Lf405 488 561 635 4x a 000

Imaging was performed on an Olympus IX81-XDC inverted microscope with a cellTIRF module, a 100X UAPO TIRF objective (NA=1.49), active Z-drift correction (ZDC) (Olympus America). Images were acquired at 20 frames per second on an iXon-897 EMCCD camera (Andor). Excitation of Alexa-647 was accomplished using a 640 nm diode laser (CUBE 640-75FP, Coherent) and 561 nm laser excitation. Laser energy density at sample is estimated to be 3kW/cm² (561 nm), 14 kW/cm² (640 nm), and 20 kW/cm² (530 nm). Excitation and emission was filtered using the quadband dichroic mirror LF405/488/561/635-4X-A-000 for 561 nm excitation, or LF405/488/561/635-4X-A-000 for 532 nm excitation (Semrock). Emission was split into two channels using a DV2 emission splitting system (Photometrics) with a T640lpxr dichroic mirror to separate emission, ET605/52m to filter near-red emission, and ET700/75m to filter far-red emission (Chroma). Samples were imaged in a buffer containing: 30 mM Tris, 100 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 50 mM glucose, 12 mM glutathione, 40 µg/ml catylase, and 500 µg/ml glucose oxidase.