Ixon 897 ultra em ccd camera

JM136 harboring pXY018 was imaged using two split channels in widefield on an Olympus IX-71 microscope. JF646 was imaged with 647 set to ~50 W/cm² and GFP-ZapA was imaged with 488 set to ~5 W/cm². The channels were split with an Optosplit II system containing 600 rdc, with a 700/55 emission filter for JF646 and 540/30 emission filter for GFP-ZapA. We used an Andor iXon 897 Ultra EM-CCD camera with an APON100×OTIRF objective (1.49 NA/oil) and engaged 1.6× optivar. Our camera’s EM-Gain was turned on to 300 with a pre-amplifier gain setting on 3 and digitizer set to 16-bit. Baseline clamp was activated, with baseline offset set to 100.

An inverted microscope (Nikon Ti-E) in combination with a CFI Apo TIRF 100 × 1.49 NA objective (Nikon) was used. Bright-field and fluorescence images were acquired using an iXon 897 Ultra EMCCD camera (Andor) coupled to an additional 2.0 × lens (Diagnostic Instruments DD20NLT). Phase-contrast imaging was performed with an Infinity 2–5 M camera (Lumenera). To track JF549 bound to HaloTag, a 553 nm laser (SLIM-553, Oxxius, 150 mW) with a power density of 3 kW/cm² on the sample plane was used in stroboscopic illumination mode with 3 ms laser pulses per 30 or 60 ms camera exposures. The microscope was controlled using the μManager software package. Data acquisition from multiple positions was performed using custom μManager plugins.