Image: live microscopy of neutrophil swarming around Zymosan bacterial target.
Analysis of neutrophil swarming imaging consumed the majority of my fourth week at MGH CEMS. As mentioned in my week 1 reflections post, in order to control the spread of infection, neutrophils perform ‘swarming,’ a process by which they cooperate to send signals and gather around pathogens, restricting potential growth (swarming imaging shown above). MGH has pioneered novel assays that allow researchers to study neutrophil swarming, and so we use this cutting-edge technique to visualize the swarming patterns of neutrophils after being treated with varying concentrations of lipopolysaccharide (LPS).
Scientists use these novel assays to analyze swarming by placing controlled quantities of target pathogens particles on a micropatterned surface. While neutrophils begin by moving randomly on the surface of the array (the “scouting” phase), as they begin to arrive at the target they guide subsequent neutrophils with mediators (the “growth” phase). As they swarm around the target, they begin to release neutrophil extracellular traps (NETs) which contain antimicrobial peptides, trapping and killing bacteria.
Our swarming experiments use 3 different factors, each with two different levels; the two targets are Candida albicans (fungus) and Zymosan (bacteria), the different treatments are low LPS and high LPS concentration, and the different medias are patient serum and fetal bovine serum (FBS), a typical media for in vitro cell culture. In order to analyze the swarming patterns and the effects of the different treatments, one must use a microscope with fluorescent imaging capabilities. The microscope has different fluorescent “channels,” each of which measures a separate feature of the swarming. The first channel, DAPI, is impermeant and marks the outside of live cells, allowing one to measure the swarming area of the neutrophils. The second channel, SYTOX, stains genetic material, so can be used to measure neutrophil death/NETosis as the cells lyse and release their cellular contents. Our final channel, brightfield, is used to measure microbial growth of the target.
For our experiment, the microscope gathered images of each well every 5 minutes for 12 hours. Once imaging is obtained from the microscope, there are few programs for automated swarming analysis. In order to gather neutrophil area data, one must manually segment the swarm area in each frame of every image sequence. To analyze the fluorescent intensity, one can download a program (called a “macro”) that reports the intensity of each frame within a given highlighted region.
The majority of my time this week has been spent doing such area and intensity analysis. It may seem like a tiring process, but the lengthy data-gathering feels extremely worth it when I get to see the fruits of my labor, the graphical analysis. Nothing is more rewarding than finally being able to see my long Excel sheets condensed into easily comprehensible visuals. To learn more about the procedure of creating, using, and analyzing neutrophil microarrays, refer to this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948161/.
Comentarios