Differences in forward angular light scattering distributions between M1 and M2 macrophages

Abstract

The ability to distinguish macrophage subtypes noninvasively could have diagnostic potential in cancer, atherosclerosis, and diabetes, where polarized M1 and M2 macrophages play critical and often opposing roles. Current methods to distinguish macrophage subtypes rely on tissue biopsy. Optical imaging techniques based on light scattering are of interest as they can be translated into biopsy-free strategies. Because mitochondria are relatively strong subcellular light scattering centers, and M2 macrophages are known to have enhanced mitochondrial biogenesis compared to M1, we hypothesized that M1 and M2 macrophages may have different angular light scattering profiles. To test this, we developed an in vitro angle-resolved forward light scattering measurement system. We found that M1 and M2 macrophage monolayers scatter relatively unequal amounts of light in the forward direction between 1.6 deg and 3.2 deg with M2 forward scattering significantly more light than M1 at increasing angles. The ratio of forward scattering can be used to identify the polarization state of macrophage populations in culture.

Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis

“…Conversely from other literature works that have tackled macrophagic polarization using spectroscopic tools addressing specific light-matter interactions ( e . g ., fluorescence lifetime imaging microscopy 26 , 27 , forward angular light scattering 28 ) for the establishment of decision support systems based on chemometry, the proposed approach exploits a simpler physical phenomenon, namely Vis-NIR reflectance 54 , for the analysis of cells in vitro . As a major advantage, our system has the potential for easy integration into routine equipment (microscopes), or even into cytometric setups, and is in principle compatible with other wide-spectrum light sources, such as cost-effective metal halide lamps.…”

“…Some of these changes, for example in the nuclear structure, in the spatial distribution and metabolic activity of mitochondria, or in the spatial distribution and overall expression of cytoskeletal proteins, can be monitored non-invasively for phenotype and functional characterization. To this end, several spectral approaches have been developed and are evolving, using different spectral ranges and molecular fingerprinting 26 – 28 . In this context, Hyperspectral Imaging (HSI) has emerged as a powerful set of techniques that integrate conventional imaging and spectroscopy 29 , 30 .…”

Single cell sequencing unraveling genetic basis of severe COVID19 in obesity

“…Though very similar to the M1 phenotype, the macrophage phenotype encountered in severe cases of COVID-19, also shows some features of M2 macrophages such as the expression of Proliferated Activated Receptor gamma agonists (PPARγ). ( Halaney et al, 2015 ).…”

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

“…This method may be used in conjunction with a recently reported label-free technique that distinguishes inflammatory and healing macrophages based on their differences in angular light scattering. 22 Our method is based on two-photon fluorescence lifetime imaging microscopy (FLIM) of NADH, and can readily be extended to in vivo studies, which is not feasible for light-scattering measurements. In addition, the phasor approach to FLIM simplifies the data analysis process to the visual inspection of clusters, especially if measured lifetimes arise from complex multiexponential decays.…”