Single-Molecule Fluorescence Imaging Techniques

Cells were spotted on a 2% agarose pad made with 0.22 µm-filtered M2G. Imaging was performed at room temperature. All images were acquired on either an N-STORM microscope (Nikon) or a custom-built setup assembled on a commercial Axio Observer D1 microscope stand (Carl Zeiss). The N-STORM microscope was equipped with a CFI Apo TIRF 100 × oil immersion objective (NA 1.49), lasers emitting at 405 nm and 561 nm (Agilent Technologies, Santa Clara, CA) and a built-in Perfect Focus system. Raw single molecule data were taken in a field of view of ∼43 × 43 µm² (256 × 256 pixels) with an Andor iXon X3 DU 897 EM-CCD camera (Andor Technology, South Windsor, CT) at a frame rate of 28.5–29 frames per second (fps). Under this acquisition condition, diffusing molecules appeared blurred and were rejected from our analysis. The custom-built microscope set up (Huang et al., 2013 (link)) was equipped with a 100 × /1.46-NA oil-immersion objective (alpha Plan-Apochromat 100 × /1.46 oil, Carl Zeiss), lasers emitting at 405 nm (50 mW, CrystaLaser, Reno, Nevada) and 568 nm (Innova 300, ∼400 mW, Coherent, Santa Barbara, CA). Fluorescence was recorded with an ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics) at a frame rate of 100 fps and a field of view of 23 × 23 μm².