Fluorescence microscopy

Overnight strains harboring Mini-CTX-clpV2-sfGFP vector for expression of the ClpV2-sfGFP fusion protein were diluted to an OD₆₀₀ of 1.0 with fresh LB medium and grown at 37°C for 1 h. The cells were then harvested, re-suspended in PBS, and were placed on 1% agarose pads and examined immediately at room temperature. A Nikon Ti2-E inverted microscope with a perfect focus system and a CFI Plan Apo Lambda × 100 oil Ph3 DM (NA 1.4) objective lens were used for imaging. Intensilight C-HGFIE (Nikon), ET-GFP (49002, Chroma) filter sets were used to excite and filter fluorescence. NIS-Elements AR 5.20.00 was used to record and manipulate the images.