Tillphotonics fei imic digital spinning disk microscope

Activation of KillerRed was conducted on the TillPhotonics/FEI iMIC digital spinning disk microscope (FEI Munich) with the widefield modus (WF Stingray F-145B) under stable environmental conditions (37 °C and 5% CO₂). Time-lapse acquisition was performed with a 60×/1.35 NA oil-immersion objective from Olympus and the Hamamatsu ORCA-R2 camera. The binning was set to 2 × 2 pixel. Photo-activation was performed with 20% laser power of a 561 nm laser diode in a diffraction-limited ROI after acquiring two pre-activation images. The dwell time was set to 1.0 ms/µm² and the line overlap to 41%. KillerRed was activated at 24 hpi and parasites were imaged overnight (16 h).

Photo-conversion experiments were conducted with HeLa cells transiently expressing LAMP1-mEos3.2 infected with PbWT (24 hpi) on the TillPhotonics/FEI iMIC digital spinning disk microscope (FEI Munich) with the widefield modus (WF Stingray F-145B) under stable environmental conditions (37 °C and 5% CO₂). Time-lapse acquisition was performed with the 60×/1.35 NA oil-immersion objective from Olympus and the Hamamatsu ORCA-R2 camera. The binning was set to 2 × 2 pixel. After acquiring two images, photo-conversion was performed with 15% power of a 405 nm laser diode in a diffraction-limited ROI. The dwell time was set to 1.0 ms/µm² and the line overlap to 75%. Fluorescence was captured every 2 min., excited with 488 nm and 561 nm wavelengths and emission collected through a multi band dichroic 405/490/561/640 nm filter. Intensities were measured with the FIJI Software. Fluorescence intensity was normalized to the first post-activation image.