Ff685 di02

When the microscope is configured for multicolor imaging, two additional filters are added to the detection path. The microscope was designed to discriminate fluorophores with strongly overlapping emission spectra by means of ratiometric spectral detection^{12 (link),34 (link)}. Two dichroic mirrors (Semrock, FF685-Di02), placed at an acute angle of 34° with respect to the fluorescence beam path, serve as polarization-selective long-pass spectral filters. The placement of the filters (DM3) is shown in Extended Data Fig. 1, and the filter spectra are shown in Supplementary Fig. 25. In this design, the ratio of s-polarized to p-polarized photons detected on the camera was ~0.6 for Alexa Fluor 647, and ~0.9 for Cy5.5, and this ratio may be used to distinguish the two dyes in the data analysis step. In the multicolor configuration, the bandpass filter (Chroma HQ700/75) was removed from the detection path.

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).