Figure 1.

Construction and characterization of a cryo-light microscopy stage. (A) An overview of the cryo-stage (red box) mounted on an Olympus IX71 fluorescence light microscope. The three labeled tubes are for liquid nitrogen input (1), liquid nitrogen overflow protection (2), and dry nitrogen gas flow to the objective lens (3). (B) Autocad drawing of the cryo-stage as viewed from top and side. Dimensions are marked in inches. (C) The design of the cryo-sample stage chamber in 3D drawing. The cartridge containing the EM grid is placed in the inner chamber (arrow). (D) An enlarged photo of the inner chamber with an actual specimen cartridge (arrowhead). (E) Cooling profile of the cryo-stage measured on the specimen cartridge. The arrows indicate the time when filling of liquid nitrogen from a pressurized tank occurred. The cartridge reaches − 171 °C within 4 minutes, and stabilizes at − 187 °C within 6 minutes. (F & G) Fluorescence images of GFP-tagged HIV-1 particles (F) and Quantum Dot (G) (acquisition time 10 s). Scale bar, 10 μm. Inserts depict the average of normalized intensity profiles of 10 particles.