Elyra 7 lattice sim microscope

M.tb strain H37Rv constitutively expressing mCherry was grown to the exponential phase in 7H9 media supplemented with 10 % OADC 0.5% glycerol and Coum-TAC, C2 or coumarin 466 were added at a concentration of 0.5 µg/mL. 18 hours later, each bacteria suspension was washed in PBS tween 0.05 % and fixed for 2 hours in PFA 4 %. An aliquot of the suspension was placed on a slide and included in Prolong Glass mounting media. Incorporation of compounds was quantified using a Zeiss Elyra 7 Lattice-SIM microscope with a Zeiss 63x NA=1.4 objective and image analysis was performed in acquired Z stacks using MicrobeJ plugin⁴⁸ in ImageJ Software. More specifically, mCherry fluorescence of M.tb was used to generate a smoothed particle contour of the bacilli using a skeletonization algorithm. Next, medial axes were generated from the particle contour and intensity profiles were calculated from the first pole (P₀) to the opposite pole (P_x) at regular levels. More than 100 individual bacteria were quantified per condition.