope (Thorlabs, USA). For dendritic analysis 2048 × 2048 pixel images with 0.25 μm/pixel resolution were captured with Z interval of 1 μm using a 20× objective lens (NA = 0.85, UPlanSApo; Olympus, Japan). For dendritic spine analysis, 2048 × 2048 pixel images with 0.025 μm/pixel resolution were captured with Z interval of 0.1 μm using a 100× objective lens (NA = 1.4, UPlanSApo; Olympus, Japan). Quantitative analysis of dendritic length and branching was performed with the help of the Sholl analysis module of a freely available Neurostudio software package [56 (link)]. Quantitative analysis of dendritic spines, including measurements of dendritic spine head area, neck length, and neck length/dendritic spine length ratio, was performed using SpineJ software [41 (link)]. Before analysis, dendritic protrusions in cultured primary hippocampal neurons were classified as headed spines, which have clearly defined head and neck, filopodia—An extremely long protrusions and stubby, relatively short protrusions without neck. Quantitative analysis was performed only on headed spines. At least 18 transfected neurons from three independent experiments were used for quantitative analysis.

The home-built SRS system used a pump laser integrated OPO (picoEmerald, APE, Germany). It provided 2 spatially and temporally overlapped pulse trains, with the synchronized repetition rate of 80 MHz. One beam, fixed at 1,064 nm, was used as the Stokes light. The other beam, tunable from 780 to 990 nm, served as the pump light. The intensity of the Stokes beam was modulated at 20.2 MHz by a resonant electro-optical modulator (EOM). The overlapped lights were directed into an inverted multiphoton scanning microscope (FV1000, Olympus, Japan). The collinear laser beams were focused into the sample by a 20× objective (UPlanSAPO, NA 0.75, Olympus, Japan). Transmitted light was collected by a condenser (NA 0.9, Olympus, Japan). After filtering out the Stokes beam, the pump beam was directed onto a large area photo diode (FDS1010, Thorlabs, USA). The voltage from photo diode was sent into lock-in amplifier (HF2LI, Zurich Instruments, Switzerland) to extract the SRS signal. Image was reconstructed through software provided by manufacture (FV10ASW, Olympus, Japan).

Live HeLa, HEK293, NIH3T3 and COS-1 cells were imaged with an Olympus IX81 inverted epifluorescence microscope 72 h after the transfection. The microscope was equipped with a 200-W metal halide arc lamp (Lumen220PRO, Prior), a 60 × 1.35 numerical aperture (NA) oil objective lens (UPlanSApo, Olympus) and an opiMOS sCMOS camera (QImaging). During imaging, cells were incubated in a cell imaging medium (Life Technologies-Invitrogen) at room temperature. The microscope was operated with a SlideBook v.6.0.8 software (Intelligent Imaging Innovations).

Fixed PfPanK2-GFP-expressing 3D7 strain P. falciparum parasites within infected erythrocytes were observed and imaged with a Leica TCS-SP2-UV confocal microscope (Leica Microsystems) using a 63× water immersion lens as described in the S1 Text. To confirm the expression of SaPanK-Ty1 in the TgPanK1-mAIDHA^+SaPanK-Ty1 line, immunofluorescence assays were performed based on the protocol described by van Dooren et al. [73 (link)]. T. gondii parasites were incubated with mouse anti-Ty1 antibodies (1:200 dilution). Secondary antibodies used were goat anti-mouse AlexaFluor 488 at a 1:250 dilution. The nucleus was stained with DAPI. Immunofluorescence images were acquired on a DeltaVision Elite system (GE Healthcare) using an inverted Olympus IX71 microscope with a 100× UPlanSApo oil immersion lens (Olympus) paired with a Photometrics CoolSNAP HQ² camera. Images taken on the DeltaVision setup were deconvolved using SoftWoRx Suite 2.0 software. Images were adjusted linearly for contrast and brightness.

For 3D imaging, brain slices were individually
mounted between two glass slides which were
surrounded by same thickness non-colorful putty that
formed a horse-shoe-like chamber (1-mm thickness
wall) to protect the tissues’ thickness from pressing
between the slides and provides a chamber for the
RI matching solution. This chamber between two
slides was filled with fresh 80% glycerol. The auto-
fluorescent vessels were imaged by an epifluorescence
microscope (BX51 with a DP72 camera, Olympus,
Japan), and CellSens imaging software (Version 1.4.1,
Olympus, Japan). After the apparatus was fixed on
the microscope stage, the specimen was imaged by an
air/dry objective lens 10× (UPlanSApo, Olympus Co.
Ltd.; numerical aperture : 0.4 and working distance:
3.1 mm) which was water immersed to increase
working distance. The EPI illumination mode and red
excitation (650 nanometers) and deep red emission
(690 nanometers) were applied for imaging. For this
purpose, selected area was imaged on a z-stack manner
(each 10-µm step) for the depth of 150 µm from the
tissue surface, automatically.

Characterization of intracellular Ca²⁺ dynamics was performed using Fura-2AM, as previously described (Jost and Hockendorf, 2019 (link)). Using a 488 nm laser for excitation (Curley et al., 2018 (link)), events were recorded at 20 to 50 fps with an EMCCD (Photometrics Cascade II 512, 512 × 512 pixels, 16-bit images) camera. Then, 10× (Olympus UPlanApo N.A. = 0.40) and 20× (Olympus UPlanSApo N.A. = 0.75) objectives were used for Ca²⁺ imaging (Frank and Vince, 2019 (link)).