Autofluorescence imaging permits label free cell type assignment in unmodified airway and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs)
- Massachusetts General Hospital, United States
- Harvard Medical School, United States
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 physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged 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 3 or 4 cell types at a time. 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 mouse tracheal explant in real-time. Furthermore, we find that this methodology for direct cell type specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of secretory and goblet cell associated antigen passages (SAPs and GAPs) enable luminal antigen sampling. Given that airway secretory cells can be stimulated to make mucous within hours, we suspect that both SAPs and GAPs are also used for luminal antigen sampling in the airway. This hypothesis is supported by our observation that secretory cells with airway SAPs are frequently juxtaposed to antigen presenting cells.
Data availability
All data is included in the manuscript and generated custom scripts are included on https://github.com/vss11/Label-free-autofluorescence.
Article and author information
Author details
Funding
Cystic Fibrosis Foundation (003338L121)
- Viral S Shah
NHLBI Division of Intramural Research (5R01HL142559)
- Charles P Lin
- Jayaraj Rajagopal
NHLBI Division of Intramural Research (RO1HL118185)
- Jayaraj Rajagopal
NHLBI Division of Intramural Research (1R01HL157221)
- Jayaraj Rajagopal
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Mice were maintained in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the Massachusetts General Hospital (MGH). All procedures were performed with Institutional Animal Care and Use Committee (IACUC)-approved protocols (#2009N000119). MGH is accredited by AAALAC International, has an assurance with the Office of Laboratory Animal Welfare (OLAW) and is registered with the United States Department of Agriculture (USDA). Euthanasia was performed via house line CO2-mediated asphyxiation and confirmatory cervical dislocation consistent with the recommendations of the American Veterinary Medical Association.
Copyright
© 2023, Shah et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Autofluorescence imaging permits label free cell type assignment in unmodified airway and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs)
eLife 12:e84375.
https://doi.org/10.7554/eLife.84375
