Plan apo 1.4 na

The amount of blue light delivered to the cells was normalized throughout all experiments using a power meter to measure the amount of light output at the 20× and 60× objectives of widefield and confocal microscopes, respectively. On the Nikon TE2000E widefield microscope using a 20× with a 0.75 numerical aperture (NA) lens and an FITC HyQ filter from chroma, blue light output was measured at approximately 12.8 microwatts (μW) at the objective. The Nikon A1R confocal microscope at 4% of total output power of the 488 nm laser delivered 12.5 to 13 μW of light through a 60× Plan Apo 1.4 NA objective lens. Nikon Elements Advanced Research imaging software (Nikon instruments) was used for the automated exposure of blue-light pulses to the cells. To administer the blue light stimulation to cells using the widefield microscope, a 30-s pulse was delivered to the entire plate. In live-imaging experiments such as uptake, the 488-nm laser was set to 4% of total power output to deliver a 30-s pulse to the cells before initiating the experiment.

Wildtype or ∆mmpL7 M. marinum expressing wasabi fluorescent protein were grown in 7H9 medium supplemented with 10% OADC, 0.2% glycerol with or without 0.005% alkyne-cholesterol or azido-PE for 48 hr. Bacteria were then washed 3x with PBS prior to detection with fluorescent probes. For azido-PE, we followed the Copper-free click chemistry of M. marinum protocol above using DIBO-647. For alkyne-cholesterol we performed a copper-click reaction with AlexaFluor-647 Azide. For copper click: 400 µM BTTP (Click Chemistry Tools) and 200 µM copper sulfate (Sigma) were dissolved in PBS and allowed to complex for 20 min. 30 µM AlexaFluor-647 Azide (Thermo-Fisher) and 1.2 mM sodium ascorbate (Sigma) were then added to the solution. Bacterial pellets in 96-well v-bottom plates were resuspended in 50 µl of the solution and were incubated at 23°C protected from light for 45 min. Bacteria were then washed 5x in PBS prior to imaging on a Nikon A1R confocal microscope with a 60x oil-immersion Plan Apo 1.4 NA objective. Nikon elements software was used to determine fluorescent intensities of wasabi and cholesterol signals calculated from line profiles drawn perpendicular to bacterial membranes at least 0.5 µm from either pole.