Rajagopal, 2022 Goblet Cell SAP

TOOLS AND RESOURCES
Autofluorescence imaging permits label-

free cell type assignment and reveals the
dynamic formation of airway secretory
cell associated antigen passages (SAPs)
Viral S Shah1,2†, Jue Hou3†, Vladimir Vinarsky2, Jiajie Xu2, Manalee V Surve2,
Charles P Lin3*, Jayaraj Rajagopal1,2,4,5*
1
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine,
Massachusetts General Hospital, Boston, United States; 2Center for Regenerative
Medicine, Massachusetts General Hospital, Boston, United States; 3Advanced
Microscopy Program, Center for Systems Biology and Wellman Center for
Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston,
United States; 4Harvard Stem Cell Institute, Cambridge, United States; 5Klarman Cell
Observatory, Broad Institute, Cambridge, 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
*For correspondence:
techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which
[email protected]
(CPL);
only allow the study of three or four cell types at a time. We report a non-invasive imaging modality
[email protected] (JR) 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
These authors contributed
of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explants
equally to this work
in real time. Furthermore, we find that this methodology for direct cell type-specific identification
Competing interest: The authors avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered
declare that no competing by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time
interests exist. physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in
Funding: See page 17 response to cholinergic stimulus. The identical process has been well documented in the intestine
Received: 22 October 2022
where the dynamic formation of SAPs and goblet cell associated antigen passages (GAPs) enable
Preprinted: 01 November 2022 luminal antigen sampling. Airway secretory cells with SAPs are frequently juxtaposed to antigen
Accepted: 29 March 2023 presenting cells, suggesting that airway SAPs, like their intestinal counterparts, not only sample
Published: 30 March 2023 antigen but convey their cargo for immune cell processing.
Reviewing Editor: Paul W

Noble, Cedars-Sinai Medical
Center, United States Editor's evaluation
The study addresses a significant impediment to deriving maximal value from living tissue culture
‍ ‍Copyright Shah, Hou et al.
This article is distributed under systems, which is simultaneously distinguishing and mapping the activity of multiple constituent cell
the terms of the Creative types over the course of experimental perturbations. The authors present a label-free approach that
Commons Attribution License, involves collecting autofluorescence and morphological features that provide distinct signatures for
which permits unrestricted use mouse tracheal epithelium and demonstrate its application for live imaging of secretory cells. This
and redistribution provided that platform may be very valuable for answering specific experimental questions about tracheal cell
the original author and source behavior in disease.
are credited.
Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 1 of 20

Tools and resources Cell Biology
eLife digest Imaging several cell types, at the same time, within a living tissue is no small endeavor.
To do so, scientists usually have to perform genetic manipulations that make certain proteins in each
cell type fluorescent and therefore easy to track. However, these approaches are cumbersome,
limited, and often not applicable to intact human tissues.
A possible alternative would be to make use of autofluorescence – the fact that certain molecules
in living cells naturally fluoresce when exposed to a particular wavelength of light. For example, this
is the case for NAD(P)H and FAD, two small molecules necessary for life’s biochemical processes, and
whose intracellular levels and locations vary depending on cell type.
In response, Shah, Hou et al. developed a new imaging technique that takes advantage of the
unique autofluorescence signatures of NAD(P)H and FAD to distinguish between the seven different
types of cells that line the surface of the airways of mice.
Using their autofluorescence approach, Shah, Hou et al. were also able to discover a new role for
secretory cells, which normally produce fluids, mucus and various proteins necessary for the lungs to
work properly. The imaging experiments show that these cells could also sample material from the
surface of the airway in a manner similar to how cells in the intestine take up material from the gut and
pass their cargo to immune cells that mediate infection control or tolerance. Further studies should
uncover more details about this new function of secretory lung cells. Other exciting discoveries may
also emerge from researchers adopting the method developed by Shah, Hou et al. to examine a
range of organs (both healthy and diseased), and attempting to apply it to human tissues.
Introduction
The airway epithelium is composed of myriad distinct cell types, many of which have only been recently
described (Deprez et al., 2020; Goldfarbmuren et al., 2020; Montoro et al., 2018; Plasschaert
et al., 2018). Studying the function of each of these cells and their contribution to the physiology
of the entire epithelial ensemble often requires cell type-specific genetic perturbation. Indeed, even
the simple identification of a given class of cells within a live tissue requires genetic labeling with a
fluorophore. Currently, cells are most commonly identified post-fixation using immunohistochemical
and immunofluorescence staining, but this prohibits the assessment of real-time cellular dynamics and
thus real-time physiology. Since post-fixation cell identification is destructive, it also precludes the
study of long-term processes or the effect of multiple serial stimuli which are both essential features of
normal organ physiology. Additionally, markers used for cell type identification in unperturbed tissue
can often be non-specific or misleading after injury. We reasoned that a methodology for identifying
and tracking diverse cell types within an organ in real time in a non-destructive fashion would permit
the study of physiologically relevant processes that are currently hidden from view.
Label-free imaging based on endogenous fluorescence of metabolic cofactors has been deployed
to measure cell metabolism in various tissues, particularly in tumors and immune cells (Katz et al.,
2002; Skala et al., 2007). NADH, NADPH, and FAD, the major endogenous fluorophores, have been
used to monitor metabolism in vitro and in vivo (Gil et al., 2019; Heikal, 2010; Huang et al., 2002;
Kasischke et al., 2004; Rocheleau et al., 2004; Sepehr et al., 2012; Tiede et al., 2007). Since NADH
and NADPH cannot be distinguished by their autofluorescence, the combination has been reported
as an aggregate quantity, NAD(P)H (Schaefer et al., 2019). To account for the effects of light scat-
tering and variation in the number of mitochondria, a metabolic ratio of FAD/(NAD[P]H+FAD) is often
employed (Chance et al., 1979). Two-photon excited fluorescence microscopy can be used to measure
this metabolic ratio in intact tissues and in vivo (Dilipkumar et al., 2019; Klinger et al., 2012; Kreiß
et al., 2020). Indeed, abundant common intestinal epithelial cell types can be distinguished based on
their morphology when imaged in this manner (Kreiß et al., 2020). Most recently, these measures of
metabolic activity have been used to identify immune cells in differing functional states during fluo-
rescence activated cell sorting (FACS) ex vivo (Lemire et al., 2022). Furthermore, immune cell interac-
tions have been visualized using autofluorescence in the murine trachea (Kretschmer et al., 2016). In
this manuscript, we describe a mouse tissue explant system that readily permits the label-free identifi-
cation of abundant epithelial cell types in living airway tissue. Furthermore, a combined assessment of
the cell resolution fine features of autofluorescence signatures can be coupled to cell morphometric

measurements to permit the identification of exceedingly rare epithelial cell types. We also use live
autofluorescence imaging to reveal the existence of secretory cell associated antigen passages (SAPs)
in the airway. The biology of such structures has only recently been dissected in the gut where they
have been referred to as both SAPs and goblet cell associated antigen passages (GAPs) (Gustafsson
et al., 2021; Gustafsson and Johansson, 2022; Knoop et al., 2015; McDole et al., 2012; Noah
et al., 2019). We describe the real-time dynamics of the formation of these structures using physi-
ologic cholinergic stimulation and their association with antigen-presenting cells (APCs). Finally, we
demonstrate that autofluorescence signatures can be used for live cell tracing. Indeed, under some
circumstances, this technique is more reliable than the use of ostensibly cell type-specific markers
whose expression can be altered by injury, physiologic stimulus, and in disease states.
Results
Label-free autofluorescence-based live imaging of an intact murine
tracheal explant
We hypothesized that we would be able to specifically assign airway epithelial cell type identity by
directly imaging specific metabolic signatures at single-cell resolution. In order to preserve subtle and
complex features of normal cellular and tissue architecture, we developed a mouse tracheal explant
setup that allows upright two-photon imaging in a physiologic chamber over days (Kwok et al.,
2022; Figure 1A and Figure 1—figure supplement 1A). To facilitate optimal detection and sepa-
ration of NAD(P)H and FAD, the samples were scanned at 730 nm and 900 nm sequentially (Huang
et al., 2002). A short pass 505 nm dichroic was used to separate the NAD(P)H and FAD signal, and
each fluorescence signal was detected using 450/100 nm (NAD(P)H) and 540/80 nm (FAD) band pass
filters, respectively (Supplement 1B). Using this setup, we ascertained NAD(P)H and FAD autofluo-
rescence emissions across the excitation spectrum (Figure 1B). Due to the curvature of the trachea,
we performed digital ‘flattening’ during image analysis where the subepithelial region was linearized
to the same Z plane. To accomplish this, we utilized second harmonic generation (SHG) imaging of
collagen fibers to identify the basement membrane (SHG signal was excited with a 900 nm laser) and
developed a MATLAB algorithm to ‘flatten’ the epithelial surface (https://github.com/vss11/Label-
free-autofluorescence, copy archived at Shah, 2023).
To verify that the measured autofluorescence signals reflect cellular metabolism in real time, we
utilized pharmacologic agents to experimentally alter NAD(P)H and FAD. Treatment with rotenone
and antimycin A, inhibitors of mitochondrial complex I and complex III, lead to a decrease in the
FAD/(NAD[P]H+FAD) fluorescence ratio at the airway surface as predicted from similar experiments
conducted in breast cancer cells (Figure 1C; Hou et al., 2016). Treatment with FCCP, a mitochondrial
uncoupler, led to an anticipated increase in autofluorescence intensity ratio, as similarly previously
documented (Figure 1D; Hou et al., 2016). In order to determine signal stability, we measured the
autofluorescence intensity ratio over 2 days of tracheal explant culture. The signal remained stable
(Figure 1E).
Autofluorescence imaging discriminates the three common airway

epithelial cell types and demarcates fine details of cell shape at the
single-cell level
As previous studies have been able to visualize common cell types in the intestinal epithelium using
autofluorescence (Kreiß et al., 2020), we first optimized our label-free imaging approach to identify
the common airway epithelial cell types: ciliated cells, secretory cells, and basal cells. We first measured
and recorded autofluorescence using live tissue and then subsequently identified cell types in that
same tissue following fixation. Specifically, autofluorescence was first imaged in live tissues, locations
were registered to subepithelial landmarks, and the same fixed tissue was subsequently stained with
conventional markers to identify cell types. Live autofluorescence signatures were then associated
with each cell that was identified by conventional staining. In order to register autofluorescence live
images to that of fixed and stained images, we imaged the SHG signals of the subepithelial collagen.
These signals served as fiduciary marks which facilitated exact localization of individual cells. We first
visualized ciliated cells with live autofluorescence imaging given their very unique morphology. As
seen in Figure 2A, ciliated cells have a robust FAD signal which is primarily located near the luminal

A B 1.5
Normalized Emission
NAD(P)H FAD
Trachea 1.0
O-ring
Semi-permeable Suture
0.5
Membrane
0.0
Insert 720 760 800 840 880 920 960
Wavelength (nm)
Antimycin (Mitochondrial Complex 1 inhibitor) +
C Baseline Rotenone (Mitochondiral Complex 3 inhibitor)
i ii iii
1.0
✱✱✱✱
0.8
0.6
Ratio
0.4
0.2
0.0
Baseline Antimycin
+
FAD NAD(P)H SHG FAD NAD(P)H SHG
Rotenone
D Baseline FCCP (Mitochondrial Uncoupler)

i ii iii
2.0 ✱✱✱✱
1.5
Ratio
1.0
0.5
0.0
Baseline FCCP
E Day 0 Day 1 Day 2

i ii iii iv 1.0 NS
0.8
0.6
Ratio
0.4
0.2
0.0
0 1 2
Days
FAD NAD(P)H SHG FAD NAD(P)H SHG FAD NAD(P)H SHG
Figure 1. NAD(P)H and FAD autofluorescence can be measured from murine tracheal epithelial cells and is stable across days. (A) Mounting of tracheal
explant for upright two-photon imaging. Semipermeable membranes (12 mm Transwell, 0.4 µm Pore Polyester Membrane Insert) were inverted and
fixed to custom 3D printed inserts in 60 mm dishes using silicone adhesive. Trachea were dissected and sutured onto O-rings for additional stability
(A inset and Figure 1—figure supplement 1A). Trachea and O-ring were then placed onto the semipermeable membrane for upright imaging and
Figure 1 continued on next page

Figure 1 continued
explant culture. Inset showing a trachea sutured to O-ring in a zoomed view. (B) Autofluorescence emission normalized to maximal emission at different
excitation wavelengths for both NAD(P)H and FAD. (C) NAD(P)H (blue), FAD (yellow), and second harmonic generation (SHG, gray) autofluorescence
images of murine tracheal explant at (i) baseline, (ii) with the addition of antimycin (10 µM – mitochondrial complex I inhibitor) and rotenone (1 µM
– mitochondrial complex III inhibitor) showing increased NAD(P)H fluorescence, and (iii) quantification of autofluorescence ratio (FAD/FAD +NAD[P]
H) demonstrating a decrease. Each point is a single cell. N=20. (D) NAD(P)H (blue), FAD (yellow), and SHG (gray) autofluorescence images of murine
tracheal explant at (i) baseline, (ii) with the addition of carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP – mitochondrial uncoupler) showing
decrease in NAD(P)H fluorescence and an increase in the metabolic ratio (iii). Each point is a single cell. N=20. (E) Time course images of NAD(P)
H (blue), FAD (yellow), and SHG (gray) of the same region of a tracheal explant at day 0 (i), day 1 (ii), and day 2 (iii) showing similar fluorescence. (iv)
Quantification of ratio (FAD/FAD +NAD[P]H) of individual cells showing stability across 0, 1, and 2 days. N=15. ****p<0.0001 by paired t-test. Scale bars
= 10 µm.
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1. Mouse tracheal explant and microscopy set up.
cell surface. Fixation and staining with acetylated tubulin (ac-tub, green) demonstrated coincidence of
this autofluorescence signature with the presence of ciliated cells identified by immunofluorescence.
Secretory cells demonstrated a more robust NADH signal (Figure 2B) that was in sharp contrast to
that of ciliated cells. Post-fixation and staining with CCSP (magenta) demonstrates that the autofluo-
rescence intensity ratio can also define secretory cell borders. Similarly, basal cells can also be iden-
tified by their autofluorescence signatures (Figure 2C) and correspond to cells positive for keratin 5
(KRT5, cyan). These differences in autofluorescence ratio can be quantified for the individual cell types
and can be used to distinguish ciliated, secretory, and basal cells (Figure 2D).
As ciliated and secretory cells show the most well separated autofluorescence ratios, we assessed
whether this difference would be maintained after pharmacologic intervention. Ciliated and secre-
tory cells could still be distinguished following the application of the aforementioned mitochondrial
poisons (Figure 1C and D), although the ratios were less well separated (Figure 2—figure supple-
ment 1). This suggests that chemical or physiologic perturbations may alter the ability to distinguish
cell types based on their autofluorescence ratios alone.
Hillocks and rare airway epithelial cell types can be distinguished using
a combination autofluorescence signatures coupled to simple metrics
of morphology
We next assessed the ability of our protocol to identify hillocks, a newly identified airway epithelial
structure (Montoro et al., 2018; Figure 3A). Due to the multilayered nature of hillocks, the structure
is raised above the surrounding surface epithelia. The unique polygonal cell shape of hillock cells
and discrete boundaries of hillocks are easily distinguishable with autofluorescence imaging. Post-
fixation staining with keratin 13 (KRT13, magenta), a protein highly expressed by hillocks, demon-
strates concordance with autofluorescence imaging.
We then took on the much greater challenge of identifying the very rare cell types of the epithe-
lium: pulmonary ionocytes, tuft cells, and neuroendocrine cells (Figure 3B–D). Ionocytes (Figure 3B)
had a characteristic shape by autofluorescence imaging which was similar to the shape of ionocytes
delineated by immunohistochemical staining with BSND (green). Additionally, ionocytes were found
to have puncta of high FAD signal toward the apical aspect of the cell (Figure 3Biv). The distinct shape
of tuft cells could be identified by an autofluorescence ratio corresponding to immunofluorescent
staining with GNAT3 (red). Of all airway epithelial cells, neuroendocrine cells labeled by PGP9 (cyan)
displayed the lowest NAD(P)H and FAD autofluorescence (Figure 3D). Interestingly, as an aggre-
gate, rare cells are characterized by very similar autofluorescence intensity ratios and could not be
distinguished by this criterion alone (Figure 3E). Given the diversity of morphologies of airway cell
types, we then quantified a number of morphometric measures to better distinguish rare cell identities
(Figure 3—figure supplement 1). Live autofluorescence imaging permits the segmentation of cell
boundaries enabling the measurement of the distance of the nucleus from the basement membrane
(nuclear position), the largest dimension in the X, Y, and Z plane (X length, Y length, and Z length),
the aspect ratios (X length/Y length) and (Y length/Z length), and the SD of fluorescence ratio inten-
sities across the cell in the Z plane (SD of autofluorescence intensity ratio above the nucleus, SD of
autofluorescence intensity ratio below nucleus, and the ratio of these two measures). We measured

Ciliated
A i ii iii
FAD NAD(P)H SHG Ac-tub FAD NAD(P)H SHG
Secretory
B i ii iii
FAD NAD(P)H SHG CCSP FAD NAD(P)H SHG
Basal
C i ii iii
FAD NAD(P)H SHG KRT5 FAD NAD(P)H SHG
D ✱✱✱✱
1.0
✱✱✱✱ ✱✱✱✱
0.8
0.6
Ratio
0.4
0.2
0.0
Ciliated Basal Secretory
Figure 2. Autofluorescence imaging of common cell types. (A–C) NAD(P)H (blue), FAD (yellow), and second
harmonic generation (SHG, gray) autofluorescence (i) coupled with cell type-specific staining (ii) with acetylated
tubulin (A) (green) for ciliated cells (B) CCSP (magenta) for secretory cells and (C) KRT5 (cyan) for basal
cells. (iii) Cell borders determined from post-fixation staining are demarcated on autofluorescence images.

Figure 2 continued
(D) Quantification of FAD/FAD +NAD(P)H for ciliated cells (N=32), secretory cells (N=32), and basal cells (N=30),
demonstrating distinct autofluorescence ratio. ANOVA test for multiple comparisons. **** p<0.0001. Scale bars =
10 µm.
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1. Autofluorescence ratios of ciliated cells and secretory cells after antimycin A + rotenone or
FCCP application.
these parameters for the common cells (basal, ciliated, and secretory), hillocks, and rare cells (iono-
cyte, neuroendocrine, and tuft). Aggregated data from a total of 30 basal cells, 32 ciliated cells, 32
secretory cells, 30 hillock cells, 21 ionocytes, 30 tuft cells, and 31 neuroendocrine cells were compiled
(Figure 3E).
Given the spread of various morphologic variables across different cell types, we performed unsu-
pervised clustering of the cell types based upon both their autofluorescence intensity ratio and their
morphological differences. In aggregate, these characteristics allowed the separation of all airway
epithelial cell types into distinct clusters (Figure 3F, Figure 3—figure supplement 2). In order to
assess the performance of machine learning algorithms designed to distinguish cell types, we divided
our data set into training and testing subsets. We utilized 75% of the total cells (154 cells) for machine
learning training, leaving 25% (52 cells) for subsequent validation. We then trained three machine
learning models using k-nearest neighbors, multinomial logistic regression, and XGBoost algorithms
to classify the different cell types. We then compared the identity assigned by the machine learning
algorithms based on the autofluorescence and morphology data to the known identities determined
by staining. K-nearest neighbor analysis and multinomial logistic regression resulted in 96% accuracy,
while XGBoost performed with 92% accuracy. This was suggestive of high-quality modeling with a
Matthews Correlation Coefficient of 0.95, 0.95, and 0.91 (Figure 3—figure supplement 3). We then
established the weight that each characteristic contributes to cell type-specific identification when
using a XGBoost machine learning algorithm (Table 1), which shows that nuclear position, aspect
YZ, NADH autofluorescence, ratio of deviation, and FAD autofluorescence are the most important
characteristics for cell type assignment. Upon excluding autofluorescence and using morphometric
criteria alone, the accuracy and model quality as measured by a Matthews Correlation Coefficient are
dramatically reduced, highlighting the importance of obtaining autofluorescence signatures for cell
type identification (Figure 3—figure supplement 4).
Autofluorescence imaging can be used to interrogate real-time

physiology and demonstrates the existence of airway SAPs
In order to assess whether this methodology is useful to assess real-time physiology, we employed a
paradigmatic physiologic stimulus and then assessed whether the tissue response could be monitored
dynamically. Cholinergic and adrenergic stimulation have historically been known to cause airway secre-
tory cell granule release (Massaro et al., 1979; Yoneda, 1977). The club cell secretory protein, CCSP
(also known as CC10, Uteroglobin, CC16, and SCGB1A1), is a major constituent of these granules and
is commonly used to assign secretory cell identity (Wong et al., 2009). We applied a classic cholin-
ergic agonist, methacholine (which is used clinically to define airway hyper-responsiveness in asthma)
to the airway explant model (Adler et al., 2013; Fischer et al., 2019; Webber and Widdicombe,
1987). Methacholine stimulation led to a 70% decrease in the CCSP immunofluorescence staining as
measured by fluorescence intensity relative to time zero (Figure 4A–B). Indeed, this stimulus-induced
reduction in CCSP, classically considered a specific cell type marker, leads to a dramatically diminished
ability to identify secretory cells.
Methacholine stimulation also alters NAD(P)H and FAD fluorescence intensities; however, these
autofluorescence intensity ratios continue to discriminate secretory cells (Figure 4C–D). Thus, we are
able to assign secretory cell identity at the single-cell level even after stimulation, thereby allowing us
to follow individual secretory cell physiologic responses over time.
To our surprise, we found that methacholine stimulation resulted in the development of non-
fluorescent spherical ‘voids’ within secretory cells (Figure 4C boxes, further magnification of these
boxes in Figure 4E). These ‘voids’ were distinct from a non-autofluorescent region that represented

Hillock
Ai ii
FAD NAD(P)H SHG K13
Ionocyte
Bi Side View
ii iii iv
FAD NAD(P)H SHG BSND FAD NAD(P)H SHG FAD NAD(P)H SHG
Tuft
Ci Side View
ii iii iv
FAD NAD(P)H SHG GNAT3 FAD NAD(P)H SHG FAD NAD(P)H SHG
Neuroendocrine
Di ii iii iv Side View
FAD NAD(P)H SHG PGP9 FAD NAD(P)H SHG FAD NAD(P)H SHG
E Standard Standard Ratio of

Nuclear Aspect Aspect
Cell Type Staining Marker FAD NADH Ratio X length Y length Z length Deviation - Deviation - Deviation
Position XY YZ
Cell Top Cell Bottom (Top/Bottom)
Basal KRT5 219.96 130.12 0.63 1.62 10.73 7.39 3.82 1.49 1.97 68.46 70.97 0.97
Ciliated AcTub 351.77 127.31 0.73 7.06 12.69 6.85 16.44 2.02 0.43 176.07 77.16 2.34
Secretory CCSP 377.55 804.19 0.33 8.05 12.41 8.26 18.09 1.54 0.46 162.88 105.85 1.65
Hillock KRT13 155.01 110.19 0.60 7.53 15.33 10.40 4.77 1.48 2.28 58.58 58.56 1.01
Ionocyte BSND 244.26 98.23 0.70 4.64 10.46 6.66 9.55 1.60 0.74 212.12 71.27 3.13
Tuft UEA1/GNAT3 240.97 119.37 0.67 5.87 18.21 7.62 10.02 2.44 0.77 73.76 79.28 0.94
Neuroendocrine PGP9 133.50 88.06 0.60 3.68 10.02 7.20 7.32 1.41 1.03 70.16 65.84 1.08
Figure 3. Rare cell detection using a combination of autofluorescence and unbiased clustering. NAD(P)H (blue),
FAD (yellow), and second harmonic generation (SHG, gray) autofluorescence in (i) of (A) hillocks, (B) ionocytes,
(C) tuft cells, and (D) neuroendocrine cells. Staining in (ii) of hillocks (K13 – magenta), ionocytes (BSND – green),
tuft cells (GNAT3 – red), and neuroendocrine cells (PGP9 – cyan). Scale bars = 10 µm in (A) and 5 µm in (B–D). (iii)

Figure 3 continued
Overlay of cell outline from (ii) onto (i). Cross sectional imaging across the Z plane (iv) showing differential
fluorescence specifically in ionocytes but absent in tuft and neuroendocrine (NE) cells. (E) Table with cell type-
specific values of both autofluorescence (FAD, NADH, and ratio) and specific morphologic parameters. ‘X-
length’=largest length in the XY plane. ‘Y-length’=smallest length in the XY plane. ‘Z-length’=largest length
in Z dimension. ‘Aspect XY’=ratio of X-length/Y-length. ‘Aspect YZ’=Y-length/Z-length. ‘SD cell top’=SD of
autofluorescence ratio from above mid height. ‘SD cell bottom’=SD of autofluorescence ratio from below mid
height. ‘Ratio of SD’ is SD cell top/SD cell bottom. (F) Unbiased clustering by UMAP from (E) demonstrating
separate clusters for each cell type. The numbers of each cell type measured were as follows: ionocytes (21), tuft
(30), hillock (30), ciliated (32), secretory (32), neuroendocrine (31), and basal (30).
The online version of this article includes the following source data and figure supplement(s) for figure 3:
Source data 1. Mean autofluorescence measurements (FAD, NADH, and ratio) and morphologic characteristics for
various cell types as described in Figure 3E.
Figure supplement 1. Morphometric parameters used in the analysis of airway epithelial cell types.
Figure supplement 2. Unsupervised clusters analysis of all airway epithelial cell types shown individually.
Figure supplement 3. Confusion matrix demonstrating the performance of three algorithms for classifying cell
identity.
Figure supplement 4. Confusion matrix demonstrating the performance of three algorithms, (i) k-nearest
neighbors, (ii) multinomial logistic regression, and (iii) XGBoost, for classifying cell identity in the absence of
NADH, FAD, and ratio autofluorescence data.
the nucleus. We performed detailed examination of the field in Figure 4C and found that ‘voids’
were present in approximately 9% of secretory cells prior to stimulation, but the fraction of secretory
cells with ‘voids’ dramatically increased to 78% following methacholine stimulation (Figure 4—source
data 1). We hypothesized that these novel structures resulted from the uptake of extracellular luminal
contents. Indeed, such a sampling process has been demonstrated in the gut epithelium during which
secretory and goblet cells internalize luminal contents. In this case, internalization results in the forma-
tion of SAPs and GAPs within the intestinal secretory and goblet cells (Gustafsson and Johansson,
2022; McDole et al., 2012; Noah et al., 2019). Given that airway secretory cells adopt a goblet cell
morphology within hours, airway SAPs and GAPs are likely equivalent (Evans et al., 2004).
To definitely evaluate our hypothesis concerning the origin of the spherical autofluorescence ‘voids’
in our system, we incubated tracheal explants cultures in 10Kd FITC-dextran. Upon stimulation with
methacholine, secretory cells showed uptake of FITC-dextran that was clearly distinguishable from
the nucleus (Figure 5A). Since autofluorescence imaging alone cannot be used to establish the pres-
ence of a membrane, we resorted to employing epithelia expressing membrane tdTomato to assess
whether the ‘voids’ were membrane bound. Indeed, we found uptake of FITC-dextran into membrane
encapsulated structures delineated by membrane tdTomato (Figure 5B). This uptake of dextran
occurs both in unstimulated and methacholine stimulated samples, though is dramatically exagger-
ated following stimulation with methacholine (Figure 5C). The increased SAP formation following
stimulation with methacholine recapitulates the induction of intestinal GAPs following cholinergic
stimulation (Knoop et al., 2015). In order to visualize this process on a faster time scale, we utilized air
liquid interface (ALI) cultures since they are significantly thinner than the native trachea, thus allowing
rapid imaging across the Z-axis. In our ALI models, we captured the fine dynamics of the formation
of intracellular membrane bound structures that housed contents that were previously located in the
airway lumen. Live imaging over 60 min demonstrates the uptake of FITC-dextran into the cell from
the apical surface, further accumulation of FITC-dextran into the cell, and a major secretion event
Table 1. Contribution of each autofluorescence or morphometric characteristic to cell type-specific

identification using XGBoost machine learning algorithm.
Ratio of
deviation
Nuclear X Y Z Aspect Aspect SD - cell SD - cell (top/
FAD NADH Ratio position length length length XY YZ top bottom bottom) Total
0.10 0.12 0.06 0.18 0.09 0.01 0.08 0.02 0.13 0.07 0.02 0.11 1.00

A Non-treated Methacholine treated B

✱✱✱✱
i ii
✱✱✱✱
1500 ✱✱✱✱
Mean CCSP Fluorescence

✱✱✱✱ ✱✱✱✱ ns
Intensity (relative units)

1000
500
0
CCSP Membrane Tomato CCSP Membrane Tomato 0 30 60 120
Time (min)
C Baseline Methacholine D
i ii
✱
200 600 0.5
✱ ✱✱
0.4
150
NAD(P)H Emission
400
FAD Emission
Ex. 2 0.3
Ratio
100
0.2
200
50
0.1
0 0 0.0
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in
in
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ol
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Ex. 1
B
B
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ha
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et
et
et
M
M
E Example 1 Example 2
Baseline Methacholine Baseline Methacholine
XY XZ XY XZ XY XZ XY XZ
i ii
* *
*
*
FAD NAD(P)H SHG FAD NAD(P)H SHG FAD NAD(P)H SHG FAD NAD(P)H SHG
Figure 4. Autofluorescence imaging reliably identifies secretory cells despite the loss of characteristic secretory cell CCSP staining following
methacholine stimulation. (A) Murine airway epithelial cells expressing membrane tomato under ROSA promoter. CCSP staining (green) using identical
imaging parameters performed on two adjacent halves of same trachea treated with (i) vehicle vs (ii) 10 µM methacholine. CCSP staining (green) is lost
following methacholine stimulation. Cell membrane denoted by membrane tomato (magenta). (B) Fluorescence intensity of CCSP staining over time
with methacholine stimulation imaged with same parameters. Each cell is represented by a dot. N=50. ****p<0.0001 by ANOVA. (C) Autofluorescence
imaging (NADH blue, FAD yellow, and second harmonic generation [SHG] gray) of trachea at baseline (i) and after methacholine stimulation (ii).
Magnified image of the region indicated by the white box is shown in (E). (D) Quantification of NADH, FAD, and fluorescence ratio (FAD/FAD +NAD[P]
H) at baseline and after methacholine stimulation at single-cell resolution (each dot represents a cell, N=8). (E) White boxed regions from (C) showing
the formation of non-fluorescent voids after methacholine stimulation in both an XY view and a cross-sectional XZ view. Non-fluorescent regions in
baseline XZ are nucleus. Asterix denotes new non-fluorescent ‘void’ after methacholine stimulation. Scale bars = 10 µm in A and C and 5 µm in E.
The online version of this article includes the following source data for figure 4:
Source data 1. Secretory cells with “voids” are present at baseline and their numbers increase following 30 minutes of methacholine treatment.
(Figure 5D and Figure 5—videos 1–3, Figure 5—figure supplement 1). This process bears a striking
resemblance to the formation of intestinal SAPs and GAPs which are generated through an endo-
cytic process that can be stimulated by acetylcholine (Gustafsson et al., 2021; Knoop et al., 2015).
Additionally, further mirroring their gut counterparts, airway secretory cells with SAPs containing
either FITC-dextran or FITC-ovalbumin closely associate with CD11c+APCs that had been recruited

Figure 5. Secretory cells take up luminal contents and form secretory cell associated antigen passages (SAPs). (A) Secretory cell demonstrating uptake
of FITC-dextran (10Kd, green) that is distinct from the nucleus stained by Draq5 (magenta). (i) Top panels are xy views and (ii) bottom panels are cross-
sectional xz view. Dotted lines outline SAPs. Scale bars = 5 µm. (B) Tracheal imaging of (i) membrane tomato (magenta) and (ii) FITC-dextran (iii) overlay
of both channels demonstrates methacholine-stimulated uptake of extracellular dextran into membrane bound SAPs (arrowheads). Scale bars = 5 µm.
(C) FITC-dextran uptake in unstimulated and methacholine stimulated tracheal explants. Trachea from a mouse expressing membrane tomato (magenta)
was bisected, and one half of the trachea was placed in media containing FITC-dextran (green) in the absence of methacholine, while the other half was
placed in the same FITC-dextran media containing 10 µM methacholine. After 30 min of incubation, dextran uptake occurs in (i) unstimulated samples
and (ii) dramatically increases following methacholine stimulation. (D) Selected images from Figure 5—video 1 demonstrating uptake of luminal FITC-
dextran and subsequent secretion of cell contents. (i) Baseline image prior to methacholine stimulation. (ii) Uptake of FITC-dextran and formation of

Figure 5 continued
SAPs (9 min after methacholine stimulation). (iii and iv) Further accumulation of FITC-dextran (31 min and 40 min after methacholine stimulation). (v and
vi) Secretion of cellular contents (53 min and 60 min after methacholine stimulation). (E) CD11c+antigen-presenting cells (APCs) interact with SAPs. (i
and i') CD11c+APCs labeled by mCherry (magenta) (ii and ii’) methacholine stimulated SAP formation with FITC-dextran uptake (ii) and FITC-ovalbumin
uptake (ii’). (iii and iii’) CD11c+APCs associate with SAPs (iv–vi and iv’–vi’) optical XZ cross sections of CD11c+APCs juxtaposed to SAPs. Scale bars =
5 µm.
The online version of this article includes the following video and figure supplement(s) for figure 5:
Figure 5—video 1. Movies of murine airway liquid interface (ALI) cultures using airway epithelial cells labeled with membrane tomato (magenta)
demonstrating dextran (green) uptake and secretion.
https://elifesciences.org/articles/84375/figures#fig5video1
Figure supplement 1. XZ optical cross-sectional view of a murine airway liquid interface culture (ALI) of airway epithelial cells expressing membrane
tomato (magenta) cultured in media containing FITC-dextran (green) demonstrating numerous examples of dextran uptake and secretion.
Figure supplement 2. CD11c+antigen-presenting cells (APC) associate with secretory cell associated antigen passages (SAPs).
to the airway epithelium following an LPS challenge (Figure 5E, Figure 5—figure supplement 2;
McDole et al., 2012). Many of these LPS-stimulated APCs are intraepithelial and possess dendriform
processes that extend toward SAPs. This suggests that airway SAPs are likely to play an important role
in the immunologic response to antigens present in the airway lumen.
Discussion
We report a label-free imaging methodology that can be used for cell type identification in live and
unmanipulated tissues. We also show that autofluorescence coupled to morphology allows one to
distinguish all the known airway epithelial cell types without the need for genetic labels. Unexpectedly,
cell identity can be established in a way that is sometimes more accurate than destructive immuno-
fluorescent approaches. This may be of particular relevance to disease states since it has been noted
that markers of cell type such as the secretory cell marker CCSP can be misleading when used for the
enumeration of cell numbers in COPD patient samples (Pilette et al., 2001), in asthma (Shijubo et al.,
1999), and in smokers (Shijubo et al., 1997) presumably due to the fact that CCSP has been secreted
into the lumen. However, despite its advantages, it should be noted that infection and inflammation
may alter autofluorescence (Dilipkumar et al., 2019). Thus, our methodology must be re-optimized in
each new experimental setting, and in each case, it is very likely that machine learning algorithms will
require re-training. However, the training set provided in this manuscript will be a useful validation for
native mouse tracheal epithelia. With these caveats in mind, we hope our methodology will eventually
find applications in assessing human airway specimens, including the real-time assessment of disease-
specific physiologic parameters and the efficacy of therapeutic interventions.
Finally, our discovery of airway SAPs demonstrates that this new methodology can be used to
define novel unappreciated physiologic processes that currently remain hidden from view. Recently,
using a new fixation procedure, luminal dextran uptake has been documented in murine airway goblet
and secretory cells, suggestive of GAPs (Tang et al., 2022). We now definitively demonstrate endo-
cytic uptake of luminal antigens, and we show that this process is stimulated by a classic cholinergic
challenge (Gustafsson and Johansson, 2022; McDole et al., 2012). However, although we see that
SAPs are associated with APCs, we have not demonstrated that APCs receive cargo from SAPs.
Thus, the actual functions of airway SAPs are yet to be discovered. Intestinal GAPs play a role
in immune tolerance (Knoop et al., 2017; Knoop et al., 2020), the imprinting of dendritic cells
(Kulkarni et al., 2020), and participate in inflammatory disease (Knoop et al., 2017; Knoop et al.,
2020). Analogous phenomenon has been documented to occur in the case of goblet cells of the eye
(Barbosa et al., 2017; Ko et al., 2018). It will be of great interest to establish the functions of airway
SAPs and their role in airway mucosal immunity, tolerance, and autoimmunity.

Materials and methods

Key resources table
Reagent
type
(species) or Source or Additional
resource Designation reference Identifiers information
Mouse monoclonal anti-tubulin,

Antibody acetylated Sigma T-6793 1:500
Antibody Rabbit monoclonal anti-BSND Abcam ab196017 1:150
Antibody Goat polyclonal anti-GNAT3 Novus NBP1-20926 1:100
Antibody Rabbit monoclonal anti-KRT13 Abcam ab92551 1:100
Antibody Goat polyclonal anti-CC10 Santa Cruz SC-9772 1:1000
Antibody Rabbit polyclonal anti-UCHL1 Proteintech 14730–1-AP 1:250
Antibody Chicken polyclonal anti-KRT5 Biolegend 905901 1:200
Chemical
compound
and drug Acetyl-β-methylcholine chloride Sigma A2251
Chemical
compound
and drug 10Kd FITC-dextran Sigma FD10S
Chemical
compound
and drug FITC-ovalbumin Thermo O23020
Chemical
compound
and drug ROCK inhibitor Y-27632 Selleckbio S1049
Chemical
compound Antimycin A from Streptomyces
and drug sp Sigma A8674
Chemical
compound
and drug Rotenone Sigma 557368
Chemical Carbonyl cyanide

compound 4-(trifluoromethoxy)
and drug phenylhydrazone Sigma C2920
Strain
and strain
background
(Mus
musculus) C57BL/6J Jax stock no. 000664
Strain
and strain
background
(Mus
musculus) MT-MG Jax stock no. 007676
Strain
and strain
background
(Mus Khanna et al.,
musculus) Cd11c-mCherry 2010
Software and https://www.mathworks.com/

algorithm MATLAB Mathworks products/matlab.html
Software and
algorithm Jupyter Notebook Jupyter https://jupyter.org/
Continued on next page

Continued
Reagent
type
(species) or Source or Additional
resource Designation reference Identifiers information
Software and Schindelin

algorithm FIJI et al., 2012 https://fiji.sc/
Software and
algorithm GraphPad Prism GraphPad https://www.graphpad.com
Semipermeable
membrane
Other 12 mm Transwell Corning 3460 support
Electron Silicone adhesive

Microscopy used for fixation
Other Sylgard 184 silicone elastomer Sciences EMS Catalog #24236–10 of transwell insert
O-ring used to
Other Silicone O-ring McMaster-Carr 1182N015 secure trachea
3D printed insert
https://github.com/vss11/ used to secure
Label-free-autofluorescence semipermeable
Other 3D printed insert This manuscript membrane
Suture used to
secure trachea to
Other 7–0 Silk Suture Ethicon 786 G O-ring
Vector
Other UEA1-dylight 649 Laboratories DL-1068–1 1:100, lectin stain
1:5000, nuclear
Other Draq5 Abcam ab108410 stain
Mouse models
C57BL/6J mice (stock no. 000664) and MT-MG (stock no. 007676) were purchased from Jackson labo-
ratory. Cd11c-mCherry mice were a gift from the Khanna lab (Khanna et al., 2010).
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. All mice were housed in an environment with controlled temperature and humidity, on
12 hr light:dark cycles, and fed with regular rodent’s chow.
Mouse tracheal explant model

As demonstrated in Figure 1—figure supplement 1A, mice were euthanized as per MGH IACUC
protocols, and trachea were dissected, cleared of connective tissue, opened longitudinally, and
bisected. The dissected trachea was then sutured onto a silicone O-ring and placed onto a custom-
made insert. The custom made insert was assembled using an inverted 12 mm Transwell, 0.4 µm Pore
Polyester Membrane Insert fixed to a custom 3D printed insert (https://github.com/vss11/Label-free-
autofluorescence) with Sylgard 184 silicone elastomer (EMS Catalog #24236–10).
Samples were incubated with Gibco DMEM/F12 (Fischer 11320033). For experiments across
multiple days, the insert was filled with media such that the meniscus was located below the trachea to
prevent explant submersion. Primocin (InVivoGen) was added to the media to prevent contamination.
Two-photon microscopy
Tracheal explants were fixed to inserts as described above. An Olympus FVMPE-RS multiphoton laser
scanning microscope was equipped with a MaiTai HPDS-O IR pulsed laser (900 nm for FAD and SHG),
an INSIGHT X3-OL IR pulsed laser (730 nm for NAD[P]H), and a 25× water immersion lens (NA 1.05;
XLPN25SWMP2) for imaging. Media was used as the submersion droplet. Configuration of the micro-
scope setup is seen in Figure 1—figure supplement 1B. A 2× optical zoom with 1024×1024 scan

size was used. A full thickness Z section was imaged from above the tracheal surface through to the
sub epithelium, with a 0.5 µm step size. Multiple regions on the same tracheal explant were imaged.
Each region was ‘registered’ using SHG features of the subepithelial collagen/cartilage/vasculature as
fiduciary marks. The insert was placed in a physiological live imaging chamber (CO2 and temperature-
controlled, TokaiHit) at 37°C and 5% CO2.
Mitochondrial poisons
Tracheal explants were incubated with DMEM/F-12 Media with Primocin (InVivoGen) and 15 mM
HEPES. After baseline imaging, samples were incubated with complex 1 inhibitor – antimycin A
(10 µM) and complex III inhibitor – rotenone (1 µM) for 10 min. Imaging was conducted as described.
Similarly, after baseline imaging, tracheal explants were incubated with media containing mitochon-
drial uncoupler, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP, 1 µM).
Immunofluorescence staining of tracheal explants

Tracheal explants were secured onto the insert setup as described above. Tracheas were rinsed in PBS
and subsequently fixed in 4% PFA in PBS for 20 min at room temperature. A glass slide was placed a
few millimeters above the tissue to form a column of liquid. Explants were subsequently washed with
PBS three times for a total of 1 hr. The explants were then permeabilized in PBS-0.3% TritonX-100
(PBST) for 30 min in a similar manner. The sample was then stained with primary antibody at 37°C for
1 hr, diluted in 1% BSA-0.3% PBST. The following antibodies and dilutions were used:
Antibody Manufacturer Dilution
Mouse anti-tubulin, acetylated Sigma T-6793 1:500
Rabbit anti-BSND Abcam ab196017 1:150
Goat anti-GNAT3 Novus NBP1-20926 1:100
Rabbit anti-KRT13 Abcam ab92551 1:100
UEA1-dylight 649 Vector Laboratories DL-1068–1 1:100
Goat anti-CC10 Santa Cruz SC-9772 1:1000
Rabbit anti-UCHL1 Proteintech 14730–1-AP 1:250
Chicken anti-KRT5 Biolegend 905901 1:200
Draq5 Abcam ab108410 1:5000
Tracheal explants were then washed with PBS for 1 hr and stained with secondary antibody at 37°C
for 1 hr. Various secondary antibodies (Jackson ImmunoResearch) were used. The samples were then
imaged using two-photon microscopy as above.
Cell autofluorescence and morphological analysis

Autofluorescence images were obtained as described above. In order to normalize the Z-axis to the
basement membrane (SHG), or ‘flatten’ the image, a MATLAB algorithm was utilized. Autofluores-
cence intensity was measured at the epithelial surface in the NAD(P)H and FAD channels using Image
J. The morphological measurements were conducted as described in Figure 3—figure supplement
1.
Dimensionality reduction and visualization

UMAP was calculated using the package in Python with the following parameters: random state = 42;
spread = 3; min; dist = 0.1. A total of 206 cells were analyzed. Data visualization was implemented
using the Matplotlib package in Python. All codes are provided in the supplemental Jupyter notebook
file.
Machine learning algorithms for classifying cell types

K-nearest neighbor, multinomial logistic regression, and XGBoost algorithms were used to classify cell
types. The Scikit-learn package was used to implement k-nearest neighbor and multinomial logistic
regression algorithms, and the XGBoost package was used to implement the XGBoost algorithm.

All the codes are provided in the supplemental Jupyter notebook file. The following features were
used to train the algorithms: 1. FAD; 2. NADH; 3. FAD/NADH ratio; 4. nuclear position; 5. X-length;
6. Y-length; 7. Z-length; 8. XY aspect ratio; 9. YZ aspect ratio; 10. SD of the fluorescence ratio from
midpoint above (cell top); 11. SD of fluorescence ratio from midpoint below (cell bottom); 12. SD
of the fluorescence ratio top/SD of fluorescence ratio bottom. 152 cells (75%) were used to train
the model, and 52 cells (25%) were used to test the models. For k-nearest neighbor algorithm, k=3
yields the highest accuracy and was therefore used to generate the confusion matrix. The Matthew’s
Correlation Coefficient was calculated using the Scikit-learn package.
Methacholine treatment and CCSP immunofluorescence staining

Tracheal explants from mice expressing membrane tomato under the ROSA promoter (stock no.
007676) were euthanized and dissected as described above. Tracheal samples were bisected as
described with one half placed in DMEM/F12 media, while the other half was placed into DMEM/F12
with 10 µM of methacholine (dry powder dissolved in DMEM/F12, Sigma A2251). After 30 min, 1 hr,
1.5 hr, and 2 hr of incubation, the samples were fixed in 4% PFA in PBS for 20 min at room tempera-
ture with agitation. Samples were then washed with PBS three times for a total of 1 hr. Samples were
permeabilized in PBS-0.3% Triton X-100 (PBST) for 30 min with gentle agitation. The samples were
stained with CC10 primary antibody (aka Scgb1a1, 1:500; SC-9772, Santa Cruz) at 37°C for 2 hr,
diluted in 1% BSA-0.3% PBST. Samples were rinsed in PBS for 30 min and incubated with secondary
antibody for 30 min with a subsequent 30 min PBS wash. As above, imaging was conducted on
the Olympus FVMPE-RS multiphoton laser scanning microscope equipped with a MaiTai HPDS-O
IR pulsed laser (900 nm for AF488) and an INSIGHT X3-OL IR pulsed laser (1050 nm for membrane
tomato). Laser power was kept constant for all images, and all tracheal samples were acquired using
the same settings. ImageJ was used for quantifying fluorescence intensity on maximal projection
images.
FITC-dextran and FITC-ovalbumin uptake assays

Explants were incubated with 10Kd FITC-dextran (Sigma FD10S) diluted into DMEM/F12 media at
a concentration of 1 mg/ml with 10 µM methacholine (dry powder dissolved in DMEM/F12, Sigma
A2251). Alternatively, explants were treated with FITC-
ovalbumin (Thermo O23020) diluted into
DMEM/F12 media at a concentration of 0.1 mg/ml with 10 µM methacholine (dry powder dissolved
in DMEM/F12, Sigma A2251). Imaging was conducted on the Olympus FVMPE- RS multiphoton
laser scanning microscope equipped with a MaiTai HPDS-O IR pulsed laser (800 nm for FITC) and
an INSIGHT X3-OL IR pulsed laser (1050 nm for Draq5 or membrane tomato) with 2× optical zoom,
1024×1024 scan size, and a step size of 0.5 µm.
APC colocalization with SAPs

Cd11c-mCherry mice were stimulated with LPS (30 µg in 40 µL PBS, delivered intranasally as a single
dose, 5 days prior to experimentation). Explants from these mice were then cultured in DMEM/F12
containing 1 mg/ml 10Kd FITC-dextran (Sigma FD10S) and 10 µM of methacholine (Sigma A2251).
Imaging was conducted on the Olympus FVMPE-RS multiphoton laser scanning microscope equipped
with a MaiTai HPDS-O IR pulsed laser (800 nm for FITC) and INSIGHT X3-OL IR pulsed laser (1050 nm
for mCherry) with 1024×1024 scan size and a step size 0.5 µm. A 15× optical zoom and line averaging
scan were used for high-resolution imaging.
Air liquid interface model

Tracheal cells were harvested and cultured as previously described (Mou et al., 2016). Briefly, cells
were cultured and expanded in complete SAGM (small airway epithelial cell growth medium; Lonza,
CC-3118) containing a TGF-β/BMP4/WNT antagonist cocktail and 5 μM ROCK inhibitor Y-27632
(Selleckbio, S1049). To generate ALI cultures, airway basal stem cells were seeded onto transwell
membranes and differentiated using PneumaCult-ALI Medium (StemCell, 05001). Air-liquid cultures
were fully differentiated after 14 days as demonstrated by the formation of air-liquid interface and the
beating of cilia.
Statistical analysis
All analysis was performed using Graphpad Prism. For analysis of experimental data after treat-
ment with mitochondrial poisons (Figure 1C–D), imaging over time (Figure 1E), and methacholine

treatment (Figure 4D), a paired t-test was used. To assess change in autofluorescence after metabolic
poisons (Figure 2—figure supplement 1), an unpaired t-test was used. For comparison of autofluo-
rescence ratio across different cell types (Figure 2D), one-way ANOVA was utilized. For comparison
of immunofluorescence staining with various durations of methacholine treatment (Figure 4B), a one-
way ANOVA was used.
Acknowledgements
We would like to thank the members of the Rajagopal lab and Lin labs for their feedback. This work was
supported by NIH-NHLBI 5R01HL142559-04 (JR, CPL), RO1HL118185-08 (JR), 1R01HL157221-01A1
(JR), the Bernard and Mildred Kayden Endowed MGH Research Institute Chair (JR), and CFF
003338L121 (VS).
Additional information
Funding
Funder Grant reference number Author
Cystic Fibrosis Foundation 003338L121 Viral S Shah
NHLBI Division of 5R01HL142559 Charles P Lin

Intramural Research Jayaraj Rajagopal
NHLBI Division of RO1HL118185 Jayaraj Rajagopal

Intramural Research
NHLBI Division of 1R01HL157221 Jayaraj Rajagopal

Intramural Research
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Viral S Shah, Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing
- original draft, Writing – review and editing; Jue Hou, Conceptualization, Formal analysis, Investiga-
tion, Visualization, Methodology, Writing – review and editing; Vladimir Vinarsky, Conceptualization,
Investigation, Methodology; Jiajie Xu, Manalee V Surve, Investigation, Methodology, Writing – review
and editing; Charles P Lin, Jayaraj Rajagopal, Conceptualization, Resources, Formal analysis, Supervi-
sion, Funding acquisition, Investigation, Methodology, Writing – review and editing
Author ORCIDs
Viral S Shah ‍ ‍http://orcid.org/0000-0002-3605-8066
Vladimir Vinarsky ‍ ‍http://orcid.org/0000-0003-1141-6434
Jayaraj Rajagopal ‍ ‍http://orcid.org/0000-0002-4122-177X
Ethics
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 confir-
matory cervical dislocation consistent with the recommendations of the American Veterinary Medical
Association.
Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.84375.sa1
Author response https://doi.org/10.7554/eLife.84375.sa2

Additional files
Supplementary files
• MDAR checklist
Data availability
All data is included in the manuscript and generated custom scripts are included on https://github.
com/vss11/Label-free-autofluorescence (copy archived at Shah, 2023).
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TOOLS AND RESOURCES

Autofluorescence imaging permits label-­

Reviewing Editor: Paul W

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  1 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  2 of 20

Autofluorescence imaging discriminates the three common airway

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  3 of 20

D Baseline FCCP (Mitochondrial Uncoupler)

E Day 0 Day 1 Day 2

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  4 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  5 of 20

FAD NAD(P)H SHG Ac-tub FAD NAD(P)H SHG

FAD NAD(P)H SHG CCSP FAD NAD(P)H SHG

FAD NAD(P)H SHG KRT5 FAD NAD(P)H SHG

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  6 of 20

Autofluorescence imaging can be used to interrogate real-time

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  7 of 20

FAD NAD(P)H SHG K13

E Standard Standard Ratio of

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  8 of 20

Table 1. Contribution of each autofluorescence or morphometric characteristic to cell type-­specific

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  9 of 20

A Non-treated Methacholine treated B

Mean CCSP Fluorescence

Intensity (relative units)

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  10 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  11 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  12 of 20

Materials and methods

Mouse monoclonal anti-­tubulin,

Antibody Rabbit monoclonal anti-­BSND Abcam ab196017 1:150

Antibody Goat polyclonal anti-­GNAT3 Novus NBP1-­20926 1:100

Antibody Rabbit monoclonal anti-­KRT13 Abcam ab92551 1:100

Antibody Goat polyclonal anti-­CC10 Santa Cruz SC-­9772 1:1000

Antibody Rabbit polyclonal anti-­UCHL1 Proteintech 14730–1-­AP 1:250

Antibody Chicken polyclonal anti-­KRT5 Biolegend 905901 1:200

Chemical Carbonyl cyanide

Software and https://www.mathworks.com/

Continued on next page

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  13 of 20

Software and Schindelin

Electron Silicone adhesive

Mouse tracheal explant model

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  14 of 20

Immunofluorescence staining of tracheal explants

Antibody Manufacturer Dilution

Mouse anti-­tubulin, acetylated Sigma T-­6793 1:500

Rabbit anti-­BSND Abcam ab196017 1:150

Goat anti-­GNAT3 Novus NBP1-­20926 1:100

Rabbit anti-­KRT13 Abcam ab92551 1:100

UEA1-­dylight 649 Vector Laboratories DL-­1068–1 1:100

Goat anti-­CC10 Santa Cruz SC-­9772 1:1000

Rabbit anti-­UCHL1 Proteintech 14730–1-­AP 1:250

Chicken anti-­KRT5 Biolegend 905901 1:200

Draq5 Abcam ab108410 1:5000

Cell autofluorescence and morphological analysis

Dimensionality reduction and visualization

Machine learning algorithms for classifying cell types

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://​doi.​org/​10.​7554/​eLife.​84375  15 of 20

Autofluorescence imaging permits label-

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 1 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 2 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 3 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 4 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 5 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 6 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 7 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 8 of 20

Table 1. Contribution of each autofluorescence or morphometric characteristic to cell type-specific

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 9 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 10 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 11 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 12 of 20

Mouse monoclonal anti-tubulin,

Antibody Rabbit monoclonal anti-BSND Abcam ab196017 1:150

Antibody Goat polyclonal anti-GNAT3 Novus NBP1-20926 1:100

Antibody Rabbit monoclonal anti-KRT13 Abcam ab92551 1:100

Antibody Goat polyclonal anti-CC10 Santa Cruz SC-9772 1:1000

Antibody Rabbit polyclonal anti-UCHL1 Proteintech 14730–1-AP 1:250

Antibody Chicken polyclonal anti-KRT5 Biolegend 905901 1:200

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 13 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 14 of 20

Mouse anti-tubulin, acetylated Sigma T-6793 1:500

Rabbit anti-BSND Abcam ab196017 1:150

Goat anti-GNAT3 Novus NBP1-20926 1:100

Rabbit anti-KRT13 Abcam ab92551 1:100

UEA1-dylight 649 Vector Laboratories DL-1068–1 1:100

Goat anti-CC10 Santa Cruz SC-9772 1:1000

Rabbit anti-UCHL1 Proteintech 14730–1-AP 1:250

Chicken anti-KRT5 Biolegend 905901 1:200

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 15 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 16 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 17 of 20

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 18 of 20

Klinger A, Orzekowsky-Schroeder R, von Smolinski D, Blessenohl M, Schueth A, Koop N, Huettmann G,

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 19 of 20

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S,

Shah, Hou et al. eLife 2023;12:e84375. DOI: https://doi.org/10.7554/eLife.84375 20 of 20