Imaging of the immune system – towards a subcellular and molecular understanding

Abstract

Immune responses involve many types of leukocytes that traffic to the site of injury, recognize the insult and respond appropriately. Imaging of the immune system involves a set of methods and analytical tools that are used to visualize immune responses at the cellular and molecular level as they occur in real time. We will review recent and emerging technological advances in optical imaging, and their application to understanding the molecular and cellular responses of neutrophils, macrophages and lymphocytes. Optical live-cell imaging provides deep mechanistic insights at the molecular, cellular, tissue and organism levels. Live-cell imaging can capture quantitative information in real time at subcellular resolution with minimal phototoxicity and repeatedly in the same living cells or in accessible tissues of the living organism. Advanced FRET probes allow tracking signaling events in live cells. Light-sheet microscopy allows for deeper tissue penetration in optically clear samples, enriching our understanding of the higher-level organization of the immune response. Super-resolution microscopy offers insights into compartmentalized signaling at a resolution beyond the diffraction limit, approaching single-molecule resolution. This Review provides a current perspective on live-cell imaging in vitro and in vivo with a focus on the assessment of the immune system.

A humanized β2 integrin knockin mouse reveals localized intra- and extravascular neutrophil integrin activation in vivo

“…In vitro studies with human neutrophils rolling on substrates of selectins and ICAM-1 (Fan et al, 2016;Yago et al, 2018) or on endothelial cells (Morikis et al, 2017) showed that arrest from rolling is associated with b 2 integrin activation. The best maps of activated integrins (Fan et al, 2016) were generated by quantitative dynamic footprinting (qDF) (Sundd et al, 2010;Wen et al, 2020), a method based on variable angle total internal reflection (TIRF) microscopy. Because the activation reporter antibodies work only for human and not for mouse integrins, all published data are for human neutrophils, lymphocytes, or cell lines.…”

Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip

“…More advanced models that also include components of the tumor stroma have been developed for evaluating drug and immune cell responses, although with limited screening capabilities ( Ayuso et al., 2021 ; Cui et al., 2020 ; Nashimoto et al., 2020 ). Despite recent developments of sophisticated cell culture tools combined with live cell imaging readout ( Ayuso et al., 2019 , 2021 ; Berg et al., 2019 ; Gopal et al., 2021 ; Lemon and McDole, 2020 ; Lin et al., 2020 ; Olofsson et al., 2018 ; Park et al., 2019 ; Shelton et al., 2021 ; Wen et al., 2020 ; Zhou et al., 2020 ), it is still rare with assays that manage to combine high-content screening over long periods of time with sufficient imaging quality to examine cellular behavior with single-cell resolution.…”

Innate immune receptor clustering and its role in immune regulation

“…The membrane clusters can be as small as a few nanometers in size, and their assembly is often transient and dynamic (Lenne et al, 2006;Sieber et al, 2007). Our understanding of receptor clusters has largely been propelled by the advancement of imaging microscopy techniques, such as super-resolution localization microscopy (Betzig et al, 2006;Dietz and Heilemann, 2019;Hess et al, 2006;Rust et al, 2006;Wen et al, 2020). The second challenge, after confirming the existence of receptor clusters, is to identify their roles in immune cell signaling.…”