Supporting Information
Biomolecular
U
ltrasound
I
maging of
P
hagolysosomal
F
unction
Bill Ling
1
, Justin Lee
2
, David Maresca
1
, Audrey Lee
-
Gosselin
1
, Dina Malounda
1
,
Margaret
B.
Swift
1
,
Mikhail G. Shapiro
1*
1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California, United States
2. Division of Biology and Bioengineering, California Institute of Technology, Pasadena, California,
United States
*Corresponding au
thor. Email: mikhail@caltech.edu
Fig
ure
S
1
: Circulation half
-
life of GVs, as measured by Doppler signal enhancement. Half
-
life
was calculated as the time
required for normalized signal enhancement to decline from its
maximum at 1 to 0.5
. Error bars represent ± SEM. N=6 (WT), 3 (PBS liposomes), 6 (
clodronate
liposomes). Welch’s t
-
test (***:
p<0.001; n.s: p>0.05).
Figure S
2
: Time courses of ultrasound contrast in the brain (blue, n = 6) and liver (red, n = 4) of
healthy C57BL/6 mice.
Ultrasound contrast is essentially transferred from the
brain to the liver,
with l
iver contrast reach
ing
its maximum (dashed line) after brain contrast dissipates. Thin lines,
individual trials; thick lines, mean.
Fig
ure
S
3
: Segmentation protocol.
A)
Non
-
overlapping
5
00
px x 500 px (approx.
200 μm
)
ROIs
were extracted from confocal micrographs of liver slices stained with anti
-
F4/80 (acquired with
20x objective, scale bar: 200 μm). We trained our Pixel Classification algorithm in ilastik by
labeling background and macrophage regions on a subset of o
ur images. Then, we segmented
the remaining images by processing with the trained network.
B)
The corresponding images in
the AF647 (GV) channel were automatically thresholded by Otsu’s method, and colocalization
was assessed in MATLAB.
Figure S4
: Ultrasound contrast is linear with respect to GV concentration.
A)
Representative B
mode and AM images of non
-
linear GVs embedded in 1% agarose. Wells are approx. 2 mm in
diameter.
B)
B mode (top) and AM (bottom) signal intensities. N = 12.
Figure S5
: U
ltrasound signal time courses used for estimating pharmacokinetic parameters.
Brain, blue lines; liver, red lines; thin lines, individual trials; thick lines, mean.
Fig
ure
S
6
:
Processing for macrophage counting. Non
-
overlapping
500 px x 500 px (approx.
400
μm
)
ROIs were extracted from confocal micrographs of liver slices stained with anti
-
F4/80
(
acquired with 10x objective,
scale bar: 500 μm). Using the Density Counting workflow in ilastik,
we annotated a subset of these images for background and cell b
odies. Then, we processed the
remaining images with our trained algorithm to predict macrophage density.
Figure S
7
: Confocal microscopy image of a liver section from a mouse treated with 30 mg/kg
clodronate demonstrating localization of GVs to the sinusoidal periphery. Scale bars: 50 μm.
Inset: 5 μm
Figure S
8
: Hepatic clearance does not saturate under experimental c
onditions.
A)
Time course
of ultrafast Doppler signal enhancement following IV injection of purified GVs at 300 s (dashed
line). Individual traces, shown as thin lines, were normalized to their respective maxima. The
thick line represents the mean of N = 4
biological replicates. Shaded area represents ± SEM.
B)
Half
-
lives of signal enhancement following IV injection of 100 μL GVs at OD30 or OD130.
Welch’s t
-
test
(n.s: p>0.05).
Fig
ure
S
9
: Hepatic macrophage activity changes with age.
A)
Time course of Do
ppler signal
enhancement in mice of different ages following GV injection. Shaded areas represent ± SEM.
N = 4
-
6
B)
Time course of liver AM signal. Shaded areas represent ± SEM. N=3
-
5
C)
Rates of
GV uptake and degradation relative to those of 8 week old mice. Error bars represent ± SD.
N=3
-
5. Welch’s t
-
test(*
:p<0.05; **:p<0.01).
Condition
Uptake Rate (min
-
1
,
±
SD)
Degradation Rate
(min
-
1
, ± SD)
k
c
(
± SD
)
0 mg/kg
clodronate
0.1
667
± 0.0
107
0.0
407
± 0.00
32
0.
7572
±
0.
2328
0.40 mg/kg clodronate
0.057
4
± 0.0
045
0.02
99
± 0.00
53
0.
6073
±
0.25
87
30 mg/kg clodronate
0.0
299
± 0.00
10
0.01
75
± 0.00
12
0.
3843
±
0.
4507
MCD: 0 weeks
0.1
667
± 0.0
107
0.04
07
± 0.00
32
0.7
572
±
0.
2328
MCD: 4 weeks
0.
1087
± 0.0
108
0.0
172
± 0.0
019
0.
7268
±
0.
2899
MCD: 7 weeks
0.
1818
± 0.0
068
0.0
612
± 0.00
17
1.0000
±
0.0
000
Age: 8 weeks
0.1
667
± 0.0
107
0.0
407
± 0.00
32
0.7572
±
0.2328
Age: 10 weeks
0.
2108
± 0.0
310
0.0
354
± 0.0
025
0.6478
±
0.0859
Age: 12 weeks
0.
2577
± 0.
0229
0.0
645
± 0.0
132
0.9576
±
0.0752
Age: 15
weeks
0.1
681
± 0.0
181
0.0
916
± 0.0
052
0.8879
±
0.1546
Table
S
1:
Constants derived from fitting pharmacokinetic model to ultrasound data.
k
1
, uptake
rate;
k
2
, degradation rate;
k
c
, conversion constant.