Mai tai broadband

Extracellular parasites were fixed, laid onto coverslips, and processed for indirect immunofluorescence as previously described (48 (link)). The coverslips were mounted in ProLong Gold (Molecular Probes) and imaged using a TCS SP5 MP-STED confocal microscope (Leica Microsystems) as described (58 ). In brief, the ATTO 647N-labeled parasites were imaged on an avalanche photodiode (APD; PerkinElmer) using an oil immersion objective (HCX PL APO CS ×100/1.40-numerical-apterature STED), a 640-nm pulsed diode laser (PicoQuant) for excitation, and a 750-nm pulse (Mai Tai Broadband; Spectra-Physics) for STED depletion.

The non-linear optical microscope used in the experiment for Two-photon Excited Fluorescence (TPEF) data acquisition was based on a confocal imaging system (LSM510Meta, Carl Zeiss, Jena, Germany) coupled to an external tunable mode-locked Ti:Sapphire laser (Mai-Tai broadband, Spectra-Physics, USA)17 (link). The laser line was tuned to a 900 nm wavelength and routed by a dichroic mirror (reflect > 700 nm, transmit < 543 nm), through an objective lens (Plan-Neofluar, 20X, NA = 0.5, Carl Zeiss, Jena, Germany) to the tissue specimen. TPEF signals were collected by the same objective lens in the epi-mode, passing through the dichroic mirror (reflect < 490 nm, transmit > 490 nm) and a 500–550 nm band-pass (BP) filter, before being recorded by a photomultiplier tube (PMT, Hamamatsu R6357, Tokyo, Japan).