Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

The overall goal of this optical imaging technique is to provide a means for rapid screening of novel bacteria-specific optical probes which may have clinical tractability for imaging infection within human lung. This method can help answer key questions in the molecular imaging field such as whether novel optical compounds are specific to their target and whether they are detectable. The main advantage of this technique is that it's translatable.

Selecting promising imaging probes using equipment that's available in the clinic provides confidence that the probes will work in that environment. Immediately before imaging, remove the human lung tissue sample from the freezer on dry ice. At room temperature, allow the tissue to thaw slightly so that it can be sliced with a scalpel into one by four millimeter sections.

Using forceps, place the sliced human lung tissue into wells of a 96-well clear flat bottom tissue culture plate. Return any unused human lung tissue immediately to the storage container and place on dry ice for transport back to the minus 80 degree Celsius freezer. Next, add 100 microliters of PBS to each lung tissue sample with a pipette to wash the blood from the sample.

Use the pipette tip to ensure all the tissue is covered in the PBS and not stuck to the walls of the well. Remove and discard the PBS. Then pipette 100 microliters of unstained Calcein AM-labeled or test probe-labeled bacteria to each well containing lung tissue.

Also, set up controls with lung tissue in 100 microliters of PBS. First clean the fibered confocal fluorescence microscopy imaging fiber connector unit with a fiber cleaner. Rub the connector on the cleaning ribbon in a forward motion to remove any dust and dirt.

Remove the protective yellow cap from the front of the laser scanning unit. Prepare the LSU hub by gently rotating the silver hub anti-clockwise until it stops. Insert the FCFM imaging fiber connector facing upwards.

Hold the fiber in place and rotate the silver hub clockwise until it clicks twice. Complete the connection by rotating the silver hub clockwise by a further 45 degrees. Next, follow the instructions that pop up for completing the FCFM imaging fiber calibration.

First, perform the FCFM imaging fiber test by pressing the start laser button on the screen. The laser will center. Select fresh vials from the calibration kit and follow the on-screen instructions.

Place the distal end of the FCFM imaging fiber into the yellow vial and watch for the increase in fluorescence on the monitor. Then place the fiber tip into the red vial without stirring. Wait for the fluorescence to disappear.

Finally, rinse the fiber tip in the blue vial. Next, perform background acquisition by placing the FCFM imaging fiber into the blue vial. Press start laser followed by calculate when it becomes an option.

Finally, perform fiber detection by placing the FCFM imaging fiber into the yellow vial. Again, press start laser followed by calculate. During the automated calibration, clean the distal end of the FCFM imaging fiber by placing in the red vial for greater than 10 seconds followed by the blue vial for at least four seconds as indicated by the software.

To begin data collection, click start on screen or press the left foot pedal to turn on the laser. Next, image each of the bacterial suspension samples. Insert the distal end of the FCFM imaging fiber and move the fiber slowly through the suspension to interrogate the sample.

Record videos of any length by pressing the right foot pedal or selecting the on-screen record controls as the fiber moves slowly around the sample. Clean the distal end of the FCFM imaging fiber with 8%hydrogen peroxide and lens cleaning tissues between samples. To image each of the lung tissue samples, insert the distal end of the FCFM imaging fiber into the sample ensuring that direct contact between the end of the fiber and the tissue is made.

Then gently move the end of the imaging fiber around to interrogate the sample. 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.

Record videos of any length by pressing the right foot pedal or selecting the on-screen record controls as the fiber moves slowly around the sample. Clean the distal end of the FCFM imaging fiber with lens cleaning tissues and 8%hydrogen peroxide between samples. To turn off the system, switch the laser off by pressing the left foot pedal or by clicking the on-screen button.

Next, disconnect the FCFM imaging fiber from the CLE device by turning the silver LSU hub anti-clockwise until it stops. Then remove the FCFM imaging fiber from the LSU hub by gently pulling the fiber connector from the LSU. Return the protective caps to the proximal end of the FCFM imaging fiber in the front of the LSU unit.

Clean and disinfect the FCFM imaging fiber with 8%hydrogen peroxide and lens cleaning tissues. Finally, place the fiber gently in the storage box. Open the software and import the files for analysis by selecting the appropriate directory on the computer through the go to icon on the software dashboard.

Double click on each video file to open them. The videos will automatically play with the optimized color lookup table and the color table adjust. Disable the automatic intensity scaling by clicking on the wand button above the intensity scale bar.

The feature is disabled when there is no black shadowing around the button. Select the desired intensity scaling by moving the minimum and maximum bars to give the best contrast. Use the histogram tool when selecting the intensity scaling to ensure the broadest dynamic range is captured.

Once the desired scaling has been achieved, right click over the dropdown menu button listed as default green. Select the option to save the lookup table. Save the lookup table to a desired location.

For each other video within the data set, apply the same lookup table by right clicking over the default green dropdown menu and selecting load lookup table. Select the appropriate file to apply consistent intensity scaling to all videos within the data set. Export the processed videos by clicking on the movie reel button.

Select the desired video format which will produce an MPG file. Then press export and choose the file location to save the video file. Labeled bacteria are visualized as green blinking dots in the video.

The lung tissue structure will be apparent as ordered fluorescence strands with alveolar space appearing black. Unlabeled Staph aureus bacteria do not fluoresce when imaged by FCFM demonstrating the requirement for optical imaging compounds. Staph aureus successfully labeled by fluorescing compounds undergoing screening are visible when imaged by FCFM.

The figure shows successfully labeled Staph aureus. Human lung tissue auto-fluoresces at 488 nanometers. The video shows human lung tissue imaged by FCFM.

Unlabeled Staph aureus were added to the tissue. They cannot be seen. However, successfully labeled bacteria can be seen against a human lung tissue background by FCFM imaging as small twinkling punctate dots.

Once mastered, this technique can be done in 10 minutes depending on the sample number. While attempting this procedure, it's important to remember to clean the fiber thoroughly when performing setup calibrations and in between sample imaging. After watching this video, you should have a good understanding of how to set up the FCFM device for imaging of biological samples and how to process the captured data.

Following this procedure, other methods like CFU plating can be performed in order to answer additional questions like determining bacterial cell number and viability post-labeling. Don't forget that working with human tissue and pathogenic bacteria can be hazardous and precautions such as the use of personal protective equipment should always be taken while performing this procedure.