R405 488 561 635

Single-molecule trace acquisition with improved throughput was performed on a custom-built wide-field fluorescence microscope built around an inverted Axiovert 200 stand (Zeiss, Germany). A 647 nm fiber laser with Gaussian-shaped emission profile (MPB Communications, Canada) was expanded to 6.0 mm and converted into a flattop beam using a beamshaper (πShaper AdlOptica Optical Systems GmbH, Germany) and further expanded to a diameter of 47 mm. The expanded beam was then guided into the microscope stand and focused on the back focal plane of a 100× 1.49 NA oil immersion objective (Apo TIRF; Nikon, Japan) objective. The variation in irradiance was below 15% across the entire illuminated area. Emitted signal was collected through the same objective, separated from excitation light using a quad-band dichroic filter (R405/488/561/635; Semrock, USA), and further filtered using a 405/488/532/635 nm notch filter (Semrock) and a 700/50 nm bandpass filter (Chroma, USA). Images were projected onto a back-illuminated sCMOS camera with a 130 × 130 µm field of view (Prime95B; Photometrics, UK). Samples were placed on a motorized stage (MS2000) and kept in focus using an autofocus system (CRISP; both Applied Scientific Instrumentation, USA). Camera and laser were synchronized using an Arduino Mega microcontroller board. All microscope components were controlled using µManager.