Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands

Abstract

Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses. Dysfunction of mitochondrial reprogramming can occur during bacterial and viral infections compromising hosts’ immune signaling. Comparative evaluation of these alterations in response to bacterial and viral ligands can provide insights into a cell’s ability to mount pathogen-specific responses. In this study, we used two-photon excitation fluorescence (TPEF) imaging to quantify reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) levels in the cell and to calculate the optical redox ratio (ORR), an indicator of mitochondrial dysfunction. Analyses were performed on RAW264.7 cells and murine bone marrow derived macrophages (BMM) stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. Responses were cell type dependent, with primary cells having significantly higher levels of FAD and higher oxygen consumption rates suggesting BMM may be more dependent on mitochondrial metabolism. Our findings also suggest that FAD-TPEF intensity may be a better predictor of mitochondrial activity and localization since it demonstrates unique mitochondrial clustering patterns in LPS vs. Poly(I:C) stimulated macrophages. Collectively, we demonstrate that TPEF imaging is a powerful label-free approach for quantifying changes in mitochondrial function and organization in macrophages following bacterial and viral stimuli.

Noninvasive morpho-molecular imaging reveals early therapy-induced senescence in human cancer cells

“…TPEF is a linear endogenous fluorescence signal arising from a third-order NLO excitation that scales linearly with the density of NAD(P)H and FAD fluorophores in the focal volume. Its signal can be exploited as an indication of the mitochondrial network location within cells, where metabolic activity is denser ( 40 ). Under near-infrared excitation (a 780-nm pump beam is used in this study), we collected endogenous TPEF signals by NAD(P)H and FAD ranging between 400 and 600 nm ( 41 ) (spectrally separated from CARS and E-CARS signals at 650 ± 20 nm).…”

Imaging immunometabolism in situ in live animals

“…The metabolic imaging of immune cells is more recent. Several studies reported the functional assessment of innate and adaptive immune cells in vitro by 2-photon microscopy of NAD(P)H and FAD [111][112][113][114][115][116][117][118][119][120] . These groups successfully performed intensity and lifetime imaging in human neutrophils, T and B cells, mouse immune cells including macrophages, neutrophils, dendritic cells, T and B cells, and several cell lines including THP-1 monocytes, RAW264.7 macrophages, and NK-92 cells, demonstrating that it is a valuable tool in immunometabolism.…”

Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy

“…A comparison of the advantages and disadvantages of each imaging technique can be found in work by Pallen et al [23]. The endogenous molecules that provide label-free multi-photon fluorescence signals in neuronal cultures are FAD and NADH [24,25]. Endogenous multi-photon fluorescence in living cells originating from NADH has already been explored to reveal the mitochondria [26].…”

“…In the present work, we took advantage of the properties of label-free non-linear microscopy (combined SHG and multi-photon autofluorescence) to monitor the different growths and differentiations of 50B11 cells on the upper and bottom sides (normal and inverted gravitational orientations) of glass substrates, comparing different time points (24,48, and 72 h).…”