Cellsense dimensions v 1.14

Single-cell static images and movies were captured on an Olympus IX83 inverted microscope using a 100×, 1.3-numerical-aperture phase-contrast objective. Fluorescence images were obtained with a Hamamatsu ORCA-R² digital charge-coupled-device camera, and the light source was the Xcite 120 light-emitting diode (Lumen Dynamics, Mississauga, Ontario, Canada). Emission filters were purchased from Chroma Technology (Bellows Falls, VT). Specific emission filters were DAPI-5060C-OMF (excitation [EX] filter, 377/50 nm; emission [EM] filter, 447/60 nm; dichroic mirror [DM], 409 nm), GFP-3035D-OMF (EX filter, 473/31 nm; EM filter, 520/35 nm; DM, 495 nm), mCherry-B-OFF (EX filter, 562/40 nm; EM filter, 641/75 nm; DM, 593 nm). Images were processed with the Olympus software package cellSense Dimensions (v 1.14). The V. vulnificus fliM-tdTomato fusion was cloned into pSU38 (27 (link)) using the Gibson assembly kit (New England Biolabs, Ipswich, MA), and expression was induced with 0.1% l-Ara (Sigma-Aldrich). Where indicated, cells were stained with DAPI for 5 min prior to the addition of 6-carboxyfluorescein (FAM)−SMAP-29D.

Single-cell static, time-lapse images and movies were captured on an Olympus IX83 inverted microscope using a 100×, 1.3-numerical-aperture phase-contrast objective. Phase-contrast and fluorescence images were obtained with a Hamamatsu ORCA-R2 digital charge-coupled-device camera, and the light source was the Xcite 120 light-emitting diode (Lumen Dynamics, Mississauga, ON, Canada). Emission filters were purchased from Chroma Technology (Bellows Falls, VT). The specific emission filters were DAPI-5060C-OMF (excitation [EX] filter, 377/50 nm; emission [EM] filter, 447/60 nm; dichroic mirror [DM], 409 nm), GFP- 3035D-OMF (EX filter, 473/31 nm; EM filter, 520/35 nm; DM, 495 nm), mCherry-B-OFF (EX filter, 562/40 nm; EM filter, 641/75 nm; DM, 593 nm). Images were processed with the Olympus software package cellSense Dimensions (v 1.14). Data from three biological replicates were analyzed for each strain. Images presented are from a single representative experiment.