(a) The time lapse microscope was constructed on the base of an Olympus IX81 microscope, using a 40x× objective lens (Air, NA 0.75). Two air-cooled electron-multiplying charge-coupled device (EMCCD) cameras were coupled to the camera port of the microscope via a magnifying relay. The cameras were simultaneously triggered using a TTL (transistor-transistor logic) pulse from an external digital-to-analog converter, with an exposure time of 50 ms and a frequency of 0.25 Hz. A dichroic mirror in the Fourier plane of the camera relay split the emission into red (fluorescence) and green (transmission bright-field imaging) channels. Each channel had a separate band pass filter. The focused laser spot was generated by expanding a collimated beam from a fiber-coupled continuous wave (CW) diode laser, injected via the epifluorescence port of the microscope and directed to the objective lens via a dichroic filter. The beam was expanded to overfill the back aperture of the objective lens to achieve a diffraction-limited spot at the focal plane. A combination of neutral density filters and a half-wave plate and polarizing beam splitter were used to adjust the power of the laser to approximately 0.01 μW. A 1:1 lens pair with one lens mounted on a Z-translation stage was used to adjust the axial position of the focus at the sample plane. A quarter-wave plate was used to circularly polarize the beam before it was injected into the microscope. The laser intensity profile is shown in (b). Bright-field trans-illumination was performed by fiber-coupling a 530 nm LED into a multimode fiber and imaging the magnified end of the fiber at the sample plane using a condenser lens. The LED was TTL triggered from via the camera acquisition to reduce light exposure to the cells, resulting in a synchronized 50 ms pulse during acquisition. A third LED (625 nm, 3 mW) was mounted close to the sample at an oblique angle to provide directional illumination for the motility assay. LED, light emitting diode.