Diaphot 200

Caulobacter cells are around 0.5-µm diameter by 2- to 4-µm long, which is on the same order of magnitude as the diffraction limit (∼250 nm). To accurately track movements of single proteins in live cells, we use superlocalization to pinpoint the location of single molecules where photobleaching was used to reduce the emitting concentration (44 (link), 48 (link), 79 (link)). Live cells were immobilized on agarose pad that was bleached overnight before the experiment. Single-molecule imaging experiments were performed on a custom epifluorescence microscope (Nikon; Diaphot 200) equipped with a Si EMCCD camera (Andor; iXon DU-897) and a high numerical aperture (N.A.) oil-immersion objective (Olympus; UPlanSapo, 100×/1.4 N.A.). Molecules were excited with either 514-nm, 1-W continuous-wave laser or 642-nm, 1-W continuous-wave laser (MPB Communications) and photoactivated by a 405-nm Obis laser at 0.8 kW/cm², 0.65 kW/cm², and 0.1 to 1 W/cm², respectively. The emission from fluorescent molecules was collected through a four-pass dichroic mirror (Semrock; Di01-R405/488/561/635) and filtered by a 514-nm long-pass filter (Semrock; LP02-514RE), a 560-nm dichroic beam splitter (Semrock; FF560-FDi01), a 561-nm notch filter (Semrock; NF03-561E), and a bandpass filter (Semrock; FF01-532/610) as previously described (48 (link), 79 (link)).

Cells were imaged on a custom epifluorescence microscope using a Nikon Diaphot 200 as core, equipped with an Andor Ixon DU-897 electron-multiplying charge-coupled device camera, a high-NA oil-immersion objective (UPlanSapo 100×/1.40 NA; Olympus), a motorized xy-stage (M26821LOJ; Physik Instrumente), and a xyz-pizeo stage (P-545.3C7; Physik Instrumente). Molecules were excited with a 642-nm, 1-W continuous-wave laser (MPB Communications Inc.). The emission was passed through a quadpass dichroic mirror (Di01-R405/488/561/635; Semrock) and filtered using a ZET642 notch filter (Chroma) and a 670/90 bandpass filter (Chroma). For 3D imaging, DH (Double Helix Optics) and Tetra6 phase masks (described in ref. 42 (link)) were inserted into the 4f-system of the microscope as described previously (43 (link)).