Imaging Bacterial Localization and Host Interaction in Human Lung Tissue

Abstract

Source: Mills, B., et al., Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy. J. Vis. Exp. (2017)

This video demonstrates real-time imaging of fluorescently labeled Staphylococcus aureus in suspension and lung tissue using fibered confocal fluorescence microscopy.

Protocol

1. Data collection with Confocal laser endomicroscopy (CLE)
   1. Following the setup, a window to select the storage location and file prefix will open. Select the desired folder for data storage, and name the prefix accordingly.
   2. Place the foot pedals so that they can be easily accessed by the operator. Left pedal: laser on/off; center pedal: pause; right pedal: record/stop.
      NOTE: The laser controls can also be accessed through onscreen controls.
   3. Click 'start' onscreen or press the left foot pedal to turn on the laser. This will start acquisition and obtain images using 100% laser power and a frame rate of 12 frames/s (default settings).
      NOTE: For other applications, these settings can be altered on screen if necessary, depending on the sample type.
   4. Image each of the bacterial suspension samples.
      1. Insert the distal end of the FCFM (Fibered confocal fluorescence microscopy) imaging fiber and move the fiber slowly through the suspension to interrogate the sample.
      2. Record videos of any length (up to 10 min) by pressing the right foot pedal or selecting the onscreen record controls, as the fiber moves slowly around the sample.
      3. Clean the distal end of the FCFM imaging fiber with 8% H₂O₂ and lens cleaning tissues between samples.​
         NOTE: Typical video lengths of 10-30 s are sufficient for in vitro imaging.
   5. Image each of the lung tissue samples.
      1. Insert the distal end of the FCFM imaging fiber into the sample, ensuring direct contact between the end of the fiber and the tissue. Gently move the end of the imaging fiber around to interrogate the sample.
         NOTE: Lifting the end of the fiber away from the tissue will remove the tissue from the focal plane; however, this may be used to image labeled bacteria that are not adhered to the tissue.
      2. Record videos of any length (up to 10 min) by pressing the right foot pedal or selecting the onscreen record controls, as the fiber moves slowly around the sample.
         NOTE: Typically, video lengths of 30 s are sufficient for ex vivo imaging on tissue.
      3. Clean the distal end of the FCFM imaging fiber with lens cleaning tissues and 8% H₂O₂ between samples.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-14 Microfuge	SciQuip	90616	Small benchtop microcentrifuge
96-well plate	Corning	3370	Assay plate
Calcein AM	Sigma Aldrich	17783	Commercial fluorescent dye
Cellvizio 488 nm Research CLE	Mauna Kea Technologies	LC-0001-488	Confocal laser endomicroscopy device
Cletop-S	Cletop	14110601	Fibre cleaner
Eppendorf (1.5 mL)	Eppendorf	30120086	1.5 mL microfuge tube
Gibco Phosphate Buffered Saline	Thermo Fisher Scientific	10010023	PBS - wash media
IC-Viewer	Mauna Kea Technologies	LW-0001	Data collection and processing software for the research CellVizio 488 nm system
Non-standard research AlveoFlex 488 nm	Mauna Kea Technologies	MP-0002-AF3	Fibred confocal fluorescence microscopy fibre
Quanti Kit 488 nm	Mauna Kea Technologies	LQ-0005	Calibration kit for the CellVizio 488 system
S. aureus ATCC 25923	ATCC	25923	Bacterial strain used in this study
Semi-micro spectrophotometry cuvette	Sigma Aldrich	C5416-100EA	For spectrophotometry
Thermomixer comfort	Eppendorf	41102422	Benchtop heater with shaking