C. reinhardtii cells, approximately 10 μm in diameter, were grown to the early exponential phase in tris-acetate-phosphate medium at 296 K on a rotary shaker (100 rpm). Five microliters of cell suspensions was dropped onto a plasma-treated Si3N4 window (200 nm thick, 1.5 mm by 1.5 mm membrane area). The window was then mounted onto a Vitrobot Mark IV plunge freezer (FEI), where the temperature and humidity were set and equilibrated at 22°C and 100%, respectively. The window was blotted for 2 s with a blot force of 0 and then immediately plunged into a liquid ethane bath cooled by liquid nitrogen (LN2). Cells were embedded in an ice layer with a thickness of 1.2 ± 0.2 μm. The cryogenically prepared samples were observed using a cryogenic light microscope and then stored in LN2 until they were retrieved for use in the x-ray microscope.

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