Tp 110r 100

All measurements were performed under an inverted microscope (IX71; Olympus) equipped with an objective lens (LUCPLFLN 40×PH, N.A. 0.6, LUCPLFLN 60×PH, N.A. 0.7 and UPLANSAPO 100×PH, N.A. 1.4; Olympus), a CMOS camera (Zyla 4.2; Andor, DMK33U174; Imaging Source, and FASTCAM Mini AX; Photoron), and an optical table (HAX-0806; JVI). The microscope was kept at 30°C by heating the room for the observation of M. pneumoniae cells, but this heating was not applied for M. penetrans and M. mobile. The microscope stage was heated with a thermoplate (TP-110R-100; Tokai Hit) for examining the temperature dependency of gliding speed. Projection of the image to the camera was acquired with imaging software that came with the CMOS camera and converted into a sequential TIFF file without any compression. All data were analyzed by ImageJ v1.48 (rsb.info.nih.gov/ij) [55 (link)] and its plugins particle tracker [56 (link)] and multitracker.

All procedures were performed at 45°C on a microscope stage heated with a thermoplate (TP-110R-100; Tokai Hit, Japan). The cell culture was poured into a tunnel chamber assembled by taping a coverslip (Nakane and Nishizaka, 2017 (link)), and both ends of the chamber were sealed with nail polish to keep from drying the sample. The position of the cell was visualized by infrared light from a halogen lamp with a bandpass filter (FBH850/40; Thorlabs) at a fluence rate of 1 μmol m⁻² s⁻¹. The cells were subjected to lateral light stimulus by an LED from the right side of the microscope stage at an angle of 5°. White LEDs at 20 and 500 μmol m⁻² s⁻¹ were used as moderate and strong light stimuli for phototaxis, respectively. Blue, teal, green, orange, red, and far-red light were applied by a monochromatic LED, M450LP1, M490L4, M530L3, M625L3, and M730L4 (Thorlabs), respectively. The LED light was collimated by the condenser lens and combined by dichroic mirrors (FF470-Di01, FF509-FDi01, FF560-FDi01, FF685-Di02; Semrock) to apply multicoloured light simultaneously. The wavelength of the resultant light was measured by a spectrometer (BIM-6002A, BroLight, China). Light intensity was measured with a power metre (Q82017A; Advantest, Japan).