All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Strains and culture conditions

Note: M. tuberculosis strain CDC1551 is used in this study, but any M. tuberculosis strain can be used in the same manner.

1. Grow the bacteria in M-OADC-TW (7H9 broth supplemented with 0.5% glycerol, 10% oleic acid dextrose complex without catalase, and 0.05% Tween-80) medium standing at 37 °C to an OD₆₀₀ of 0.5 (~ 0.2 x 10⁷ colony-forming unit or CFU).
2. Dilute the culture in M-OADC-TW (series of 1:10 dilutions of the bacteria). Plate the dilutions of the bacteria (10⁵, 10⁶, 10⁷, 10⁸) in triplicate onto selective 7H11 plates to allow determination of CFU.
3. Incubate the plates for four weeks at 37 °C or until colonies can be accurately counted.
4. Centrifuge the bacterial inoculum at 8,534 x g for 5 min to obtain a pellet. Wash the pellet once with 10 mL of saline (0.9% NaCl) and re-suspend the pellet in 15 mL of saline (0.9% NaCl).

2. Aerosol infection of mice using a Madison Chamber

1. Allow mice to acclimate to the new surroundings for a week.
2. Weigh the mice before loading them into the chamber.
3. Plug three cords into the power strip: the main chamber power, the vacuum pump, and the air compressor, in that order.
4. Carefully unscrew the glass jar. Bring the glass jar to the biosafety cabinet and add the challenge inoculum suspended in 15 mL of saline (0.9% NaCl) to achieve ~ 10⁴ – 10⁶ CFU of bacteria in the lungs.
5. Close the lid of the glass jar in the biosafety cabinet. Attach the jar to the nebulizer unit and adjust the vertical stainless steel tube so that the lower (intake) end is about a quarter of an inch below the level of the fluid in the jar.
6. Load all the animals needed for the experiment (the chamber can hold up to 90 mice) into the chamber and close all the latches on the door.
7. Check the main (room) air flow meter. Ensure that the center of the float (ball) runs about 50 L/min as measured on the scale on the left.
8. Press the Start button on the control panel of the chamber. Set the airflow rate through the compressor air flow meter (the smaller meter on the left) as 4 L/min on the scale. Check visually to ensure that the challenge inoculum is being nebulized.
9. After 15 min, when the red light on the front of the control panel appears and an audible signal indicates the end of the run, press the Reset button on the lower right corner of the control panel to reset the timers.
10. Hold down the small red button on the door of the chamber to release the vacuum.
11. Open the chamber door and remove the animals. Place the mice back into their cages.
12. Remove the glass nebulizer jar and place it in a sealed, leak-proof transport container. Place the container inside the bio-safety cabinet and discard the challenge suspension into a designated waste container.
13. Place the used nebulizer jar inside a biohazard bag and seal the bag for transport to the autoclave.
14. At the end of the infection procedure, spray the inside of the chamber with buffered phenol and 70% ethanol and allow the chamber to sit for 10 min.
15. Then, wipe down all accessible interior surfaces very thoroughly. Dispose of all contaminated paper towels, etc. in the biohazard trash. Autoclave the trash, waste containers, and used nebulizer jars.
16. Place the animals back in the containment room until the imaging time-point.
17. On the day of imaging, transfer the animals in a secondary container to the imaging room.

3. Animal anesthesia

1. Anesthetize the mice with isoflurane using a customized gas anesthesia system.
2. Weigh each of the two charcoal filter canisters located on top of the anesthesia unit.
3. Replace with a new canister if the weight is 50 g above the initial weight.
4. Check the vaporizer unit to ensure sufficient isoflurane for the procedure.
5. Place the nose cone holder inside the imaging chamber. Place the number of nose cones required for the procedure and seal the remaining openings with the nose cone blockers.
6. Turn on the oxygen supply from the high-pressure cylinder and set it at 55 psi.
7. Turn on the evacuation pump located in front of the anesthesia unit and set it to 8 L/min.
8. Turn on the oxygen toggle located in front of the anesthesia unit.
9. Turn on the gas flow to the anesthesia induction chamber to set the flow of gas at 1.5 L/min. Turn the gas flow off.
10. Turn on the gas flow to the imaging chamber to set the flow of gas at 0.25 L/min. Turn the gas flow off.
11. Turn on the isoflurane vaporizer and set it at 2 – 2.5%. Adjust the isoflurane level according to the number and weight of animals being used for the experiment by rotating the dial on the isoflurane vaporizer (2 – 2.5% for a mouse weighing ~20 g; 4% for a guinea pig weighing 300 g).
12. Place the mice in the anesthesia chamber and close the lid. Turn on the gas flow to the anesthesia induction chamber.
13. Leave the mice in the anesthesia induction chamber for 5 – 10 min until completely anesthetized.
14. Once the mice are anesthetized, apply an optical ointment to the eyes to protect them while imaging.
15. Place the mice in ventral or sternal recumbency such that their noses are placed in the nose cone to facilitate the anesthesia of mice during the imaging procedure.

4. Reporter enzyme fluorescence (REF) imaging

1. Inject the substrate (20 µM, 2.5 µL/g of weight) by intraperitoneal injection into the infected as well as the control mice. The mice are under anesthesia inside the imaging chamber for less than 1 minute.
2. Start the imaging system.
3. Initialize the system by clicking on Initialize. Wait until the temperature bar turns green.
   NOTE: The imaging chamber consists of a heated platform to maintain the body temperature of the animal while imaging.
4. For imaging acquisition setting, select Fluorescent | Trans-illumination for whole animal or Epi-illumination for lung tissues, Structure and Overlay in Acquisition Control Panel.
5. Set the Field of view to B for a single mouse and the lamp level to high.
6. Set the exposure time to auto, medium binning, 2 – 3 for the f/stop, and excitation filter at 745 nm and emission filters from 780 nm to 840 nm.
7. For sequence setup, click on Sequence Setup, and select 9 – 12 Trans-illumination points in the lung area.
8. Click on Acquire for image acquisition.
9. Place the mouse back into the cage post-imaging.
10. Monitor the mice until fully recovered or sacrifice them for quantifying CFU if imaging at a time-point of the experiment. Perform a modified Karnofsky score to monitor the well-being of mice post-imaging.