888 emccd camera

Macrophages and bacteria were imaged using an Andor integrated (Andor, Belfast, UK) Metamorph-controlled (Molecular Devices, Sunnyvale, CA) Nikon TiE motorized microscope (Nikon Corporation, Tokyo, Japan) with a 20x, 0.75 NA phase objective. For Mtb RFP and mCherry fluorescence, excitation source was a 561 laser line and emission was detected through a Semrock Brightline 607 nm filter (Semrock, Rochester, NY). Images were captured using an 888 EMCCD camera ((Andor). Temperature (37°C), humidity and CO₂ (5%) were controlled using an environmental chamber (OKO Labs, Naples, Italy). Approximately 40 fields of view were captured every 10 min, one phase contrast image and one fluorescent image per field at every time point. Fluorescence readings after death were confirmed by widefield microscopy (data not shown), but fluorescence readings before and particularly at the point of cell death were strongly influenced by cell movement in the z-plane and are not included in the analysis. For imaging data after cell death, the fluorescence signal starting 4 hr after the cell death event was used for analysis. For DRAQ7 (BioStatus, Leicestershire, UK) entry, excitation source was a 640 nm laser and emission collected through a Semrock Brightline 685 nm filter For pH dye pHrodo detection, excitation source was a 488 laser while emission was collected through a Semrock 525 nm filter.

Macrophages and bacteria were imaged using an Andor (Andor, Belfast, UK) integrated Metamorph-controlled (Molecular Devices, Sunnyvale, CA, USA) Nikon TiE motorized microscope (Nikon Corporation, Tokyo, Japan) with a 20x, 0.75 NA phase objective. Images were captured using an 888 EMCCD camera (Andor). Temperature and CO₂ were maintained at 37°C and 5% using an environmental chamber (OKO Labs, Naples, Italy). For timelapse protocols, images were captured once every 10 min for the duration of the time-lapse. For each acquisition, images were captured at wavelengths applicable to fluorophores used in the analysis including transmitted light (phase contrast), 561 nm (RFP), and 640 nm (DRAQ7, lysotracker). Image analysis was performed using custom written matlab script. Single cell segmentation was manually carried out prior to fluorescent signal quantification. For each cell, fluorescent signal in each channel was quantified as pixel intensity.

Cell density was reduced to 7x10⁴ cells/ml and cells were attached to ploy-l-lysine (Sigma-Aldrich) coated 6-well optical plates (MatTek). Cell-free and coculture infections were imaged in tandem using a Metamorph-controlled Nikon TiE motorized microscope with a 20x, 0.75 NA phase objective in a biosafety level 3 facility. Excitation sources were 488 (GFP, YFP), 561 (mCherry), or 640 nm (CTFR) laser lines and emission was detected through a Semrock Brightline quad band 440–40 /521-21/607-34/700-45 nm filter. Images were captured using an 888 EMCCD camera (Andor). Temperature (37°C), humidity and CO₂ (5%) were controlled using an environmental chamber (OKO Labs). Fields of view were captured every 30 minutes and a minimum of 1000 target cells were acquired per condition. Threshold for detection of the onset of HIV gene expression was set so that no positive cells were detected in the uninfected control. Cells with above threshold expression were scored as positive.