Label free autofluorescence imaging permits comprehensive and simultaneous assignment of cell type identity and reveals the existence of airway secretory cell associated antigen passages (SAPs)

Abstract

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a properly orchestrated physiologic response. Thus, modern approaches to dissect physiologic mechanism would benefit from an ability to identify specific cell types in live tissues and image them in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 2 or 3 cell types. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in real time. Furthermore, we find that this methodology for direct cell type specific identification avoid potential pitfalls with the use of ostensibly cell type-specific markers that can be altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate realtime physiology using a clinically relevant model of cholinergic stimulation and identify dynamic secretory cell associated antigen passages (SAPs) that are highly reminiscent of previously reported goblet cell associated antigen passages (GAPs) in the intestine.

Single-cell transcriptomics of a dynamic cell behavior in murine airways

“…However, a similar approach can take advantage of different methods of cell labeling and live imaging, including genetic labeling of single cells with fluorescent reporters ( Figure 1—figure supplement 1b ). The requirement for fluorescence is another constraint of this approach, although label-free imaging approaches may be used to visualize the behavior of individual cells in native tissues ( Shah et al, 2022 ). Another consideration is that long-term live imaging itself can influence gene expression and cell behavior.…”

Label free metabolic imaging to enhance the efficacy of chimeric antigen receptor T cell therapy

“…FAD displays an opposite trend, with free and protein-bound FAD having a long and short lifetime, respectively (29)(30)(31). OMI provides single-cell resolution to characterize heterogeneity within population and identify important cell subsets while offering non-invasive, label-free, and real-time readouts of cell metabolism ( 26,32 ). Here, we test whether OMI measurements can inform manufacturing conditions that improve gene transfer efficiency and metabolic fitness of CRISPR-edited anti-GD2 CAR T cells for neuroblastoma treatment.…”

Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy

“…FAD displays an opposite trend, with free and protein-bound FAD having a long and short lifetime, respectively (27)(28)(29). OMI provides single cell resolution to characterize heterogeneity within population and identify important cell subsets (24,30), while offering non-invasive, label-free, and real-time readouts of cell metabolism.…”