Label-free single cell metabolic imaging of neutrophil activation

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Presentation Time: 02:15 PM - 03:30 PM
Poster Board Number: B704
Abstract ID: 5334

Presenting Author:
Rupsa Datta , Associate Scientist at Morgridge Inst. for Res.

Abstract:

Upon activation, neutrophils combat infiltrating pathogens through a variety of effector functions, including oxidative burst, phagocytosis, and neutrophil extracellular traps (NETs). Metabolic pathways within neutrophils can be pharmacologically affected to modify immune outcomes. However, there is a lack of techniques to assess neutrophil metabolism in a label-free, non-destructive manner at single cell level. To address this, human neutrophil metabolism was investigated using two-photon microscopy of intrinsic fluorophores reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and flavin adenine dinucleotide, FAD. We image the optical redox ratio (ORR), [NAD(P)H / (NAD(P)H + FAD)] and the fluorescence lifetimes of NAD(P)H. Single-cell metabolic profiles of neutrophils were generated using an automatic cell segmentation pipeline. We found that activation of neutrophils either pharmacologically or by pathogen infection induced a significant metabolic change characterized by an increase in ORR and decrease in NAD(P)H lifetime, indicating an increase of the NAD(P)H pool, which suggests increases in glycolysis and the pentose phosphate pathway. Neutrophil activation was confirmed by measuring effector functions including oxidative burst. Importantly, identical metabolic changes were detected in the neutrophils of transgenic zebrafish larvae following activation, confirming the validity of these findings in vivo.