Obm1 100

Coffee beans were “Azabu blend” bought from Azabu Kobo in Japan. 240 mL of boiled water at around 80 °C was poured into 20 g of the coffee powders in a virgin pulp coffee filter (HARIO, Japan). To make a sweet coffee, 4 g of white caster sugar (Nissin Sugar Co., Ltd., Japan) was added to 60 mL of coffee. As 97.8% of the sugar is sucrose, the sucrose concentration is about 200 mM. The drop (0.5 μL) was evaporated on a ceramic dish plate (Lasagna dish L, Aeon, Japan) pre-cleaned by water. During the evaporation, temperature and relative humidity were kept within 298 ± 5 K and 30 ± 5%, respectively. The evaporative deposits were imaged by a Olympus SZXV upright microscope with SZ2-LGB illuminator and acquired by a camera (NY-X6i, Canon). The length was measured using a calibrated ocular micrometer (OBM1/100, Olympus).

COS-7 cells were transfected with sfGFP-P2A-Rep-miRFP-Sec61β/pcDNA3.0. sfGFP was used as a transfection marker to identify transfected cells and expression level. The single-molecule imaging system was based on an inverted microscope (ECLIPSE Ti, Nikon) equipped with an sCMOS camera (ORCA-Flash4.0, Hamamatsu Photonics), a quad-band 405/488/561/640 dichromic mirror and a 732/68 and 525/45 emission filter (Semrock). The ORCA-Flash4.0 sCMOS camera was controlled by HCImageLive 4.3.1.33 (Hamamatsu Photonics). Objective-based HILO illumination was achieved using a 640 nm laser (OBIS 640 nm LX 100 mW, Coherent) and an Apo TIRF 100×/1.49 oil-immersion objective lens (Nikon). Single-molecule image track was acquired with an exposure time of 200 ms at a frame rate of 5 Hz. The irradiance of 640 nm laser at the sample plane was 28.9 W cm⁻². Laser power was measured with a laser power meter (power meter console, PM400, Thorlabs; photodiode sensor, S120VC, Thorlabs). Size of the illumination area was measured with an objective micrometer (OB-M#, 1/100, Olympus).