Ccd axiocam icm1

Cultures of B. divergens at 30% parasitemia were stained with MitoTracker Red FM mitochondrial stain (Thermo Fisher Scientific, Eugene, OR) at a final concentration of 500 nM and according to the manufacturer’s instructions. Then, 10⁵ fluorescence-stained cells were deposited on the surface of Au-G200F1 finder grids coated with holey carbon (R2/2; Quantifoil) and functionalized with poly-l-lysine (Merck, Germany) and fiducial gold markers (100 nm; BBI Solutions, UK) used for tomographic alignment purposes. To conserve the cellular structures and membrane arrangements in close-to-native conditions, cells attached to the grids were cryo-fixed by plunge freezing in liquid ethane using a Leica EM CPC plunge freezer (Leica Microsystems, Germany). Vitrified grids were transferred in liquid nitrogen to the cryo-correlative cooling stage (CMS196 stage; Linkam Scientific Instruments, UK) to hold samples at a stable −190°C during analysis. The cryo-stage was inserted into an AxioScope A1 (Carl Zeiss, Germany) epifluorescence microscope with an N-Achroplan 10×/0.25 Ph1 objective and imaged with a CCD AxioCam ICm1 (Carl Zeiss). Cryo-fluorescence correlative microscopy was used to preselect vitrified samples and map cell coordinates. Selected samples were then transferred to ALBA synchotron (Barcelona, Spain) at liquid nitrogen temperature.

MCF-7 cells were cultured on Au-EM finder grids or Au-HZBII special grids for ALBA and HZB-BESSYII, respectively, coated with holey carbon (R 2/2; Quantifoil). When cells reached 70 % confluence, they were incubated with 0.25 mg ml⁻¹ SPION for different times. LysoTracker Red DND-99 and DAPI were added 10 min before in vivo imaging of the grids. An automatic map of the grid was generated using a Leica DMI6000B epifluorescent microscope with an incubation system, using a 20×/0.4 Fluorotar L dry objective with an OrcaR2 monochrome digital camera; 1–3 min were needed to generate a grid-map for Au-HZBII or Au-EM finder grids, respectively.
After in vivo imaging (<1 min), culture grids were fixed by plunge-freezing with an EM CPC vitrification unit (Leica Microsystems). Vitrified grids were transferred in liquid nitrogen to the cryo-correlative cooling stage (Linkam Scientific Instruments) to hold samples at a stable −190 °C during analysis. The cryo-stage was inserted into an AxioScope A1 (Carl Zeiss) epifluorescence microscope with a N-Achroplan 10×/0.25 Ph1 objective and imaged with a CCD AxioCam ICm1 (Carl Zeiss).
Cryo-fluorescence correlative microscopy was used to pre-select vitrified samples and map the position of cells containing nanoparticles. Selected samples were then transferred to the synchrotrons at liquid nitrogen temperature.

After infection, HeLa cells were fixed by plunge-freezing using a Vitrobot Mark IV (Thermo Fisher Scientific). Prior to vitrification, samples were slightly fixed with 1% PFA for 5 min to inactivate bacteria and imaged using an Axiovert Z1 driven by ZEN Blue 2.3 software (Carl Zeiss) epi-fluorescence microscope or a confocal microscope LSM710 (Carl Zeiss) driven by ZEN 2010 software. To stain for mitochondria, samples were incubated with 100 nM of Mitotracker Red (Invitrogen) for 30 min before vitrification. In all cases, grids were incubated with 100 nm fiducial gold nanoparticles (that would help during the alignment of the tilt series) for 30 s before vitrification. Following vitrification, grids were shipped to the Mistral beamline at the ALBA synchrotron (Barcelona, Spain). Vitrified grids were then transferred in liquid nitrogen to the cryo-correlative cooling stage Linkam CSM196 (Linkam Scientific Instruments) to hold samples at a stable −190°C during analysis. The cryo-stage was inserted into an AxioScope A1 (Carl Zeiss) epifluorescence microscope with a N-Achroplan 50×/0.6 Ph1 objective and imaged with a CCD AxioCam ICm1 (Carl Zeiss).
Cryo-fluorescence microscopy was used to pre-select vitrified samples and map the position of cells. Selected samples were then transferred to the Mistral synchrotron beamline at liquid nitrogen temperature.