Frappa system

Fluorescence microscopy was carried out essentially as described by Ringgaard et al. (2013) (link), Heering and Ringgaard (2016) (link), Alvarado et al. (2017) (link), and Heering et al. (2017) (link). Bacterial strains for fluorescence microscopy analysis were inoculated in LB medium and cultivated at 37°C and shaking to an OD₆₀₀ = 0.5–0.6. Cells were then spotted on a pad of 1% agarose in 50% PBS + 10% LB on a microscope slide, covered with a coverslip and imaged immediately. All microscopy was performed on a Nikon Eclipse Ti inverted Andor spinning-disk confocal microscope equipped with a 100x lens and an Andor Zyla sCMOS cooled camera and an Andor FRAPPA system. Microscopy images were analyzed using ImageJ imaging software¹ and Metamorph Offline (version 7.10.2.240, Molecular Devices). FlhF-sfGFP fusion was imaged at 400 ms exposure, and sfGFP-FlhG at 1000 ms for all backgrounds. Demographs were constructed by measuring the fluorescence intensity profiles in Fiji and processing the data in R (3.0.1, R Foundation for Statistical Computing), using a script described by Cameron et al. (2014) (link), Alvarado et al. (2017) (link), Heering et al. (2017) (link), and Muraleedharan et al. (2018) (link).

Live-cortical neurons were imaged in ACSF at 34 ˚C on an Olympus IX71 equipped with a spinning disc scan head (Yokogawa) with a 60x NA1.4 objective. Excitation illumination was delivered from an AOTF controlled laser launch (Andor) and images were collected on a 1024 × 1024 pixel Andor iXon EM-CCD camera. Data acquisition was performed with Metamorph (Molecular Devices) or Andor IQ software. For some experiments a spinning disk Marianas live-cell imaging system (3i) running Slidebook software (3i) was used. Photoconversion of mEOS3.2 expressing cells was carried out using targeted illumination (FRAPPA system, Andor) with the 405 nm laser (1 s dwell time 5% laser power). For most experiments, a 7 µm Z-stack (0.5 µm step-size) was acquired at each time point.