Zyla 4.2 plus

In vitro fertilization assay was performed as previously reported^{5 (link)}. 10 μl of blood from mouse infected with 28R/GTA strain was diluted and incubated in a gametogenesis inducing medium for 15 min or overnight, at 21 °C. The precipitated cells in the medium were recovered after incubation, and observed using a fluorescence microscope (AxioImager M2, Zeiss). The movement of mature male gametes was captured and recorded using a high-sensitivity CCD camera Zyla 4.2 PLUS (Andor).

As shown in Figure 1(c), we installed a CMOS camera (Zyla 4.2 Plus, Andor, USA) for fluorescence imaging and a CMOS camera (DFK 33UX264, Imagingsource, USA) for microsphere tracking on an upright microscope (BX51WI, Olympus, Japan) with a 60x objective lens (NA: 0.7, Olympus, Japan). A white light source was combined with a blue bandpass filter (FL488-10, Thorlabs, USA) to act as an excitation source, and the fluorescence camera was combined with a green bandpass filter (FB530-10, Thorlabs, USA) to receive the fluorescence light from the sample. To capture both images simultaneously, we installed a 50:50 beam splitter (CCM1-BS013, Thorlabs, USA) at the intersection point between the two cameras.

The morphology of ZnO and ZnO-DOPC nanoparticles was studied by Field Emission Scanning Electron Microscopy (FESEM, Auriga and Merlin, Karl Zeiss, Oberkochen, Germany). The diluted samples were spotted on a silica wafer and coated by a thin layer of Pt for further imaging. The particle size and Zeta potential of the three samples were determined using the Dynamic Light Scattering (DLS) technique (Zetasizer Nano ZS90, Malvern, Worcestershire, UK), while the crystalline structure was analyzed by X-ray diffraction with a X’Pert diffractometer in configuration θ–2θ Bragg-Brentano using a Cu-Kα radiation (λ = 1.54 Å, 40 kV and 30 mA).
To confirm the formation of the supported lipid bilayer on the surface of ZnO-DOPC nanoparticles, fluorescence co-localization experiments were performed. The DOPC shell was labeled with 1% Bodipy-DHPE lipid by incubating this dye (0.2 µg per mg of lipids) with the dispersed DOPC lipids prior to assembly. ZnO nanoparticles, after amine functionalization using APTMS, were labeled with Atto550-NHS ester dye (2 µg per mg of NPs) overnight under stirring at RT and then washed twice with fresh ethanol. A fully-motorized wide-field inverted microscope Nikon Eclipse TiE (Nikon, Tokyo, Japan), in combination with a high resolution sCMOS camera (Zyla 4.2 Plus from Andor) and an immersion 60× oil objective was used.

Microscopic inspection and image acquisition were performed using an Eclipse Ti-E inverted microscope (Nikon) equipped with 40x/(0.95 N.A.) air objective. A LED light source (SOLA SE II, Lumencor) was used for fluorescence excitation. YFP fluorescence was excited with a 470/40 filter, and emission was collected with a T495lpxr dichroic mirror and a 525/50 filter. Propidium iodide fluorescence was excited with a 560/40 filter, and emission was collected with a T585lpxr dichroic mirror and a 630/75 filter (filters and dichroic mirror from Chroma). A motorized encoded scanning stage (Märzhäuser Wetzlar GmbH) was used to collect multiple stage positions. In each well, five xy positions were randomly chosen, and 5 × 5 adjacent fields of view (with a 5% overlap) were scanned. Images were acquired with an SCMOS camera (ZYLA 4.2PLUS, Andor). NIS Elements 5.02 software was used for acquisition and basic image processing.

12-well plates were mounted on a stage top without warming (room temperature) during image acquisition. Images were collected using an Eclipse Ti-E inverted epi-fluorescence microscope (Nikon) equipped with a Plan Apo 20x/0.75 NA air objective and the Perfect Focus System for maintenance of focus. An LED light source (SOLA SE II, Lumencor) was used for fluorescence excitation. Rhodamine B-marked fluorescent beads were excited through a 560/40 filter, and emission was collected with a T585lpxr dichroic mirror and 630/75 filter (filter cube #49008, Chroma). Images were acquired with a SCMOS camera (ZYLA 4.2PLUS, Andor Technology, Ltd.) controlled with NIS Elements 5.02 software (Nikon Instruments Inc.). 10 × 10 adjacent fields of views (covering a total area of 2.82 × 2.82 mm) were monitored per each well. Multiple stage positions were collected using a motorized encoded scanning stage (SCANplus IM 130 × 85, Märzhäuse).