Brightline hc

Imaging was done with a widefield imaging system based on an Olympus IX83 inverted microscope body equipped with a home-made image splitter coupled to a sCMOS camera (ORCA Flash V2, Hamamatsu) as sketched in Fig. 1. Excitation was done in epifluorescence mode by a supercontinuum white light laser (Fianium) coupled to a high power AOTF (Fianium), which was controlled through an FPGA-RT unit (National Instruments) coded with Labview. This unit synchronized the alternated laser excitation with the camera acquisition. Images were acquired at 37 °C with Micromanager and a 40x objective. The donor fluorophore was excited at 442nm (power 200 muW), the acceptor at 515nm (power 240 muW). The fluorescence emission was first separated from the excitation via a triple line beamsplitter (Brightline R442/514/561 Semrock) in the microscope body. The fluorescence emission was further splitted with a beamsplitter at 510nm (Chroma) and filtered with a 475/50 filter (BrightLine HC, Semrock) for the donor channel and a 519/LP longpass filter (BrightLine HC, Semrock) for the acceptor channel. Hence, in two camera snapshots, four images were obtained with all combinations of donor/acceptor excitation and donor/acceptor emission.

Dual imaging was performed using an upright microscope (BX51WI; Olympus) and a 10 x objective (UplanSApo, NA 0.4; Olympus). For bright-field imaging, light emanating from a near-infrared (780 nm) LED (M780LP1 and driver LEDD1B; Thorlabs) was filtered using a (785/62 BrightLine HC; Semrock) and projected onto the sample via the bright-field illumination condenser. To excite fluorescence, the Teal line from an LED lamp (Spectra X light engine; Lumencor) was filtered (513/17 BrightLine HC; Semrock) and projected onto the sample using a 520 nm long-pass dichroic (FF520-Di02; Semrock). Transmitted and emitted light were filtered using a 532 long-pass filter (BLP01-532R; Semrock). To simultaneously record images in bright-field and fluorescence, a dual-camera device was used (DC²; Photometrics). Light was split into two channels using a 695 long-pass dichroic mirror (695DCXRUV; Photometrics) and images were projected into two cameras (acA3088-57um; BASLER). Fluorescent light was band-pass filtered (550/49 Brightline HC; Semrock) before reaching the camera sensor. The exposure time (6ms) of one camera served to synchronize the acquisition of the second camera and the Lumencor light engine. Individual worms were manually tracked using a 3-axis motorized stage (X-LSM150A; Zaber).

An inverted wide-field microscope (Leica DMI6000B) setup equipped with a high numerical aperture lens (HCX PL APO 100×/1.47 OIL CORR TIRF) and an EMCCD-camera (Andor Technology) was used for single-molecule imaging. The image size was set to 120 × 120 pixels with a pixel size of 160 nm. Measurements were done by illumination for 10 ms with a 515 nm laser beam (Cobolt) at an intensity of 2 kW cm⁻². For trypanosomes expressing HASP::eYFP or kinesin-MORN::eYFP pre-bleaching was performed if necessary until single molecules could be monitored. 8000–10,000 consecutive images were recorded at 28 Hz. Signals were detected by the camera using appropriate filter combinations (zt405/514/633rpc (dichroic, Chroma) and 550/49 BrightLine HC (emission filter, Semrock)). eYFP signals were singled-out by exploiting the intrinsic blinking properties of eYFP at high illumination intensities (Fölling et al., 2008 (link), Lee et al., 2011 (link)).