Microfluidic-Based Cell Sorting of <i>Francisella tularensis</i> Infected Macrophages Using Optical Forces

TLDR

The study examined whether brief exposures to focused 1064‑nm laser light during optical deflection cause photoinduced damage in mouse macrophages. The microfluidic fluorescence‑activated cell sorter uses an infrared laser to laterally deflect hydrodynamically focused cells, with activation assessed by ERK phosphorylation and NF‑κB translocation, and is applied to isolate fluorescently labeled Francisella‑infected macrophages. The µFACS system sorted green‑labeled macrophages at 22 cells/s with 93 % purity and 60 % recovery, and 10,738 infected cells at 11 cells/s with 93 % purity and 39 % recovery, while brief laser exposure did not affect cell viability or function.

Abstract

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (µFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-κB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10 738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.