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Going Deeper: Biomolecular Tools for Acoustic and Magnetic
Imaging and Control of Cellular Function
Dan I. Piraner
1
,
Arash Farhadi
2
,
Hunter C. Davis
1
,
Di Wu
3
,
David Maresca
1
,
Jerzy O.
Szablowski
1
, and
Mikhail G. Shapiro
1,*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125, USA
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
3
Division of Engineering and Applied Sciences, California Institute of Technology, Pasadena, CA
91125, USA
Abstract
Most cellular phenomena of interest to mammalian biology occur within the context of living
tissues and organisms. However, today’s most advanced tools for observing and manipulating
cellular function – based on fluorescent or light-controlled proteins – work best in cultured cells,
transparent model species or small, surgically accessed anatomical regions. Their reach into deep
tissues and larger animals is limited by photon scattering. To overcome this limitation, we must
design biochemical tools that interface with more penetrant forms of energy. For example, sound
waves and magnetic fields easily permeate most biological tissues, allowing the formation of
images and delivery of energy for actuation. These capabilities are widely used in clinical
techniques such as diagnostic ultrasound, magnetic resonance imaging, focused ultrasound
ablation and magnetic particle hyperthermia. Each of these modalities offers spatial and temporal
precision that could be used to study a multitude of cellular processes
in vivo
. However,
connecting these techniques to cellular functions such as gene expression, proliferation, migration
and signaling requires the development of new biochemical tools that can interact with sound
waves and magnetic fields as optogenetic tools interact with photons. Here, we discuss the exciting
challenges this poses for biomolecular engineering, and provide examples of recent advances
pointing the way to greater depth in
in vivo
cell biology.
Length scales for studying cellular function
in vivo
Before discussing technologies for cellular imaging and control, it is useful to think about
the length scales on which these techniques must operate. Consider three representative
*
Correspondence should be addressed to MGS: mikhail@caltech.edu, Phone: 626-395-8588, 1200 E. California Blvd, MC 210-41,
Pasadena, CA 91125.
AUTHOR CONTRIBUTIONS
All authors wrote the manuscript.
COMPETING INTERESTS
The authors declare no competing financial interests.
HHS Public Access
Author manuscript
Biochemistry
. Author manuscript; available in PMC 2018 October 03.
Published in final edited form as:
Biochemistry
. 2017 October 03; 56(39): 5202–5209. doi:10.1021/acs.biochem.7b00443.
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biological systems: the mammalian microbiome, the adaptive immune system and the brain
(Figure 1a). A microbe’s life in the mammalian gastrointestinal tract is intricately linked to
its location along the length of the tract, its radial position within the lumen, and its place
within a microenvironment such as the colonic crypt
1
. These locations are associated with
length scales of centimeters, millimeters and microns, respectively. Likewise, adaptive
immunity is a multi-scale phenomenon. Antigen presentation and recognition occur at sub-
micron immunological synapses while immune cel recruitment from blood and lymphoid
organs, proliferation and regulatory signaling occu on the scale of millimeters to
centimeters. Similarly, neura signaling is organized a length scales ranging from sub-micron
synapses to millimeter-sized brain region and centimeter-scale axonal projections.
In all three systems, key biological questions involve the function of particular cell types
within a certain spatially-defined anatomical context. For example, which microbes can
successfully colonize the small intestine? Which genes do T-cells express after migrating
into a tumor and recognizing a neoantigen? How does the activity of excitatory neurons in a
certain part of the hippocampus relate to the development of seizures? Each of these
questions involves ensemble cellular behaviors occurring on the millimeter scale, which are
difficult to recapitulate in
in vitro
models. Studying biology at this scale complements the
understanding gained by examining cells at the single-cell and sub-cellular level, and
requires a dedicated set of experimental tools.
Forms of energy for biological imaging and control
The key elements of any technology for cellular imaging and control are the form of energy
applied to or measured from the sample and the molecular mechanisms connecting this
energy to a biological process of interest (Figure 1b). Since the work of van Leeuwenhoek,
the dominant energy type used to study biological phenomena has been visible light, with
modern microscopy taking advantage of an impressive array of molecular tools to optically
visualize and perturb cellular processes. Unfortunately, visible light gets scattered within
approximately one millimeter in most tissues, limiting its use to
in vitro
specimens and
shallow or surgically accessed anatomical regions.
On the other hand, sound waves and magnetic fields are capable of penetrating deep into
tissues. Ultrasound at MHz frequencies permeates through several centimeters, enabling
imaging or focused energy deposition with a wavelength-dependent resolution down to
approximately 100 μm
2
. This is further improved to below 10 pm with recently developed
super-resolution techniques
3
. Due to this excellent performance, ultrasound imaging is
widely used in the clinic and in pre-clinical research. In addition, ultrasound can be focused
at depth to deliver mechanical forces or localized heating
4
. These capabilities are used
clinically for non-invasive ablation of diseased tissues.
Likewise, magnetic fields experience minimal tissue attenuation. They can be used to
produce high-contrast images of many organs by exploiting the context-dependent magnetic
resonance behavior of nuclear spins, with a spatial resolution on the order of 100 μm. In
addition, static or time-varying magnetic fields can produce mechanical forces or heat in
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tissues containing magnetic nanomaterials
5
, which can be localized in to the millimeter scale
using field-free point scanning techniques
6
.
Based on their tissue penetration and spatiotemporal resolution, sound waves and magnetic
fields are well-suited to imaging and controlling the function of cells
in vivo
(Figure 1b). All
that is needed is a set of biomolecular tools that can link these forms of energy to specific
cellular functions such as gene expression and signaling. Developing such tools presents an
exciting challenge to biomolecular engineers. Just as the discovery of the green fluorescent
protein stimulated the development of hundreds of reporters, sensors and actuators through
creative protein engineering, recent developments in acoustically and magnetically active
proteins may allow us to engineer a similar variety of biological tools for ultrasound and
magnetic resonance. Initial inroads towards this goal are described in the following sections.
Biomolecular tools for ultrasound imaging
Diagnostic ultrasound uses the scattering of sound waves to delineate tissue boundaries,
monitor the motion of organs such as the heart and quantify the velocity of blood flow
(Figure 2a). Until recently, the prospect of using ultrasound to image the function of specific
cells was remote due to the lack of suitable molecular reporters. Conventional ultrasound
contrast agents are micron-sized synthetic bubbles that resonantly scatter sound waves.
Although these microbubbles can be targeted to specific endovascular targets for molecular
imaging in the bloodstream, their size and longterm instability makes it difficult to use them
in labeling and monitoring the function of specific cells
7
. Alternatively, scattering synthetic
nanoparticles have been explored as ultrasound contrast agents with the potential for cell
labeling and extravascular interrogation
8
,
9
.
To connect ultrasound more closely with molecular and cellular biology, we recently
adapted a unique class of gas-filled proteins, called gas vesicles or GVs, as the first
biomolecular acoustic reporters. GVs evolved in aquatic photosynthetic microbes as a means
to regulate buoyancy for optimal access to sunlight and other nutrients
10
. Despite their
name, gas vesicles contain no lipids; they comprise a 2 nm-thick protein shell enclosing a
hollow interior with dimensions on the order of 250 nm (Figure 2, b-c). Their shell allows
gases dissolved in the surrounding media to freely permeate in and out of their interior,
while their hydrophobic inner surface prevents the formation of a liquid aqueous phase. GVs
are encoded by clusters of 8–14 genes, including two primary structural proteins and several
minor constituents, chaperones and regulators.
In 2014, we showed that GVs can produce ultrasound contrast in purified form, inside cells
and
in vivo
, establishing them as the first acoustic biomolecules
11
. Since this initial
discovery, considerable advances have been made in understanding the acoustic properties of
GVs and improving the ability of ultrasound to detect them with greater sensitivity and
specificity. One key finding was that GVs undergo nanoscale buckling deformations under
ultrasound (Figure 2d), resulting in non-linear scattering and allowing amplitude-modulated
pulse sequences to detect GVs with greater specificity against background tissues (Figure
2e) in a process analogous to two-photon microscopy
12
,
13
. Another key finding was that the
acoustic properties of GVs can be engineered at the genetic level. In particular, a key
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component of the GV shell called GvpC influences the response of GVs to pressure, setting
thresholds for buckling and irreversible collapse
14
. Tuning GvpC at the genetic level enables
modulation of GVs’ nonlinear signals, as well as multiplexed imaging of GV variants with
differential pressure sensitivity
14
(Figure 2f). Additionally, fusions of GvpC with other
polypeptides enable the tailoring of GV surface properties such as charge or affinity for
molecular imaging targets
14
.
A major effort is also underway to express GVs heterologously as genetically encoded
reporters. As an initial target, we have developed genetic constructs to express GVs in model
commensal and pathogenic microbes such as
E. coli
and
S. typhimurium
(Figure 2g)
15
.
Imaging these and other microbes in mammalian hosts could enable new studies of the
microbiome and the tracking of engineered probiotic therapies. Cells expressing the current
generation of acoustic reporter genes can be visualized at densities below 10
8
cells/ml,
corresponding to a volume fraction of 0.005% – a level compatible with imaging microbes
in the GI tract or tumors (Figure 2h)
15
.
An alternative mechanism by which ultrasound can facilitate the visualization of cells
in
vivo
is photoacoustic imaging, a technique wherein optical excitation is absorbed and
converted into thermoelastic pressure waves, which are detected by ultrasound transducers
16
.
This enables the use of light to image deeper structures because photons are allowed to
scatter
en route
to their target, with spatial information provided by ultrasound. The major
advantage of photoacoustic imaging compared to pure ultrasound is its ability to leverage
existing molecular tools developed for optical imaging, including fluorescent proteins or
light-absorbing pigments such as melanin (Figure 2i)
17
,
18
. However, this technique is still
difficult to employ at depths beyond one to two centimeters without causing tissue
phototoxicity.
Biomolecular tools for ultrasonic actuation
In addition to imaging, ultrasound can be used to deliver energy to focused regions of tissue,
with targeting on the scale of a single millimeter. Depending on beam intensity and pulse
duration, this energy can be used to apply mechanical forces, drive resonant cavitation of
bubbles, or deposit heat (Figure 3a)
4
. These capabilities are used clinically for non-invasive
surgery
19
. If they could instead be harnessed, at lower intensities, to modulate the activity of
specific cells
in vivo
, this would facilitate the study of cellular function within relevant
anatomical contexts.
Several nascent approaches have been proposed to enable this possibility. For example, the
ability of ultrasound to controllably heat tissue within the well tolerated range of 37–42°C
can be coupled to natural or engineered temperature-dependent signaling pathways. This
approach has been used to remotely activate transcription driven by the heat shock promoter,
pHSP70, in cultured mammalian cells and live mice
20
. While this approach is highly
effective in certain contexts, the thermal set-point and activity level of heat shock promoters
varies between cell types, is not easily tunable, and responds to other stimuli in addition to
temperature
21
.
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In bacteria, endogenous heat shock promoters have only modest activation in response to
this range of temperatures, necessitating the development of engineered thermal bioswitches.
To address this need, we recently introduced two families of orthogonal, tunable
temperature-dependent transcriptional repressors for remote control of bacterial function
22
.
These bioswitches are based on the TlpA transcriptional repressor from
S. typhimurium
and
a variant of the cI repressor from the Lambda bacteriophage (TcI). Unlike the ~ 10-fold
thermal induction of heat shock promoters, the expression of genes downstream of TlpA and
TcI operators is turned on by more than 100-fold in response to mild heating. We showed
that TlpA and TcI can be engineered through directed evolution to actuate at different
desired temperatures, as required by a given application (Figure 3b). In addition, they can be
used in combination to build thermal logic circuits, for example to turn on two different
functions at two different temperatures. We have demonstrated that these switches can be
used to spatially pattern gene expression in plated bacterial cells (Figure 3c) and also in
bacteria implanted
in vivo
(Figure 3d).
Besides heating, ultrasound is also able to apply mechanical forces to tissues. These forces,
which are amplified by acoustically active structures such as microbubbles, could be coupled
to signaling elements such as mechanosensitive ion channels, allowing non-invasive control
of cellular signaling. This concept was recently demonstrated
in vitro
by combining
synthetic microbubbles with mammalian cells heterologously expressing the
E. coli
mechanosensitive ion channel MscL
23
. Microbubbles were similarly used to stimulate the
endogenous mechanosensor Trp4 in
C. elegans
24
. Unlike thermal stimuli, which are
typically associated with timescales on the order of seconds, mechanical effects can be
produced on the order of milliseconds, potentially allowing more rapid control of cellular
signaling. However, techniques requiring microbubbles are limited in their application to
mammals due to the difficulty of delivering bubbles to relevant tissues.
In addition to directly controlling cellular function, ultrasound can be used to spatially target
the delivery of genetically encoded tools or treatments. In the brain, such delivery can be
targeted non-invasively by opening the blood-brain barrier reversibly at a specific location
using focused ultrasound and intravascular microbbubles
25
. This technique allows the
delivery of adeno-associated virus (AAV) vectors to targeted regions with millimeter
precision
26
.
Biomolecular tools for magnetic resonance imaging
Like ultrasound, MRI derives contrast from both, endogenous variation in the properties of
tissue and molecular contrast agents. Taking advantage of the rich behavior of nuclear spins
under various physical and chemical conditions has enabled the development of several
classes of biomolecular MRI reporters
27
,
28
. One major class comprises proteins that contain
paramagnetic metals, such as iron or manganese, or lead to the accumulation of these ions in
tissue. Proteins in this class include ferritin, bacterial cytochromes, the transferrin receptor
and other transporters (Figure 4, a-b). Paramagnetic species in these proteins produce T
1
contrast through spin exchange of coordinated water protons and T
2
contrast by distorting
the magnetic field near the protein. Another class of reporters includes proteins with large
numbers of exchangeable protons – the nuclear spin most commonly imaged with MRI.
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These protein-bound protons resonate at a distinct frequency (chemical shift) relative to
water-bound protons, and can be selectively saturated with radiofrequency pulses, quenching
their MRI signal. By applying such saturation while these protons exchange rapidly with the
aqueous pool, the signal of the entire pool can be substantially reduced. This “catalytic”
contrast scheme is called chemical exchange saturation transfer, or CEST. Proteins
detectable with this method include a synthetic lysine-rich protein
29
and human
protamine
30
.
While these pioneering reporter types have been used to demonstrate the imaging of
genetically defined cells using MRI, they are generally limited by their low molecular
sensitivity (requiring protein concentrations on the order of μ
Μ
) or the requirement of metal
cofactors, which may not always be bioavailable. Recent efforts have therefore been focused
on developing alternative classes of reporters that are more sensitive and do not require
metals. For example, we recently introduced aquaporin 1 as a biomolecular reporter for MRI
based on its ability to enhance the diffusion of water across cell membranes (Figure 4c)
31
.
Aquaporins are transmembrane channels that passively conduct water with exquisite
selectivity at rates of up to one billion water molecules per channel per second. We showed
that the overexpression of this autologous, non-toxic, metal-free molecule produces contrast
in diffusion-weighted MRI at concentrations below 500 nM, allowing non-invasive imaging
of gene expression
in vitro
and
in vivo
(Figure 4d). In addition to aquaporin 1, other water-
permeable channels such as the urea transporter can produce diffusion-based contrast, albeit
with lower channel selectivity
32
.
To push the molecular and cellular sensitivity of MRI even further, recent work has focused
on directly addressing a fundamental physical limitation of conventional magnetic
resonance: the weak magnetic alignment of nuclear spins under thermal equilibrium. This
low polarization results in overall MRI signals approximately 10
5
times weaker than they
could be if all the available spins aligned with the applied magnetic field. This limitation can
be overcome with hyperpolarization – an advanced technique in which nuclei are prepared
via physical methods in a state of non-equilibrium polarization that is up to 10,000-fold
stronger than baseline
33
. Hyperpolarized nuclei such as the noble gas
129
Xe can then be
delivered to the body by inhalation to be imaged during their polarization half-life of a few
seconds. Because each hyperpolarized atom carries a much stronger signal than thermally
polarized molecules, MRI reporters acting on these nuclei are detectable at much lower
concentrations than their conventional counterparts. The first biomolecular reporters for
hyperpolarized xenon MRI were GVs, the aforementioned gas-filled protein nanostructures.
GVs allow xenon dissolved in surrounding media to exchange in and out of their gaseous
compartment, producing MRI contrast through CEST at picomolar concentrations (Figure 4,
e-f)
34
. Other proteins active as contrast agents for
129
Xe MRI and other hyperpolarized
nuclei have also been reported
35
,
36
.
In addition to reporters connected to gene expression, biomolecules have also been
engineered as MRI sensors – allowing dynamic tracking of processes such as
neurotransmission and kinase signaling. One class of such sensors, inspired by pioneering
synthetic approaches
37
, comprises iron-containing metalloproteins in which the accessibility
of an open iron coordination site to water is modulated by the binding of small molecules,
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thereby altering T
1
contrast
38
. Directed evolution allows the tuning of this small molecule
binding site for selective interactions with neurotransmitters, such as dopamine and
serotonin. The resulting reporters have been used to image the dynamics of neurotransmitter
release and reuptake in rodent brains
39
. Other biomolecular sensor constructs, based on T
2
and CEST mechanisms, have been developed to image signals such as kinase and protease
activity
40
.
Biomolecular tools for magnetic control
Magnetic fields exert forces on magnetically active materials such as superparamagnetic and
ferromagnetic particles
5
. Depending on the particle type, these forces can be sufficient to
guide the movement of materials or cells in the body and actuate receptor signaling (Figure
5a). In addition, rapidly alternating magnetic fields can generate heat in particles whose
magnetization oscillates with the applied field, which in turn can be used to control
temperature-dependent processes (Figure 5b).
Most strategies for magnetic control of cell function have relied on synthetic magnetic
nanoparticles as transducers of the magnetic field. For example, superparamagnetic iron
oxide nanoparticles have been used to cluster cell surface receptors or apply directly
actuating forces on integrin and notch
41
(Figure 5c). In addition, superparamagnetic particles
have been used in combination with alternating fields to activate temperature-sensitive ion
channels such as TRPV1
42
(Figure 5d). This approach enabled remote control of neural
signaling
in vivo
in mice surgically implanted with such particles
43
(Figure 5, e-f).
Additionally, cells containing iron oxide particles have been concentrated at certain locations
in vivo
44
,
45
.
Translating these approaches into more versatile, fully-genetic constructs is challenging due
to the unsolved problem of heterologous biosynthesis of strongly magnetic nanomaterials.
Although superparamagnetic and ferromagnetic iron oxide nanocrystals are made by
magnetotactic bacteria
46
, the genes encoding their specialized organelle machinery for such
synthesis have so far been transferred only to their close genetic relatives. The magnetic
nanostructures formed in commensal microbes and mammalian cells, such as ferritin, tend to
be paramagnetic or weakly superparamagnetic. Efforts to increase the magnetic strength of
ferritin through genetic engineering have so far come up short of qualitatively altering its
magnetic character.
Despite this physical limitation, some groups have reported that fusions of ferritin to
temperature- and mechanically-sensitive ion channels allow neurons expressing these
channels to be activated remotely using both alternating and static magnetic fields
47
,
48
.
These reports are somewhat surprising based on classical theoretical estimates of the forces
and temperatures that could be produced by ferritin
49
. However, it is possible that as-yet
unknown alternative mechanisms are at play.
Outlook
The development of biomolecular tools for non-invasive cellular imaging and control is in its
infancy. Inspired by the history and impact of fluorescent and optogenetic proteins, many
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opportunities exist to develop acoustic and magnetic technologies connected to a variety of
cellular processes. For example, biomolecular ultrasound imaging is a new field animated 3
years ago with the development of GVs as its first biomolecular reporter. Much remains to
be learned about the acoustic properties of these molecules and how they can be tuned at the
genetic level to increase imaging sensitivity, or engineered to respond dynamically as
sensors of cellular signaling. In addition, more work is needed to transfer the machinery
encoding GVs to a greater number of species. In particular, expressing GVs in mammalian
cells represents a major unsolved challenge in genetic engineering, given the need to
functionally transfer a large operon driving the self-assembly of a complex macromolecular
structure between two domains of life. This work takes place against a backdrop of other
exciting developments in ultrasound, exemplified by super-resolution imaging
3
and the
recent invention of functional ultrasound (fUS), a technique for imaging neural activity non-
invasively with improved spatiotemporal resolution compared to functional MRI (< 100 μm
and < 10 ms) using transducers that can be mounted on freely moving animals
50
.
Biomolecular MRI is only slightly more mature, with an expanding variety of contrast
mechanisms but no clear frontrunner to become the go-to molecule for
in vivo
cellular
imaging. Recently developed aquaporin-based reporter genes offer unique advantages in
terms of their simplicity and biocompatibility, while GVs have the potential to bring the
advantages of hyperpolarization to boosting the
in vivo
sensitivity of cellular MRI.
Engineering both of these molecules and accompanying
in vivo
imaging methods for
maximum sensitivity and potential use as dynamic sensors are major avenues for future
research. In addition, an outstanding grand challenge is the engineering of heterologous
magnetite biosynthesis, which would provide powerful MRI contrast, as well as
opportunities for actuation. In parallel with these molecular efforts, progress is being made
on improving the information content of MRI images. For example, we recently used
nitrogen vacancy diamond magnetometry, an optical technique for imaging magnetic fields,
to map the nanoscale magnetic field in cells containing iron oxide nanoparticles and connect
these maps to the T
2
contrast seen by MRI
51
. This study demonstrated experimentally that
the spatial arrangement of magnetic materials inside cells strongly influences contrast,
guiding the development of magnetic cellular reporters and sensors and imaging parameters
for their specific identification
in vivo
.
Complementing these evolving imaging technologies, much additional work is needed on
genetically encodable agents to control cellular responses with acoustic or magnetic energy.
For example, there is a lack of mammalian thermal bioswitches that are orthogonal to
pleotropic heat shock pathways and tunable to different temperature thresholds analogously
to the system we developed for microbial remote control. In addition to regulating gene
expression, tools are needed to connect thermal inputs directly to signaling pathways.
Similarly, for ultrasound actuation based on mechanical forces, use in mammals will require
eliminating the need for synthetic microbubbles to produce constructs that can be fully
genetically encoded. Likewise, the synthetic magnetic particles uses in well-accepted
magnetic control techniques using thermal, mechanical or clustering mechanisms must be
replaced with genetically encodable materials. The potential use of ferritin for this purpose
requires further study to reconcile its encouraging empirical performance and predicted lack
of efficacy based on previously studied physical properties.
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In summary, going deeper into the body to study cellular function within its native
in vivo
context requires engineering interactions between deeply penetrant forms of energy and
biomolecules to enable non-invasive imaging and control. Several recent advances have
provided exciting proofs or concept for this approach, and inform the development of new
classes of biomolecular tools. Many depths remain to be plumbed.
ACKNOWLEDGEMENTS
We thank members of the Shapiro Laboratory for helpful discussions. Related work in the Shapiro laboratory is also
supported by the Heritage Medical Research Institute, the National Institutes of Health, the Defense Advanced
Research Projects Agency, the Jacobs Institute for Molecular Engineering in Medicine, the Caltech Center for
Environmental Microbial Interactions, the Human Frontiers Science Program, the Burroughs Wellcome Fund, the
Pew Scholarship in the Biomedical Sciences, the Sontag Foundation, the Packard Fellowship for Science and
Engineering. D.M. is supported by the Human Frontiers Science Program Cross Disciplinary Postdoctoral
Fellowship (Award No. LT000637/ 2016). A.F. is supported by the Natural Sciences and Engineering Research
Council of Canada PGSD.
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Figure 1 - Modalities for in vivo imaging and control of cellular function.
(a) Diagram of the length scales of several biological processes of interest
in vivo
, and the
degree to which these length scales are accessible by imaging technologies. (b) Approximate
length scales and maximal tissue penetration depths accessible by optical, acoustic, or
magnetic imaging.
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Figure 2 - Biomolecular tools for ultrasound imaging.
(a) Illustration of sound propagation in the imaging medium and received echo used to form
the ultrasound image. (b) GVs are hollow protein nanostructures that freely allow diffusion
of dissolved gas through their shell but exclude water
11
. GVs are encoded by operons
consisting of 814 genes. (c) Representative transmission electron micrograph of purified GV
from Halobacterium
11
. (d) Simulation illustrating nanoscale deformation of GVs under
ultrasound leading to nonlinear backscattered echo
12
. (e) Amplitude-modulation pulse
sequence reveal GVs in mouse colon
13
(Reprinted from Maresca, D et al (2017). Nonlinear
ultrasound imaging of nanoscale acoustic biomolecules. Applied Physics Letters, 110(7),
73704, with the permission of AIP Publishing). (f) Multiplexed imaging of genetically
engineered GVs
14
. (g) Heterologous expression of GVs in E. coli using an optimized GV
gene cluster
15
. (h) Ultrasound image of E. coli expressing GVs or the non-echogenic
luminescence reporter, luciferase
15
. (i) Photoacoustic imaging of tumor expressing
tyrosinase, and surrounding blood vessels
52
.
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Figure 3 - Biomolecular tools for acoustic control.
(a) Ultrasound can be focused at depth in tissue and apply several forms of energy to
interface with cells. (b) Schematic of the gene circuit utilized to gate a GFP reporter gene
with a temperature-sensitive repressor (TSR), and a panel of tuned variants of temperature-
sensitive repressors
22
. (c) MRI thermometry imaging demonstrates a spatial temperature
gradient induced by FUS on a plate of bacterial cells, resulting in spatially targeted gene
expression
22
. (d) E. coli were injected into both hindlimbs of a nude mouse; after FUS
application to the right hindlimb, reporter gene expression is significantly enriched at the site
of heating
22
. (e) Diagram of mechanism by which microbubble cavitation can result in
membrane deformation leading to mechanoreceptor activation. (f) Microbubbles attached to
cultured retinal pigment epithelium cells
23
. (g) Uptake of membrane-impermeable dye into
retinal pigment epithelium cells expressing MscL and functionalized with microbubbles
23
.
(h) C. elegans worm motor response to ultrasound in a bath of microbubbles
24
.
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Figure 4 - Biomolecular tools for magnetic resonance imaging.
(a) Metalloproteins interact magnetically with aqueous
1
H nuclear spins, leading to T
1
or T
2
MRI contrast. (b) Migrating neuroblasts expressing ferritin produce a hypointense track
(arrow) in T
2
weighted MRI
53
. Asterisks denote adenovirus injection sites. (c)
Overexpression of aquaporin enhances passive diffusion of water across the cell membrane,
resulting in contrast on diffusion weighted MRI. (d) AQPi expression in mouse xenograft
shows significant contrast compared to contralateral GFP expressing xenograft after
expression is induced with doxycycline
31
. (e) GVs interact with hyperpolarized xenon
dissolved in biological media, producing contrast in
129
Xe MRI. (f) Genetically distinct GVs
produce different chemical shifts in
129
Xe MRI, enabling multiplexed imaging
34
.
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