of 27
1
Table of Contents
Table S1. Exposure time for experiments reported in this work
................................
................................
..
2
Table S
2
. List of primers
................................
................................
................................
.............................
2
Table S
3
. List of plasmids
................................
................................
................................
...........................
3
Table S
4
. List of strains
................................
................................
................................
..............................
3
Figure S1. Amplitude and phase image stack of fluorescent beads
................................
.............................
4
Figure S
2
. Point
spread function of CFAST fluorescence imaging
................................
.............................
5
Figure S
3
. Simulation validation of
CFAST
3D fluorescence imaging and comparison to other methods
..
6
Figure S4. Evaluating the effectiveness of digital refocusing using 1.25
-
micron beads
..............................
7
Figure S5.
Digitally refocused amplitude images of a Siemens star using CFAST
................................
.....
8
Figure
S
6
. Validating CFAST bacteria imaging with dual color widefield fluorescence imaging
...............
9
Figure
S
7
. Amplitude and 3D fluorescence images of additional sections on the maize root
......................
9
Figure
S
8
. Quantitative phase images of roots
................................
................................
..........................
10
Figure S9. Comparison of digital and physical defocusing in root Imaging
................................
...............
11
Figure S10. Average raw data and amplitude reconstruction at
various
physical defocus distances
..........
12
Figure S11. Digital refocusing using the amplitude
and
quantitative phase image stack
..........................
13
Figure
S
12
. CFAST imaging of
Brachypodium distachyon
root mixed with
P. syn
-
mNG
.........................
14
Figure
S13
.
Comparison between confocal and CFAST imaging
................................
.............................
15
Figure
S14
.
Additional confocal and CFAST images
.
................................
................................
...............
16
Figure
S15
.
CFAST imaging of
Brachypodium distachyon
root alone
and
mixed with
P. syn
..................
16
Figure
S
16
. Domain segmentation for identifying bacteria colony based on CFAST images
...................
17
Figure
S
17
. Planktonic 96
-
well plate assays of
P. syn
strains
................................
................................
....
18
Figure
S
18
. Demonstration of autofocusing using CFAST
................................
................................
.......
19
Figure
S
19
. Photodamage to the bacterial biofilm caused by exposure to the laser
................................
..
19
Figure
S
20
. Surface area and fluorescence level of
P. syn
P
phzA
-
mNG
colony biofilm over time
...............
20
Figure
S
21
. Custom
-
made imaging chamber design
................................
................................
.................
20
Figure
S
22
. Design schematic of a CFAST setup that is more photon efficient
................................
........
21
Figure
S
23
. Quantification of
background uniformity overtime
................................
...............................
21
Note S1. Impact of aperture design on depth of field and imaging efficiency
................................
...........
22
Note
S2
.
Photon efficiency and signal
-
to
-
noise ratio evaluation using CFAST
................................
........
23
2
References
................................
................................
................................
................................
................
27
Table
S1. Exposure time for
experiments reported in this work. The numbers reported for timelapse bacteria
imaging are at the 12
-
hour mark.
Object
Maize roots
P. syn
-
mNG
P. syn
-
mNG +
P. gram
(1:5)
P. syn
-
mNG +
P. gram
(1:20)
P. syn
-
mNG +
P. orien
(1:5)
Exposure
time (s)
1
2
2
4
1
Object
P. syn
-
mNG +
P. orien
(1:1)
P. syn
P
phoA
-
mNG
(P lim)
P. syn
P
phoA
-
mNG
(P rep)
P. syn
P
ph
z
A
-
mNG
(P lim)
P. syn
P
ph
z
A
-
mNG
(P rep)
Exposure
time (s)
1
1
8
8
8
Table S
2
.
List of primers
Code
S
equence 5
́
-
3
́
Overhang
Amplicon
oREA011
atggtctccaagggcga
blunt
pJM220_mNeonGreen
_bb_fwd
oREA012
TGAGCTCATGCATGATCGAATT
blunt
pJM220_mNeonGreen
_bb_rev
oREA013
TTGAAGCTAATTCGATCATGCATGAGCT
CAcgaattcgagctcggtacc
pJM220 FRT
PA1/04/03_RBS_fwd
oREA014
attatcttcctcgcccttggagaccatgcttaatttctcctctttaattct
agatgtg
pJM220
mNeonGreen
PA1/04/03_RBS_rev
oREA015
TTGAAGCTAATTCGATCATGCATGAGCT
CAtttgaacacctataacactccaagg
pJM220 FRT
pPhzA_2
-
79_F
oREA016
catattatcttcctcgcccttggagaccataggcagtgttctccttag
ttga
pJM220
mNeonGreen
pPhzA_2
-
79_R
oREA019
TTGAAGCTAATTCGATCATGCATGAGCT
CAaaagttaatcttttcaacagctg
pJM220 FRT
pPhoA_Ecoli_F
oREA021
gcccttggagaccatgcttaatttctcctctttaatacattaaaaaata
aaaacaaagcg
pJM220
mNeonGreen
pPhoA_Ecoli_R with
RBS
3
Table S
3
. List of plasmids
Plasmid code
D
escription
S
ource
pJM220
-
T0T1
miniTn7 delivery plasmid with rhaSR
-
PrhaBAD inducible
promoter driving mNeonGreen expression
Modified
DKN 2097
pJM220_mod_1
pJM220 modified with a P
PA10403
promoter and mNeonGreen
this study
pJM220_mod_2
pJM220 modified with a P
phzA
promoter and mNeonGreen.
P
phzA
is the promoter for the
phzA
-
G
operon. It was obtained
from
P. synxantha
2
-
79
this study
pJM220_mod_3
pJM220 modified with a P
phoA
promoter and mNeonGreen.
P
phoA
is the promoter of an alkaline phosphotase. It was
obtained from
E. coli
MG1655
this study
Table S
4
. List of strains
Name
Description
Source
P. synxantha 2
-
79
wild type
D. Mavrodi
P. synxantha
P
PA10403
-
mNeonGreen
constitutive promoter (P
PA10403
) driving mNeonGreen
expression
this study
P. synxantha
P
phzA
-
mNeonGreen
inducible promoter (P
phzA
) driving mNeonGreen
expression
this study
P.
synxantha
P
phoA
-
mNeonGreen
inducible promoter (P
phoA
) driving mNeonGreen
expression
this study
P. synxantha
P
tac
-
mCherry
inducible promoter (P
tac
) driving mCherry expression
D. Mavrodi
Paraburkholderia
graminis
wheat rhizosphere isolate
DKN 2398
Pseudomonas orientalis
wheat rhizosphere isolate
DKN 2418
E. coli
DH10B
used for routine cloning
DKN 1965
E. coli
SM10 λpir
Helper strain (carries pTNS1)
DNK 1298
E. coli
MG1655
wild type
DKN 1369
4
Figure S1.
(
a) Amplitude and phase image stack of fluorescent beads in Figure 1d generated by digitally
propagate the reconstructed field to different axial planes. Physical focal plane
=
0
μm. Scale bar: 50
μm. (b) Line profiles of measured amplitude and phase of three fluorescent beads. Measurements with
physical focal plane positions
=
0
μm (solid lines) and
=
10
μm (dashed lines) are consistent
,
confirming the accuracy of the amplitude and phase measurements.
5
Figure S
2
. Point spread function
(PSF) of CFAST fluorescence imaging at different depth in for spinning
disk orientation (2) in Figure 1. Color bar, normalized intensity. Scale bar, 10 microns.
6
Figure S
3
. Simulation validation of 3D fluorescence imaging using CFAST and
comparison to other
methods. (a) Simulated 3D line structure on a 15
-
micron (radius) sphere. (b) Four fluorescence images
corresponding to spinning disk orientation (1)
-
(4) in Figure 1 using CFAST; the reconstruction matches the
ground truth. (c
)
The CFAST setup can also image half pupil each time and reconstruct the complex field
and 3D fluorescence using two frames. However, it comes at a cost of reduced depth of field as shown in
the reconstruction. The algorithm fails to reconstruct the 3D obj
ect using
(d) the quadrated PSF
1
and (e) the
double
-
helix PSF
2
. Color bar, depth in microns. Scale bar, 10 microns.
7
Figure S
4
.
Evaluating the effectiveness of digital refocusing using 1.25
-
micron beads.
(a) Raw CFAST
images for a 1.25
-
micron bead with different focal plane positions
. (b) Reconstructed amplitude and
phase of 9 beads after digital refocusing. Scale bar: 5 microns in (a), 2 microns in (b).
D
igital refocusing
successfully restores relatively circular shape
s
for most beads when the physical defocus is within ±50
microns.
M
inor shape distortions become apparent when the physical defocus extends to 100 microns.
8
Figure S5. Digitally refocused amplitude images of a Siemens star using CFAST. Images were captured
with physical defocus ranging from
-
200 to 200 microns. (a) The raw images, (b) reconstructed images and
(b) line profiles near the resolution limit. Scale
bar: 20 microns. No significant degradation in reconstruction
quality was observed within the ±200 microns defocus range.
9
Figure
S
6
. Validating CFAST bacteria imaging with dual color widefield fluorescence imaging. (a) Images
of a mixture of
P. syn
-
mNG
and
P. synxantha
P
tac
-
mCherry
(
P. syn
-
mCherry
). (i) CFAST fluorescence
image, (ii) CFAST phase image, (iii) widefield fluorescence image where red represents P. syn
-
mCherry
and green represents
P. syn
-
mNG
. (b,c) The zoomed regions of interest in (a). The (ii) CFAST phase
highlights both strains with single bacteria sensitivity while the (i) CFAST fluorescence only shows
P. syn
-
mNG.
Colo
r bar: depth in (i), phase in (ii). Scale bar: 10 microns.
Figure
S
7
. Amplitude and 3D fluorescence images of additional (a) thin and (b) thick sections on the maize
root. Color bar, depth in microns. Scale bar, 50 microns.
10
Figure
S
8
.
Quantitative phase images from CFAST 3D imaging of (a) maize roots and
(b)
fluorescent
bacteria on
Brachypodium distachyon
roots. Scale bar, 50 microns.
11
Figure S9
.
Comparison of digital and physical defocusing in root
i
maging. The digital
refocusing stack is
reconstructed from raw images captured with a physical focal plane at z = 0 micron. Orange and purple
arrows indicate areas where the left and right sides of the root are in focus, respectively. The digitally
defocused images align well
with the physically defocused images. Scale bar: 100 microns.