Mondragón‐Palomino et al. Supplement p. 1
Supplemental information for
Three-dimensional imaging for th
e quantification of spatial pat
terns in
microbiota of the intestinal mucosa
Octavio Mondragón-Palomino
1,β
, Roberta Poceviciute
1
, Antti Lignell
1,2,α
, Jessica A. Griffiths
2
, Heli Takko
1
, and
Rustem F. Ismagilov
1,2
*
1
Division of Chemistry and Chemic
al Engineering, California Inst
itute of Technology
2
Division of Biology and Biological Engineering, California Inst
itute of Technology
1200 E. California Blvd., Pasadena
, CA, United States of Americ
a
* Correspondence to:
rustem.admin@caltech.edu
.
α
Current address: Department of C
hemistry, University of Helsin
ki, Helsinki, Finland
β
Current address: Laboratory of Parasitic Diseases, National In
stitute of Allergy and Infectious Diseases, Bethesda,
MD, United States of America
Contents:
Supplementary Materials and Methods
Composition of acrylamide monomer mix
Tissue preservation and clearing for imaging
HCR staining of bacterial 16S rRNA
Media for bacterial culture
Optimization of lysozyme treatment for HCR
Controls for in situ HCR
In vitro assay to find formamide concentrations fo
r stringent hybridization of taxon-specific HCR probes,
as well as to quantify their sensitivity and specificity
Processing and analysis of in situ imaging
DNA extraction, sequencing, bioinformatics analyses, and absolute quantification
Supplementary Figures S1-S28
Supplementary Video captions S1-S4
Supplementary Tables S1-S8
Supplementary References
Contributions of non-corresponding authors
Mondragón‐Palomino et al. Supplement p. 2
Supplementary Materials and Methods
Composition of acrylamide monomer mix
The following reagents were used for the preservation of expose
d intestinal tissue: Acrylamide solution 40% in
water (#01697, Sigma Aldrich, Saint Louis, MO, USA), 2% Bis (#1
610142, Bio-Rad, Hercules, CA, USA),
Paraformaldehyde 32% (#15714-S, Electron Microscopy Sciences, B
risbane, CA, USA), Polymerization thermal
initiator VA044 (#
NC0632395,
Wako Chemicals, Richmond, VA, USA). The acrylamide monomer mix
for the
protective gel layer on the mucosa requires the crosslinker bis
-acrylamide to become rigid. The final
concentrations of reagents for the gel layer were: 4% Acrylamid
e, 0.08% Bis-acrylamide, 4% Paraformaldehyde,
2.5 mg/mL VA044, 1X PBS. The final concentrations of reagents f
or acrylamide embedding of the rest of the
sample were: 4% Acrylamide, 0.0% Bis-acrylamide, 4% Paraformald
ehyde, 2.5 mg/mL VA044, 1X PBS.
Tissue preservation and clearing for imaging
To prepare tissue samples for imaging of the mucosal microbiota
(Fig. 1-5), 4 mice of 20–21 weeks of age (strain
C57BL/6J) and 1 germ-free mouse (Fig. 2) of 11 weeks of age wer
e euthanized and their GIT tissues harvested.
Mice for the antibiotic-challenge experiments (Fig. 6-7) were 1
4-15 weeks of age (strain C57BL/6J) at day one
of the experiment. Each mouse received an intraperitoneal injec
tion of 220 μL of a 10X dilution of the sedative
Fatal-Plus (Vortech Pharmaceuticals, Dearborn, MI, USA). Once a
mouse was anesthetized, we performed
transcardial perfusion with sterile, ice-cold 1X PBS for 20 min
at a rate of 4-5 mL/min to euthanize the mouse
and clear its vasculature of blood. During perfusion, the expos
ed viscera were kept wet with sterile 1X PBS, and
covered with a small bag of ice. After perfusion, the viscera w
ere quickly removed and kept in a dry, sterile, tube
in ice. In a biosafety cabinet, the GIT was isolated from the m
esentery, liver and attached fat. The jejunum and
duodenum were also removed and discarded. To preserve the exter
nal muscle layer of the intestines in its
distended form, we fixed the remaining GIT (from ileum to rectu
m) for 3 min in ice-cold 4% paraformaldehyde
(15714-S Paraformaldehyde 32%, Electron Microscopy Sciences, Ha
tfield, PA, USA) and then washed it in ice-
cold 1X PBS for 3 min to stop fixation. After fixation, the dis
tal colon and the ileum were removed and discarded.
The cecum and the proximal colon were separated and kept in ste
rile containers on ice.
In a large Petri plate on an ice-cold surface, the cecum and th
e proximal colon were cut open longitudinally and
the bulk contents cleared with sterile tweezers. The remainder
of the GIT contents were removed by gently
dripping sterile, cold 1x PBS on the exposed surfaces. Any inte
stinal contents that remained attached to the
tissue surface after PBS treatment were retained. The proximal
colon was then cut into two segments. One
contained all the folds and the other segment was a transition
from the cecum to the colon and contained no
folds. The cecum tissue was split into four segments: the end t
ip, the middle, the top left, and the top right. Each
segment was placed into a pool of PBS (0.5 mL) on a glass slide
, which was contained by a silicon isolator
(#666503; Grace Bio-Labs, Bend, OR, USA) to keep the tissue in
place and prevent tissue desiccation. Glass
slides with tissue samples were kept in a large Petri dish in a
n ice box.
Next, in a chemical safety cabinet, tissue samples from each mo
use were put in a petri dish, placed in an ice
box, and fixed for 1 h by adding 1 mL of ice-cold 4% paraformal
dehyde (PFA) to each PBS pool. We replaced
with fresh 4% PFA every 15 min. For the tissues used in the ant
ibiotic-challenge experiments, we improved this
step by closing the chamber with a glass slide and fixed tissue
s for 60 min without replenishing PFA. After
fixation, tissues were flipped over onto the pool of 4% PFA (so
that the muscle side was facing up). To increase
the volume of the pools in which the tissues were submerged, we
stacked onto each slide an additional two
silicon isolators. We added more 4% PFA, covered each pool with
a silicon membrane to avoid evaporation,
placed them in Petri dishes and transferred the slides into an
ice box. The ice box was then placed in an
anaerobic chamber along with the bis-acrylamide monomer mix (
SI Appendix, Composition of acrylamide
monomer mix
). In the anaerobic chamber, we removed the 4% PFA in which the
tissues were floating and
substituted it with 2 mL of the monomer mix. The tissues were l
eft in the monomer mix on ice for about 15 min
so that the components of the mix could penetrate the bacterial
biofilms and other contents on the tissues. The
monomer mix was removed using a pipette and substituted with 1
mL of fresh mix. Finally, we removed 900 μL.
We covered the pools with plastic membranes (#664475; Grace Bio
-Labs), and added a Kimwipe imbibed with
Mondragón‐Palomino et al. Supplement p. 3
PBS to maintain the humidity in the petri dish. We sealed each
petri dish with parafilm and put all the Petri dishes
into an incubator set to 37 °C in the anaerobic chamber for 3 h
to allow the acrylamide layer to form at the glass-
tissue interface. We removed the Petri dishes from the incubato
r and the anaerobic chamber and added a few
droplets of 1X PBS onto each tissue to keep them humid. The Pet
ri dishes were refrigerated (4 °C) overnight.
The next day, the Petri dishes containing the tissue samples we
re put in a box with ice and brought back into
the anaerobic chamber, where each tissue was embedded in an acr
ylamide matrix without bisacrylamide. This
step is necessary to turn the tissue into a hydrogel. Embedding
lasted 3 h, after which the excess acrylamide
mix was removed and the tissue was polymerized for 3 h at 37 °C
. The tissues were taken out of the incubator
and the anaerobic chamber, and stored at 4 °C with a few drople
ts of sterile PBS.
Tissue samples were removed from the glass slides with a steril
e razor-blade and glued (Gluture 503763; World
Precision Instruments, Sarasota, FL, USA) onto a piece of semi-
rigid plastic (Polypropylene film 160364-46510;
Crawford Industries, Crawfordsville, IN, USA) that was previous
ly cleaned of RNAse (RNaseZap, AM9780;
ThermoFisher Scientific), sterilized with 70% ethanol, and trea
ted with oxygen plasma for 3 min to enhance
adherence.
After samples were turned into a hydrogel and before they were
passively cleared, we permeabilized bacteria
according to the parameters prescribed by the optimization of l
ysozyme treatment (
SI Appendix,
Optimization of
lysozyme treatment for HCR
). Samples were pre-incubated in 10 mM Tris-HCl (#AM9856, Invit
rogen, Carlsbad,
CA, USA) for 1 h at room temperature, then treated with lysozym
e at a concentration of 5 mg/mL in 10 mM Tris-
HCl, pH = 7.9, at 37 °C for 7 h for thin samples without much m
aterials left on their surface, and for 13h for
samples with abundant contents left on their surface. Lysozyme
treatment was stopped by washing excess
enzyme overnight in 1X PBS at room temperature in gentle shakin
g. Permeabilized samples were enclosed in
tissue cassettes and cleared for 4 d in 8% w/v sodium dodecyl s
ulfate (SDS) in PBS, pH = 8.3 at 37 °C. SDS
was vigorously stirred. pH was adjusted daily. SDS was removed
by washing in stirred 1X
PBS for 2 d
at 25 °C.
Total DNA was stained with DAPI (3 μg/mL in PBS) for 1 d. Host
mucus was stained by submerging samples in
a solution of WGA in 1XPBS at a concentration of 50 μg/mL.
Distal ileum samples for imaging (Fig. 3G-I) were obtained from
one 9-month-old C57BL/6J male mouse and
were processed similarly to tissues from the cecum and proximal
colon.
HCR staining of bacterial 16S rRNA
To fluorescently tag 16S rRNA transcripts from mucosal bacteria
, we incorporated HCR labeling of RNA to the
workflow
1,2
. HCR is executed in two stages: detection and amplification. I
n the detection stage, one or multiple
HCR probes hybridize to homologous RNA transcripts. In the ampl
ification stage, a unique initiator sequence
encoded in each probe selectively hybridizes to matching DNA ha
irpin pairs. The HCR seeded by the initiator
sequence concatenates the matching fluorescently labeled hairpi
ns into a long double-stranded DNA molecule.
Independent probes and orthogonal hairpins enable multiplexed f
luorescent labeling of RNA markers.
We designed HCR probes (
SI Appendix,
Tables S1-S2) and used them to image the location of total bac
teria
and specific taxa on intestinal tissue. We used the eubacterial
probe eub338-B4 and B4-Cy3B hairpins. Samples
were “pre-hybridized” in a solution of 2xSSC (#V4261, saline so
dium citrate, Promega Corp., WI, USA) and 10%
dextran sulfate sodium (#D8906, Sigma, MO, USA) for 1 h at room
temperature. Next, we treated samples for
16 h at 46 °C in a buffer consisting of 2xSSC, 10 %w/v dextran
sulfate sodium, 15% formamide (#BP227100,
Fisher Scientific, NH, USA) and probe eub338-B4 at a final conc
entration of 10 nM. The unbound probe was
washed off for 1 h in a solution of 2xSSCT (2xSSC, 0.05 % Tween
20) and 30% formamide, followed by another
wash in 2xSSCT for 1 h. Samples were pre-amplified with in a bu
ffer of 5xSSC and 10 % w/v dextran sulfate for
1 h. The pre-amplification time was used to aliquot, heat-shock
(90 s at 95 °C), and cool down hairpins to room
temperature (at least 30 min in the dark). The amplification st
ep was carried out in a buffer that consists of a
solution with 5xSSC, 10 % w/v of dextran sulfate and a final co
ncentration of 120 nM of each hairpin (B4-Cy3B-
H1, B4-Cy3B-H2 for Fig. 1-5, and B1-A514-H1, B1-A514-H2 for Fig
. 6-7). The amplification reaction lasted 16-
20 h at room temperature in the dark. Hairpins that did not par
ticipate in the reaction were washed out in 2xSSCT
for at least 1 h at room temperature with gentle shaking.
Mondragón‐Palomino et al. Supplement p. 4
Multiplexed fluorescent labeling of 16S rRNA transcripts of muc
osal bacteria by HCR was executed analogously
to the monochromatic staining. However, because taxon-specific
probes have different melting temperatures,
hybridization reactions were executed over 3 d, starting with t
he probes that required the highest formamide
concentration and finishing with the probes that required the l
east formamide. Because the detection sequence
cfb560 is degenerate and usually produces a low signal, we only
considered the sequences (cfb560a, cfb560b)
that target bacteria we found through sequencing. Hybridization
reactions for multiplexed imaging (Fig. 5) were
as follows: muc1437-B4 and the suite lgc354a-b-c-B5 at 10% form
amide, clept1240-B3 at5% formamide, and
cfb560a-B2, cfb560b-B2 and lac435-B1 at 0% formamide. Similarly
, for cecal tissues of mice unexposed to and
recovered from ciprofloxacin, formamide concentrations were: lg
c354a-b-c-B5, eub338-B1, and clept1240-B3 at
10% formamide, lac435-B3 and muc1437-B4 at 5% formamide, and cf
b560a-B2 and cfb560b-B2 at 0%
formamide. Samples were “pre-hybridized” in a solution of 2xSS
C (#V4261, saline sodium citrate, Promega
Corporation) and 10% dextran sulfate sodium (#D8906, Sigma) for
1 h at room temperature. Next, samples were
treated for 16 h at 46 °C in a buffer consisting of 2xSSC, 10 %
w/v dextran sulfate sodium, formamide at the
specified concentration (#BP227100, Fisher Scientific) and each
probe at a final concentration of 10 nM.
Unbound probes were washed off for 1 h in a solution of 2xSSCT
(SSC with % Tween 20) and 30% formamide,
followed by another wash in 2xSSCT for 1h (or 3 h for the last
wash on day 3). Probe clept1240-B3 was used at
a 2X concentration because it has one degenerate base. After al
l probes were hybridized to samples, the
amplification stage was carried out in a single step. HCR hairp
in pairs were assigned to fluorophores as follows:
B1-A514, B2-A647, B3-A594, B4-Cy3B, B5-A488. The HCR hairpin pa
irs for the ciprofloxacin challenge
experiments were as follows: B1-A514, B2-A546, B3-A594, B4-A647
, B5-A488. The amplification buffer consists
of a solution of 5xSSC, 10% of dextran sulfate and 120 nM of ea
ch hairpin. The amplification reaction was run
for 20 h at room temperature in the dark. Hairpins that did not
participate in the reaction were washed out in
2xSSCT for 4 h at room temperature with gentle shaking.
HCR probes were ordered as individual 250 nmol scale DNA oligos
purified by standard desalting (Integrated
DNA Technologies, IA, USA). Hairpins were ordered from Molecula
r Instruments, a Caltech facility within the
Beckman Institute. All the solutions were made with DNase/RNase
-free distilled water (#10977023, Invitrogen).
Media for bacterial culture
The following media were used to culture bacteria for
in vitro
assays.
Escherichia coli
was cultured in LB media
(LB Broth, #240230, Difco, Becton, Dickinson and Company, NJ, U
SA) and LB agar.
Clostridium scindens
was
cultured in a mix of 50% Shaedler media (#cm0497, Oxoid, Thermo
Fisher, Waltham, MA, USA) and 50% MRS
media (Lactobacilli MRS Broth, #288130, Difco), and in Schaedle
r agar.
Lactobacillus AN10
was cultured in a
mix of 50% Shaedler media and 50% MRS media, and in MRS agar.
Bacteroides fragilis
and
Faecalibacterium
prausnitzii
were cultured in LYBHI
3
media (brain-heart infusion medium supplemented with 0.5% yeas
t extract,
Difco, Detroit, USA), and in LYBHI agar.
Akkermansia muciniphila
was cultured in LYBHI media supplemented
with hog-mucus.
Harvesting of tissues for sequencing
Four 4-month-old adult male and female specific-pathogen-free (
SPF) mice were euthanized by CO
2
inhalation
according to approved IACUC protocol #1646 and #1769 and follow
ing all guidelines and standard operating
procedures (SOPs) of the Caltech Institutional Animal Care and
Use Committee (IACUC). The gastrointestinal
tract, from the stomach to the rectum, was dissected and stored
in a sterile container on ice. The cecum of
mice was cut open with sterile instruments on an ice-cold steri
le surface inside a biosafety cabinet. The bulk of
cecal contents was removed with sterile tweezers, stored in ste
rile tubes, and kept at -20 °C. The cecal tissue
was kept flat on a cold and sterile surface while it was cleane
d with ice-cold and sterile 1X phosphate-buffered
saline. PBS 1X was obtained from a 10X dilution of phosphate bu
ffered saline 10X (Corning, 46-013-CM) in
ultra-pure DNase/RNase-free distilled water (10977023; ThermoFi
sher, Waltham, MA, USA). After removing
contents from the cecum, the cecal mucosa was harvested by scra
ping it with sterilized microscopy glass
Mondragón‐Palomino et al. Supplement p. 5
plates. Samples were stored in sterile tubes at -20 °C. Cecal c
ontents and tissue scrapings were sent to Zymo
Research (Irvine, CA, USA) for 16S rRNA gene sequencing and bio
informatics analyses (
SI Appendix, DNA
extraction, sequencing, bioinformatics analyses, and absolute quantification
).
For the antibiotic challenge experiment, fecal pellets for sequ
encing of bacterial 16S rRNA gene were collected
from all mice at days 0 (no antibiotic), 4 (end of antibiotic a
dministration) and 14 (end of 10-day recovery). For
each time point, the samples of two mice per cage were weighted
and processed as described elsewhere
4
to
obtain DNA extracts, which were
sent to Zymo Research (Irvine,
CA, USA) for 16S rRNA gene sequencing and
bioinformatics analyses.
OPTIMIZATION OF LYSOZYME TREATMENT FOR HCR
A. Preparation of acrylamide gels pads with embedded bacteria
Bacteria were cultured to exponential phase at 37 °C in anaerob
ic conditions. From this culture, a dense (10
9
–
10
10
cells/mL) suspension of cells was prepared in PBS. This suspen
sion was spiked into the monomer mix with
bisacrylamide (
SI Appendix,
Composition of acrylamide monomer mix
) to the final cell density of ~ 5 x 10
7
cells/mL. The mix of monomer and cells sat on ice for 15 min be
fore being dispensed into pools made of silicone
isolators (13 mm diameter x 0.8 mm depth; #666507; Grace BioLab
s) glued to microscope slides. We pipetted
106 μL into each pool, and polymerized at 37 °C for 3 h in anae
robic conditions. The next day, the original
silicone isolators were replaced with larger ones (20 mm diamet
er x 2.6 mm depth; #666304; Grace-Bio Labs).
The new pools with the gels were filled with a monomer mix with
no bisacrylamide (
SI Appendix,
Composition of
acrylamide monomer mix
) and incubated on ice for 3 h in anaerobic conditions. Next, t
he monomer mix was
removed and the gels were polymerized at 37 °C for 3 h in anaer
obic conditions. Bacteria in the gel pads were
predigested in lysozyme buffer (10 mM Tris, pH=8.0) at room tem
perature for 1 h, and then digested with
lysozyme (1, 2.5 or 5 mg/mL lysozyme in 10 mM Tris, pH = 8.0) a
t 37 °C for 6 h. Lysozyme was washed away
with PBS at room temperature overnight. The gel pads were clear
ed with 8% SDS in 1xPBS,
pH = 8.3, at 37 °C
for 2 d following a 1x PBS wash at 25 °C for another 2 d. Bacte
ria were hybridized with a eubacterial HCR probe
(eub338-B5) in hybridization buffer with 15% formamide and ampl
ified for 16 h. Finally, DNA was stained with
DAPI (5 μg/mL) overnight.
B. Imaging of gel-embedded bacteria
Gel pads were mounted in 1x PBS and imaged with an upright lase
r-scanning confocal microscope (LSM880,
Carl Zeiss AG, Germany) using a long-working-distance water-imm
ersion objective (W Plan Apochromat 20X/1.0
DIC Korr UV Vis IR, #421452-9700; Carl Zeiss AG). Fluorophores
were excited using two lasers with λ = 488
nm and λ = 405 nm. Imaging settings were the same across all ge
l pads. Images were processed in commercial
software for 3D image analysis (Imaris, Bitplane AG, Switzerlan
d). Cell surfaces were identified by their
fluorescence in the 405 nm channel. Finally, for the identified
cells, mean cell fluorescence intensity in the 488
nm channel was computed.
C. Results
The Gram-positive bacterium
Clostridium scindens
was efficiently permeabilized in gel pads subjected to the
treatment of 5 mg/mL of lysozyme (Fig. 2B). To assess whether l
ysozyme treatment may affect HCR staining of
Gram-negative bacteria, a lysozyme treatment optimization exper
iment was carried out using the model Gram-
negative bacterium
Bacteroides fragilis
(
SI Appendix, Fig. S1
). Exponential phase
B. fragili
s cells were
embedded into acrylamide gel pads, treated for 6 h with four co
ncentrations of lysozyme (no lysozyme control,
1.0 mg/mL, 2.5 mg/mL, and 5.0 mg/mL) and cleared with 8% SDS fo
r 2 d. 16S rRNA was stained by HCR using
universal detection probe eub338, and DNA was stained with DAPI
. Bacteria in gel pads were imaged in a
confocal microscope (LSM 880, Carl Zeiss AG) from the surface o
f the gel down to 600 μm into each gel.. A 3D
rendering of confocal images in the DAPI channel (
SI Appendix, Figs.S1a, S1d, S1g and S1j
) showed that DAPI
staining did not require permeabilization of the peptidoglycan
layer, justifying the choice of using the DAPI
channel to define the surface of bacterial cells. When lysozyme
treatment was omitted,
B. fragilis
cells at the
surface of the gel were stained poorly by HCR (
SI Appendix, Fig. S1b
). Image analysis was in agreement with
Mondragón‐Palomino et al. Supplement p. 6
these visual inspections; ECDF c
urves shifted progressively to
higher Signal/Background with depth (
SI
Appendix, Fig. S1c
). Across all 100-μm thick slices, a substantial fraction of ce
lls (>20%) were fainter than the
set background value (
SI Appendix, Fig. S1c
). The lowest lysozyme concentration (1 mg/mL) was sufficient t
o
improve HCR staining of
B. fragilis
(
SI Appendix, Fig. S1e-f
). Although cells at the surface of the gel pad
appeared brighter, >99% of all cells across the entire 600 μm w
ere brighter than the set background value (
SI
Appendix, Fig. S1f
). Lysozyme concentrations 2.5 and 5.0 mg/mL did not deteriorat
e HCR staining (
SI Appendix,
Figs. S1h-i and S1k-l
). These results showed that a treatment of 5 mg/mL of lysozyme
for 6 h pearmeabilized
the peptidoglycan layer of Gram-positive and Gram-negative bact
erial cells; thus, we used this treatment as a
reference for
in situ
experiments.
IN VITRO
ASSAYS TO FIND FORMAMIDE CONCENTRATIONS FOR STRINGENT HYBRIDIZATION OF
TAXON-SPECIFIC HCR PROBES, AS WELL AS TO QUANTIFY THEIR SENSITIVITY AND SPECIFICITY
We created an
in vitro
assay to determine the adequate formamide concentration for th
e hybridization of each
HCR probe to its ideal target (
SI Appendix, Table S1 and Fig. S3
), as well as to test the probes’ sensitivity and
specificity. The assay consists of regularly spaced shallow acr
ylamide gels on a glass slide. Bacteria are
embedded in the gels, which are then surrounded by individual s
ilicone wells.
A. Preparation of bacteria embedded in shallow acrylamide gels
Bacteria were grown anaerobically at 37 °C to OD600 0.2–0.24 fr
om overnight cultures (
SI Appendix,
Media for
bacterial culture
). Cultures were pelleted and resuspended in a preparation of g
el mix with bisacrylamide (
SI
Appendix,
Composition of acrylamide monomer mix
). In anaerobic conditions, 3.8 μL of the acrylamide with
bacteria were pipetted into each well of a SecureSeal
imaging spacer (#470352; Grace BioLabs) that had been
glued to a clean glass slide. Wells were sealed with a silicone
membrane (#664475; Grace Bio-Labs). The slide
was flipped upside down for 5 min so that bacterial cells could
settle on the surface, and then the slide was
placed in a sealed petri dish and placed in an anaerobic incuba
tor for 2 h at 37 °C. Once gels solidified, a silicone
isolator (#665101; Grace BioLabs) was added to each slide to cr
eate a pool around each gel. Next, bacteria
were treated with a solution of 1 mg/mL lysozyme in 10 mM Tris
balanced to pH = 8 for 2.5 h at 30 °C. Gels were
washed twice with 1x PBS for 10 min and 30 min. In agreement wi
th clearing methods, the glass slide was
submerged in a solution of 4% SDS in 1x PBS at 37 °C for 2 h. T
he silicone wells were removed and the SDS
solution was gently rinsed with 27 °C 1x PBS. Slides were furth
er washed in 1xPBS for 10 min and overnight at
room temperature. Slides were dried out and another silicone is
olator was applied around gels. Probes were
hybridized in 2x SSC (saline sodium citrate) with 10% dextran s
ulfate, 0-60% formamide, and final probe
concentration of 10 nM. The hybridization buffer was pipetted i
nto the silicone isolator wells, covered with a
hybridization film (#716024; Grace BioLabs) and put in a sealed
petri dish. Glass slides were incubated at 46 °C
for 12 h. Unbound probes were washed three times with 2x SSCT (
2x SSC, 0.05% Tween 20), and 30%
formamide for 10 min. Three additional 10 min long washes were
done in a buffer of 2x SSCT.
In the amplification step, hairpins were heat-shocked at 95 °C
for 90 s and cooled down at room temperature for
30 min. Gels were covered with the amplification buffer of 2x S
SC, 10 % w/v dextran sulfate and hairpins to a
final concentration of 120 nM, and covered with a hybridization
film. The amplification reaction was carried out
at room temperature for 12 h. Unbound hairpins were washed out
in a solution of 2x SSCT three times for 10
min. Three additional 10-min-long washes were done in a buffer
of 2x SSC. Finally, bacterial DNA was stained
with DAPI for 1 h, followed by a 30 min wash in 1x PBS. Slides
were dried out and another imaging spacer was
applied around the gels (#654008; Grace BioLabs). Gels were mou
nted in 1x PBS and covered with a glass
coverslip. Bacteria on the upper surface of the gels were image
d with an oil-immersion objective (Plan
Apochromat 63X/1.4 Oil DIC, #420782-9900-799; Carl Zeiss AG) in
an upright confocal microscope (LSM 880,
Carl Zeiss AG).
B. Formamide curves
We next established the range of formamide concentrations that
would yield stringent hybridization of taxon-
specific HCR probes. Two slides of gels were prepared for each
target bacterium (
SI Appendix,
Fig. S3
). One
slide was used to quantify the efficiency of hybridization for
concentrations of formamide in 15% steps from 0-
Mondragón‐Palomino et al. Supplement p. 7
60 %. The second slide was used to refine the coarse measuremen
ts in 5% steps around the maximum of the
coarse curve. Each slide was prepared once. We obtained one st
ack of images from each gel. Stringent
hybridization was obtained around the concentration of formamid
e that produced the strongest average
fluorescence. To determine the optimal formamide concentration
for the hybridization of probe muc1437 for
A.
muciniphila
, we tested the concentrations 0%, 5%, and 10%. We found that 5
% formamide provided stronger
signal than 0% and 10%. As a result, we set the concentration o
f formamide at 5% in hybridization reactions for
the antibiotic challenge experiments, as opposed to the 10% tha
t was previously used.
C. Sensitivity and specificity of taxon-specific HCR probes
To quantify the sensitivity and specificity of taxon-specific H
CR probes, one multi-species glass slide was
prepared for each tested probe (
SI Appendix,
Fig. S3
). Hybridization was carried out at a formamide
concentration within the intervals prescribed by the formamide
bar plots (gam42a: 5%, eco630: 10%, lac435:
0%, lgc354: 5%, cfb560: 0%, lab158: 10%, clept1240: 0%.) . Each
hybridization experiment was carried out in
one gel. One stack of images was stained from each gel. We test
ed the specificity of lac435 , lgc354 , cfb560 ,
lab158 , clept1240 against
A. muciniphila
, without any detectable overlap.
D. Image Processing
Images were analyzed using commercial software (Imaris, Bitplan
e, Belfast, UK). Image stacks were 3D-
rendered and surfaces were created over individual bacteria usi
ng the fluorescent signal from the DAPI stain.
Because bacterial DNA is found throughout the cell, surfaces de
rived from DAPI fluorescence encompass entire
cells. For each cellular volume, the software computed the aver
age fluorescent intensity for two channels: the
eubacterial channel (eub338-B5/Alexa488) and the channel for a
taxon-specific sequence (B4/Cy3B).
Formamide plots (
SI Appendix,
Fig. S4
) were obtained by plotting the mean and standard deviation of
the
fluorescence intensity in the Cy3B channel for each concentrati
on of formamide.
To quantify the sensitivity and specificity of taxon-specific p
robes (Fig. 5B), images were processed as described
in the previous paragraph. For each probe we set a fluorescenc
e detection threshold such that 85% of the ideal
target bacterium was detected (e.g., the lac435 probe’s ideal t
arget was
C. scindens
). For each non-ideal target
(for example lac435 should not target
B. fragilis
although it may bind to a small number of cells), off-target
hybridization is quantified as the fraction of bacteria above t
he fluorescence detection threshold.
ACQUISITION, PROCESSING AND ANALYSIS OF
IN SITU
IMAGING
A. Objectives and laser wavelengths
For large-scale acquisition, we used either of two objectives:
Plan-Neofluar 5X/0,15 (#440320, Carl Zeiss AG),
or EC Plan-Neofluar 5X/0.16 (#420330-9901, Carl Zeiss AG). For
imaging at 20X magnification, we used one
CLARITY optimized objective with an adjustable correction colla
r for compensation of spherical aberrations: Clr
Plan-Neofluar 20x/1.0 Corr nd=1.45 M32 85mm (#421459-9970-000,
Carl Zeiss AG). Fluorophores were excited
with laser light of the following wavelengths: 405 nm, 488 nm,
561 nm, 633 nm. During acquisition, the power of
lasers was adjusted to avoid saturation and photobleaching due
to prolonged excitation of fluorophores with
laser light. Spectral acquisition was used only for imaging sam
ples with multiplexed HCR.
B. Imaging of the host-microbiota interface in the proximal col
on
In one sample of the proximal colon (
SI Appendix,
Fig. S5
), four areas corresponding to the tops of intestinal
folds were imaged. The resulting image stacks contained three c
hannels: one for DNA (DAPI), one for bacteria
(HCR staining), and one for mucus (WGA lectin). To quantify the
thickness of the layers of mucus at the top of
intestinal folds, image stacks were 3D rendered in a commercial
software (Vision4D 3.0, Arivis AG, Germany).
Next, the maximum intensity projections of two digital cross se
ctions (7 μm), along and across the longitudinal
axis of the folds, were obtained. The thickness of the internal
mucus layer was measured (n = 85) from the edge
of the epithelium to the edge of the internal mucus layer. The
thickness of the external mucus layer was
measured (n = 75) from the end of the internal mucus layer to t
he edge of the external mucus layer (Fig. 3B).
Mondragón‐Palomino et al. Supplement p. 8
A second sample of the proximal colon from a different mouse wa
s imaged to show the procedure is repeatable
and produces consistent results (
SI Appendix,
Fig. S9
). Bacteria colonized profusely the outer mucus barrier
between luminal contents and the epithelium. Bacteria were most
ly segregated to the outer mucus layer, but
manage to contact the epithelial layer at points where the inne
r mucus layer is thin. The outer mucus layer was
interspersed by spherical objects that are consistent with DAPI
staining of mammalian cells (
SI Appendix,
Fig.
S9e-g,
cyan circles in panels), and larger objects that we hypothesize
are food particles (
SI Appendix,
Fig. S9e-
g,
cyan shade on top of outer mucus-bacteria layer).
C. Linear unmixing of spectral imaging
Computational linear unmixing of spectral imaging was performed
to determine the relative contribution from
each fluorophore for every pixel of
in situ
multiplexed imaging of bacteria. Linear unmixing requires the
emission
spectrum of every fluorophore that was employed in the staining
of samples including DAPI and the suite of
Alexa fluorophores of HCR. Spectra were acquired independently
but in similar optical conditions as described
in our
in situ
imaging of bacteria. We used
E. coli
bacteria embedded in thick acrylamide gels that were prepared
as previously described (
SI Appendix,
Optimization of lysozyme treatment for HCR
). Two gels of 13-mm diameter
were split into six smaller gels that were taken through our st
andard HCR protocol. Each gel with
E. coli
was
hybridized with a different probe. Each HCR probe consisted of
the eubacterial detection sequence eub338 and
a different initiator sequence. Each initiator sequence matched
a different hairpin/fluorophore set. Probes:
eub338-B1 (A514), eub338-B2 (A647), eub338-B3 (A594), eub338-B4
(A546), eub338-B5 (A488) and eub338-
B4 (Cy3B). Bacterial DNA was not stained with DAPI. The emissio
n spectrum of DAPI was acquired directly from
the tissue samples. Gels were mounted in a RIMS solution with n
~ 1.46. Imaging of bacteria in gels was carried
out using a laser-scanning confocal microscope with parallel sp
ectral acquisition (LSM880, Carl Zeiss AG), and
with the same objective as imaging of tissue samples (Clr Plan-
Neofluar 20x/1.0 Corr nd=1.45 M32, Carl Zeiss
AG). We extracted the spectral references from the imaging of b
acteria using commercial software (Zen 2.3 SP1,
Carl Zeiss AG). Finally, the spectral references and the same s
oftware were used to perform linear unmixing of
in situ
images.
D. Multiplexed imaging of cecal crypts
The size of cecal tissue samples was approximately ¼ of the tot
al size of the cecum. Because there are
thousands of crypts in a sample, it was not practical to image
them all at 20X magnification (20 crypts/field of
view, 425x425 μm
2
). Inst
ead, we examined samples thoroughly at 20X magnification and im
aged only the areas
where crypts were colonized, making the best effort to capture
most colonized crypts. Therefore, imaged crypts
were representative of the region of the cecum that we imaged a
nd we believe that ¼ of the cecum may be
representative of the rest of the organ, but that would need to
be further investigated. We imaged the top portion
of the cecum, which we have found to be consistently colonized
by bacteria in C57BL/J6 mice of ages 13-20
weeks. In the context of the antibiotic challenge experiments,
in the control group, we imaged 296 colonized
crypts (2 mice) and in the recovery group we imaged 199 coloniz
ed crypts (3 mice). In unexposed mice, we
quantified 160 crypts (3 mice) after 4 days of ciprofloxacin. H
owever, the number of crypts that were visually
examined during image acquisition is perhaps two orders of magn
itude larger. It is also important to mention that
we focused on a very specific form of colonization of the mucos
a, namely the colonization of individual crypts,
but the mucosa is colonized much more abundantly in the crevice
s that are formed when multiple crypts merge
at the luminal side (Fig. 1D).
Multiplexed confocal spectral images of cecal mucosa at 20X mag
nification were taken through linear unmixing
and analyzed computationally to measure the abundance and locat
ion of bacterial taxa that were labelled by
HCR. The resulting data files contained image stacks with seven
channels. Five channels corresponded to the
probe/fluorophore pairs that were used in HCR (lcg354/A488, lac
435/A514, muc1437/Cy3B, clept1240/A594
and cfb560/A647, or eub338/A514, lcg354/A488, lac435/A594, muc1
437/A647, clept1240/A594 and
cfb560/A546), one channel corresponded to the fluorescent DNA m
arker DAPI, and one channel stored pixels
that were not assigned to any of the other six channels in line
ar unmixing and thus captured undefined content.
Multiplexed imaging for the antibiotic challenge experiment als
o included a channel to remove unspecific HCR
signal, whose spectrum was defined within each image. Image sta
cks were uploaded to commercial software
Mondragón‐Palomino et al. Supplement p. 9
Vision4D (Vision4D 3.0, Arivis AG) and saved in the native
sis
format. Because tissue was sometimes very tilted
with respect to the plane of imaging, image stacks were rotated
so that crypts were approximately aligned with
the spatial z axis. Rotated stacks were cropped manually to rem
ove areas without data.
For the initial small data set in SPF antibiotic-naïve mice, th
e spatial analysis included ~60 crypts from three
fields of view obtained from one cluster of crypts in a sample
of the cecum. For the antibiotic challenge
experiments, the spatial analysis included 296 colonized crypts
from two untreated mice and 199 colonized
crypts from three antibiotic-treated mice that were imaged afte
r 10 days of recovery. The internal volume of
crypts was segmented manually using the “Draw Objects Tool.” Th
e manual segmentation of crypts was guided
by the DAPI channel, which showed the location of nuclei on the
epithelial wall of crypts. To restrict the analysis
to bacteria inside crypts, we used the segmented internal volum
es of crypts as a mask on the channels with
HCR staining (i.e., the fluorescence intensity value of voxels
outside crypts was set to zero in the five HCR
channels). Next, bacterial channels were segmented with an “Int
ensity Threshold” filter. In the output of this
operation, a bacterial cell or group of bacteria in each channe
l (a segment) was defined as a set of contiguous
pixels with intensities that fe
ll within a range (minimum and m
aximum bounds, hereafter Min and Max) where at
least one pixel had an intensity equal to a core value (require
d core intensity, RCI). For the initial small data set
in SPF antibiotic-naïve mice, segmentation parameters (Min, Max
and RCI) were estimated by measuring the
intensity of a subset of pixels in each channel throughout ever
y stack and defined RCI as the mean of pixel
intensities, and Min as the difference between the mean and the
standard deviation of intensities. Max was set
equal to the maximum intensity of bacteria in the channel. Next
, we filtered out segments that were <18 voxels.
Channel cfb560/A647 required further manual curation to remove
segments that were not likely to be bacteria
due to their size and location. Finally, to determine which bac
terial segments were located within each crypt we
combined all bacterial segments into a single list and used the
“Segment Colocalization” operation. Bacterial
segments were considered the “Subjects,” and the manually segme
nted crypts were used as “References.” The
“Colocalization Measure” required that “Subjects” (bacteria) we
re completely within the “References” (crypts).
The identities of bacterial segments and their crypt-specific a
ssignment were stored at the end of the pipeline.
The final result of the image-processing pipeline is shown in S
upplementary Video S4. For the antibiotic
challenge experiments, segmentation parameters (min, max and RC
I) were set manually for each image stack
and stored in the segmentation pipelines saved within the corre
sponding analysis pipelines. In the eubacterial
channel, we filtered out segments that were <13 voxels. A size
filter was not applied to the rest of the channels.
Minimal manual curation of segmentation in the eubacterial chan
nel was required in a reduced number of crypts.
These crypts typically had the lowest amounts of bacteria and t
he poorest signal/noise ratio, and were
constrained to one sample. Objects that were segmented in the t
axon-specific channels were validated as
bacteria if they co-localized with an object in the eubacterial
channel (
SI Appendix, Fig. S6-8
). To do this, we
used the “Colocalization Measure” that “Subjects” were partiall
y within the eubacterial object “References”. The
exception to this rule was the channel (muc1437/A647) because t
he rRNA of
A. muciniphila
is poorly hybridized
by the eubacterial probe eub338.
The “Intensity Threshold” filter produced large bacterial segme
nts that spanned multiple fields of view. We
exported the identity (imaging channel), volume (voxel count),
center of mass (z coordinate in μm), and first/last
plane of all segments (bacteria and crypts) into a MATLAB-reada
ble file. From this output, we obtained the
abundance (voxel count) of bacteria for each crypt, as well as
the position (center of mass) of each bacterial
segment in the framework of the corresponding crypt. The spatia
l reference z = 0 μm in each crypt was set at
the luminal end of the crypt segment.
Tissues obtained at the end of the administration of ciprofloxa
cin (day 4) were stained with a eubacterial probe
and all crypts there seemed to be empty except for a few. Three
of these crypts were found in the sample from
the cage that was less harshly affected by ciprofloxacin (by th
e total load of bacteria in feces) (
SI Appendix, Fig.
S11a-b
). However, fluorescent signal was also observed underneath the
epithelium. To clarify this, we performed
two-color HCR tagging with the
eubacterial eub338 probe in the
2 samples from the other 2 cages (
SI Appendix,
Fig. S11c-f
). To thoroughly quantify the abundance of bacteria in antibiot
ic-treated tissues, we picked randomly
~5 crypts per field of view from all antibiotic-treated samples
for a total of 160 crypts, and processed the imaging
of the eubacterial signal in the same way as we did for coloniz
ed crypts in other tissues. The crypts with putative
Mondragón‐Palomino et al. Supplement p. 10
bacteria (
SI Appendix, Fig. S11a-b
) were included. The image processing pipeline confirmed that m
ost crypts
were empty and did not yield any segmented objects within most
crypts, and in the crypts where objects were
found, no overlap between segmented objects in the eub338-A514
and eub338-A633 channels was found.
Therefore, we concluded that the signal in the first sample (
SI Appendix, Fig. S11a-b
) was not of bacterial origin,
but noise in the A633 channel and set the volume of bacteria in
crypts as zero (Fig. 6D).
Controls for
in situ
HCR
We performed HCR
in situ
to quantify the intensity of fluorescence due to nonspecific d
etection and amplification
(Fig. 2D-F). We used an HCR probe with a nonspecific eubacteria
l detection sequence (non338) (
SI Appendix,
Table S1
) on one tissue sample from the proximal colon of a mouse with
a microbiota (specific pathogen free,
SPF), and an HCR probe with a eubacterial detection sequence (e
ub338) on one tissue sample from the proximal
colon of a germ-free (GF) mous
e. The HCR reactions for these co
ntrol experiments followed the same steps as
the procedure to stain mucosal bacteria with a single eubacteri
al probe.
Statistics
When proportional abundances obtained from sequencing of 16S rR
NA genes are combined with absolute
counts of 16S rRNA gene copies (qPCR), the variance of each dat
a set combines to produce a compounded
experimental error for the taxon-specific absolute abundances (
Fig. 6C). This error can be calculated from the
knowledge of the error from each data set by standard or Monte
Carlo error propagation methods. Unfortunately,
due to the use of an external vendor for sequencing and microbi
al quantification, we did not have access to all
of the data on variance and measurement errors in the commercia
l methods and therefore we could not perform
error propagation. Nevertheless, key results like the recovery
of total bacterial load and the lack of
Muribaculaceae
at 10 days post-antibiotic are unlikely to change drastically
due to systematic error of the
absolute and relative abundances. More subtle changes in the ab
solute abundance of other microbial families
might be corrected after the propagation of systematic error.
We calculated the silhouette score for the hierarchical cluster
ing analysis (Fig. 7B). The score for each crypt and
the distribution of scores for each crypt community class (A-F)
(
SI Appendix, Fig. S27
). The silhouette score
shows that hierarchical clustering and the binning method can s
egregate data points reasonably well. Most crypt
communities have a positive score (446 out of 468) and for each
bin (A-F), 94%, 56%, 94%, 45%, 38%, and
40% of crypt communities have a silhouette score higher than 0.
5.
DNA extraction, sequencing, bioinformatics analyses, and absolute quantification
A. Origin of samples and DNA extraction
Cecal contents from four mice (Fig. 5A) were sent to Zymo Resea
rch LLC (Irvine, CA) for DNA extraction,
sequencing and informatic analysis. For the antibiotic challeng
e experiments (Fig. 6-7), feces were obtained
from six mice of cohort A (two mice from each of three cages) a
t 0, 4 and 14 days. Approximately 19 mg of frozen
feces from each sample were used for extraction of DNA using th
e ZymoBIOMICS® DNA Miniprep Kit (Zymo
Research, Irvine, CA). Similarly, feces were obtained from six
mice of cohort B (three mice from each of two
cages) that were not exposed to antibiotic. Approximately 8 mg
of frozen feces from each sample were used for
extraction of DNA using the ZymoBIOMICS® DNA Miniprep Kit. DNA
eluates from the samples of cohorts A and
B were sent to Zymo Research for sequencing, absolute quantific
ation of bacterial load, and informatics
processing through the ZymoBIOMICS Targeted Sequencing Service
(Zymo Research, Irvine, CA), as described
below after the report provided by Zymo Research.
B. Targeted Library Preparation
Bacterial 16S ribosomal RNA gene targeted sequencing was perfor
med at Zymo Research using the Quick-
16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). Zymo Res
earch proprietary bacterial 16S primers
Mondragón‐Palomino et al. Supplement p. 11
amplified the V3-V4 region of the 16S rRNA gene. PCR reactions
were performed in real-time PCR machines to
control cycles and therefore limit PCR chimera formation. The f
inal PCR products were quantified with qPCR
fluorescence readings and pooled together based on equal molari
ty. The final pooled library was cleaned with
the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irv
ine, CA), then quantified with TapeStation
(Agilent Technologies, Santa Clara, CA) and Qubit (Thermo Fishe
r Scientific, Waltham, WA).
C. Controls
The ZymoBIOMICS Microbial Community Standard (Zymo Research, Ir
vine, CA) was used as a positive control
for each DNA extraction. The ZymoBIOMICS Microbial Community DN
A Standard (Zymo Research, Irvine, CA)
was used as a positive control for each targeted library prepar
ation. Negative controls (i.e. blank extraction
control, blank library preparation control) were included to as
sess the level of bioburden carried by the wet-lab
process. The final library was sequenced on Illumina MiSeq™ wit
h a v3 reagent kit (600 cycles). The sequencing
was performed with 10% PhiX spike-in.
D. Bioinformatics Analyses
Unique amplicon sequences variants were inferred from raw reads
using the DADA2 pipeline
5
. Potential
sequencing errors and chimeric sequences were also removed with
the Dada2 pipeline. Chimeric sequences
were also removed with the DADA2 pipeline. Taxonomy assignment
was performed using Uclust from Qiime
v.1.9.1 with the Zymo Research Database, a 16S database that is
internally designed and curated, as reference.
Composition visualization, alpha-diversity, and beta-diversity
analyses were performed with Qiime v.1.9.1
6
. If
applicable, taxonomy that have significant abundance among diff
erent groups were identified by LEfSe
7
using
default settings. Other analyses such as heatmaps, Taxa2ASV Deo
mposer, and PCoA plots were performed
with Zymo Research internal scripts.
E. Absolute Abundance Quantification
A quantitative real-time PCR was set up with a standard curve.
The standard curve was made with plasmid DNA
containing one copy of the 16S prepared in 10-fold serial dilut
ions. The primers used were the same as those
used in Targeted Library Preparation. The equation generated by
the plasmid DNA standard curve was used to
calculate the number of gene copies in the reaction for each sa
mple. The PCR input volume was used to
calculate the number of gene copies per microliter in each DNA
sample.
Mondragón‐Palomino et al. Supplement p. 12
Figure S1
.
Lysozyme treatment optimization using
Bacteroides fragilis
as a model Gram-negative bacterium.
Exponential-phase
B. fragilis
was embedded into four acrylamide gel pads and each pad was tr
eated with lysozyme at a di
fferent concentration (a-c: no-
lysozyme control;
d-f: 1.0 mg/mL for 6 h, g-i: 2.5 mg/mL for 6 h, and j-l: 5.0 mg
/mL for 6 h). HCR was used to stain 16S rRNA using a
eubacterial detection sequence (a, d, g, j) and DAPI was used t
o stain DNA (b, e, h, k
)
. In each gel pad, one field of view was imaged
from the surface of the gel down to 600 μm. Image stacks were b
inned in 100 μm slices by depth. In each bin, empirical cumulat
ive
distributions functions (ECDF) of HCR-stained
B. fragilis
(c, f, i, l) were computed. Cell
surfaces were defined by sett
ing a threshold in
Mondragón‐Palomino et al. Supplement p. 13
the DAPI channel, and mean cell fluorescence in the HCR channel
was computed for each cell. Ce
lls were binned into six 100-μm
thick
slices by depth. For each slice,
the ECDF of the signal/backgro
und ratio was plotted. Signal was defined as the mean cell fluo
rescence
intensity in the HCR channel, and background was defined as the
99th percentile of voxel fluore
scence values in the HCR channe
l from
a control gel pad with no-bacteria. A given ECDF value on the c
urve corresponds to the fraction
of cells having a signal/backg
round ratio
less than or equal to the signal/ba
ckground ratio specified on
the horizontal axis.
Figure S2. Empirical cumulative distributions functions (ECDF)
of HCR staining of Bacteroides fr
agilis over entire image stack
s
(600 μm deep).
Bacteria were embedded in four acrylamide gel pads, each treat
ed with a concentration of lyso
zyme (0.0, 1.0, 2.5, or 5.0
mg/mL).
Mondragón‐Palomino et al. Supplement p. 14
Figure S3. Experimental workflow to obtain the formamide hybrid
ization curves (SI Appendix, Figure S4) of HCR probes with
taxon-specific detection seque
nces ( SI Appendix, Table S1).
Cells of target bacteria were embedded in shallow acrylamide g
els
and subjected to a sequence of t
reatments analogous to the in s
itu method.. Microbial cells wer
e treated with different formam
ide
concentrations to determine the
optimal range of concentrations
for stringent hybridization to the bacterium’s specific detect
ion sequence.
Mondragón‐Palomino et al. Supplement p. 15
Figure S4. Formamide bar plots for the hybridization of taxon-s
pecific HCR probes to t
heir ideal targets (
SI Appendix,Table S1
).
From these plots we estimated the
range of formamide concentrat
ion to use in the detection step of HCR as follows: gam42a: 5-1
5%,
eco630: 10-15%, lac435 0-10%, lgc354: 0-10%, cfb560: 0-5%, lab1
58 0-10%, clept1240 0-10%. For the eubacterial detection sequen
ce
eub338 we used 15-20% and for mu
c1437 we used 5-10% formamide.
Mondragón‐Palomino et al. Supplement p. 16
Figure S5. Large-scale imaging of the proximal colon and the di
stal ileum.
(a) Maximum intensity projection of tiled images from the
proximal colon of an adult mouse. DNA (blue) was stained with D
API, and mucus (green) was stained with wheat germ agglutinin l
ectin
conjugated to A488 fluorophore. Bacteria were stained by HCR bu
t were not imaged at low magnification in this sample. The imag
e was
obtained by stitching together multiple fields of view acquired
at 5X magnification. The folded
topography of the proximal col
on is clearly
visible near the distal end of the sample, which was not covere
d by mucus but merely contained a large mucus thread. The proxi
mal side
of the sample was originally cov
ered with luminal contents; the
se were carefully removed befor
e the application of our method,
however
some contents remained. Scale b
ar: 5 mm. (b) Maximum intensity
projection of tiled images from the distal ileum of an adult mo
use
processed by our method. DNA (bl
ue) was stained with DAPI, mucu
s (green) was stained with wheat germ agglutinin lectin conjuga
ted
to A488 fluorophore, and bacteria (orange) were stained by HCR
with a eubacterial probe (eub338). The image was obtained by st
itching
together multiple fields of view acquired at 5X magnification.
Particles and materials that adhered to the tissue during clean
ing were
retained. Labels
A-C
indicate conglomerates of bacteria-colonized food particles, m
ucus and biofilms that adhered to the ileal epithelium.
Scale bar: 5 mm.
Mondragón‐Palomino et al. Supplement p. 17
Figure S6. Representative digital cross
section (1μm thick) of cecal tissue that was
hybridized with a eubacterial probe (eub338
/ A514) and a taxon-specific probe for
phylum Bacteroidetes (cfb560 / A546).
The
fluorescence from the DAPI stain for total DNA
is shown in grey. The fluorescence from the
eubacterial signal (cyan) was segmented and
used to mask the fluorescence from the cfb560
/ A546 channel ( yellow). Only the fluorescence
from the cfb560 / A546 channel that was
matched by the eubacterial channel was
considered to originate from bacteria. All scale
bars correspond to 100 μm.
Mondragón‐Palomino et al. Supplement p. 18
Figure S7. Representative digital cross section (1μm thick) of
cecal tissue that was hybridized with a eubacterial probe (eub3
38
/ A514) and a taxon-specific probe for order Bacilli (lcg354 /
A488).
The fluorescence from the DAPI stain for total DNA is shown in
grey. The fluorescence from the eubacterial signal (cyan) was s
egmented and used to mask the fluor
escence from the lcg354 / A4
88
channel (green). Only the fluorescence from the cfb560 / A546 c
hannel that was matched by the eubacterial channel was consider
ed to
originate from bacteria. All sca
le bars correspond to 100 μm.
Mondragón‐Palomino et al. Supplement p. 19
Figure S8. Representative digital cross section (1 μm thick) of
cecal tissue that was hybridized with a eubacterial probe (eub
338
/ A514) and taxon-specific prob
es for families Lachnospiraceae
and Ruminococcaceae (lac435-clept1240/ A594).
The
fluorescence from the DAPI stain for total DNA is shown in grey
. The fluorescence from the eubac
terial signal (cyan) was segme
nted and
used to mask the fluorescence from the lac435-clept1240/ A594 c
hannel ( magenta). Only the fluor
escence from the lac435-clept1
240/
A594 channel that was matched by the eubacterial channel was co
nsidered to originate from bacteria. All scale bars correspond
to 100
μm.
Mondragón‐Palomino et al. Supplement p. 20
Figure S9. Images of the spatial structure of the host-microbio
ta interface of the proximal colon from a sample that was
processed as the sampl
e shown in Fig. 3A-F.
(a) The 3D rendering of confocal imaging (20X) of the crest of
a fold in the proximal
colon. The epithelium (blue) is covered by a mix of mucus (gree
n) and bacteria (red). (b-d) Maximum intensity projections of a
digital
cross-section (5 μm) depicted in (
a) with a dashed line. (e-g)
Maximum intensity projections o
f a digital cross section (12
μm) from a
second image stack that was obtained from the same sample. All
scale bars correspond to 100 μm.
Mondragón‐Palomino et al. Supplement p. 21
Figure S10
.
Representative images of the mucosal microbiota of the cecum of
2 mice (a and b) that were not exposed to
antibiotic ciprofloxacin (cohort B, Fig. 6A).
(a-b) Maximum intensity projecti
ons of digital cross-sections
(12.45 μm) from 3D imaging.
In a cluster of colonized crypts (host tissue in grey from tota
l DNA staining), adjacent cavities are colonized by bacterial c
olonies (cyan)
of variable volume and depth.
All scale bars correspond to 100
μm.
Mondragón‐Palomino et al. Supplement p. 22
Figure S11. Representative images of the mucosal microbiota of
the ceca of 3 mice (a-b, c-d, e-f) from 3 cages that were expos
ed
to the antibiotic ciprofloxacin
for 4 days (cohort A, Fig. 6A).
Approximately 200 crypts were
imaged from each mouse. (a)-(f)
Maximum
intensity projections of digital
cross-sections (12.45 μm) from
3D imaging. All scale bar
s correspond to 100 μm.
Mondragón‐Palomino et al. Supplement p. 23
Figure S12. Representative images of the mucosal microbiota of
the cecum of 3 mice (a, b, and c) from 3 cages that were
administered antibiotic ciprof
loxacin for 4 days and whose micr
obiota was allowed to recover
for 10 days (cohort A, Fig. 6A).
(a)-(c) Maximum intens
ity projections of digital cross-sections
(12.45 μm) from 3D imaging. All
scale bars correspond to 100 μ
m.
Mondragón‐Palomino et al. Supplement p. 24
Figure S13. Maps of the location of crypts that contained a bac
terial colony (colored dots) on the cecal mucosa of two mice (a
and b) that were not exposed t
o ciprofloxacin (Fig. 6A, cohort
B).
Clusters of bacterially colonized crypts were identified
computationally based on the dist
ance between them. Two coloniz
ed crypts were considered as part of the same cluster if the di
stance
between their center of mass on t
he (x,y) plane was less than o
r equal to 150 μm, which is approx
imately two times the median
distance
between the center of contiguous
colonized crypts. Crypts withi
n the same cluster were given the same color.
Mondragón‐Palomino et al. Supplement p. 25
Figure S14. Maps of the location of crypts colonized by bacteri
a (colored dots) on the cecal mucosa of three mice (a, b and c)
whose microbiota was allowed to recover for 10 days after a 4-d
ay administration of ciprofl
oxacin (Fig. 6A, cohort A).
Two
colonized crypts were considered as part of the same cluster if
the distance between their center of mass on the (x,y) plane w
as less than
or equal to 150 μm, which is approximately two times the median
distance between the center of c
ontiguous colonized crypts. Cr
ypts
within the same cluster were given the same color.
Mondragón‐Palomino et al. Supplement p. 26
Figure S15. Scatter plots of crypt communities in untreated and
recovery mice obtained through UMAP and tSNE dimensionality
reduction algorithms.
Community types (A-F) were def
ined by the clusters identified
in a hierarchica
l clustering ana
lysis (Fig. 7B).
Mondragón‐Palomino et al. Supplement p. 27
Figure S16. Maps of the location of crypts that were colonized
by bacteria, on the cecal mucosa of two mice (a and b) who were
not exposed to ciprofloxacin (Fig. 6A, cohort B). The location
of crypts is colored according
to the taxonomic makeup of the
associated bacterial commu
nity (Fig. 7
B). .
Mondragón‐Palomino et al. Supplement p. 28
Figure S17. Map of the location of crypts that were colonized b
y bacteria on the cecal mucosa of a mouse whose intestinal
microbiota was allowed to recover for 10 days after interruptin
g a 4-day long administration of
ciprofloxacin (Fig. 6, cohort
A).
The map was split into two fragments (a and b) to remove the ar
eas of the sample where we did not find clusters of colonized
crypts. The location of crypts is
colored according to the tax
onomic makeup of the associated b
acterial communi
ty (Fig. 7B) .
Mondragón‐Palomino et al. Supplement p. 29
Figure S18. Map of the location of crypts colonized by bacteria
on the cecal mucosa of a mouse
whose intestinal microbiota
was allowed to recover for 10 days after interrupting a 4-day l
ong administration of ciprofloxaci
n (Fig. 6, cohort A). The loc
ation
of crypts is colored according
to the taxonomic makeup of the a
ssociated bacterial community (Fig. 7B). The map was split into
three fragments (a, b, and c) to r
emove the areas of the sample
where we did not find clus
ters of colonized crypts.
Mondragón‐Palomino et al. Supplement p. 30
Figure S19. Map of the location of crypts colonized by bacteria
on the cecal mucosa of a mouse whose microbiota was allowed
to recover for 10 days
after interrupting a
4-day long administ
ration of ciprofloxacin (Fig. 6
, cohort A). The location of cry
pts is
colored according to the taxonomic
makeup of the associated bac
terial community (Fig. 7B). The map was split into two
fragments (a and b) to remove the
areas of the sample where we
did not find clusters of colonized crypts.