Cm100 transmission electron microscope

Mature gametocytes of NF54 WT and the 7-Helix-1-KO line 2E6 were enriched via Percoll purification and activated as described above. Samples were collected at 0 and 30 min p.a. and fixed with 1% v/v glutaraldehyde and 4% w/v paraformaldehyde/PBS (pH 7.4) overnight at 4°C. Post-fixation of the specimens was performed with 1% v/v osmium tetroxide and 1.5% w/v K₃Fe(CN)₆ in PBS for 2 h at RT, followed by incubation in 0.5% w/v uranyl acetate for 1 h. For dehydration of the specimens, increasing concentrations of ethanol (70%/80%/95%/100%) were used, followed by an incubation step for 1 h in propylene oxide and another 1 h incubation step in a 1:1 mixture of propylene oxide and Epon (Sigma-Aldrich). Subsequently, specimens were embedded in Epon at 60°C for 48 h. Ultrathin sections were cut with a Leica ultramicrotome Ultracut UCT and post-stained with 1% w/v uranyl acetate for 30 min and 0.2% w/v lead citrate for 15 s. Examination of the sections was performed using a CM100 transmission electron microscope (FEI) and images were recorded digitally with a Quemesa TEM CCD camera and iTEM software (Olympus Soft Imaging Solutions). Alternatively, samples were analysed with a Zeiss EM10 transmission electron microscope and the photographs taken were scanned and processed using Adobe Photoshop CS software.

Phage samples for electron microscopy were prepared from phage stocks (originated from a single plaque) according to previously published protocols [22 ]. Phage suspensions were subsequently precipitated with polyethylene glycol (PEG) and purified using centrifugation in a CsCl gradient [22 ]. Bacteriophage particles were visualized using a negative staining method. Briefly, phage suspensions (5 μl) were placed on glow discharge activated carbon-coated grids [28 (link)]. After an adsorption period (30 s), the unabsorbed liquid material was removed using filter paper. The electron-microscopic grids were then stained with 10 μl of 1% phosphotungstic acid, 2% uranyl acetate, or 2% ammonium molybdate for 10–30 s; excess staining solution was removed using filter paper. Samples were viewed using a MORGAGNI 268D (FEI, Hillsboro, OR, USA) or a Philips CM 100 transmission electron microscope (FEI).

Spheroids were incubated for 24 h with 3.67 µM mTHPC or 50 nm M-Lipidots and irradiated for 1 min as described above. One hour after light treatment they were washed and fixed and sequentially treated with OsO₄ and uranylacetate. After dehydration they were embedded in Epon/Araldite and sections were contrasted with uranyl acetate and lead citrate. They were examined with a CM100 transmission electron microscope (FEI, Eindhoven, The Netherlands) or with an Auriga 40 scanning electron microscope (Zeiss, Oberkochen, Germany). For a more detailed description see Additional file 1.