Hydrogel-Tissue Chemistry: Principles and Applications
Viviana Gradinaru
1
,
Jennifer Treweek
1
,
Kristin Overton
2
, and
Karl Deisseroth
2,3,4
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,
California 91125, USA; viviana@caltech.edu
2
Department of Bioengineering, Stanford University, Stanford, California 94305, USA;
deissero@stanford.edu
3
Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California
94305, USA
4
Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA
Abstract
Over the past five years, a rapidly developing experimental approach has enabled high-resolution
and high-content information retrieval from intact multicellular animal (metazoan) systems. New
chemical and physical forms are created in the hydrogel-tissue chemistry process, and the
retention and retrieval of crucial phenotypic information regarding constituent cells and molecules
(and their joint interrelationships) are thereby enabled. For example, rich data sets defining both
single-cell-resolution gene expression and single-cell-resolution activity during behavior can now
be collected while still preserving information on three-dimensional positioning and/or brain-wide
wiring of those very same neurons—even within vertebrate brains. This new approach and its
variants, as applied to neuroscience, are beginning to illuminate the fundamental cellular and
chemical representations of sensation, cognition, and action. More generally, reimagining
metazoans as metareactants—or positionally defined three-dimensional graphs of constituent
chemicals made available for ongoing functionalization, transformation, and readout—is
stimulating innovation across biology and medicine.
Keywords
CLARITY; hydrogels; metareactant; HTC; hydrogel-tissue; clearing
INTRODUCTION
In the study of complex biological systems, a powerful experimental approach is that of
analysis or disassembly (removing components, such as a particular type of cell or complex
DISCLOSURE STATEMENT
All protocols, software, and other information regarding these methods is freely available from the authors and online, and
disseminated via free hands-on training courses (
clarityresource-center.org
and
clover.caltech.edu
). V.G. and K.D. have disclosed
intellectual property regarding HTC methods to Caltech and Stanford, some of which has been licensed to ClearLight Diagnostics,
which is exploring applications for cancer diagnostics, and with which there are consulting arrangements and equity; V.G. and K.D.
each also have grant support from the US federal government (National Institutes of Health and National Science Foundation) to
further develop, apply, and disseminate these methods.
Published as:
Annu Rev Biophys
. 2018 May 20; 47: 355–376.
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HHMI Author Manuscript
of molecules, from the native context for further study). For example, the current revolution
in cancer treatment was in part enabled by reductionist molecular and cellular-level analysis
of isolated cancer cells and of specific immune-system cells that play a role in suppressing
tumor growth. The success of this analytical paradigm has, in part, extended to neuroscience
as well; studies of isolated neurons and axons have facilitated elucidation of the fundamental
logic of single-neuron information processing.
However, for systems like the intact vertebrate brain (composed of 10
7
—10
11
interconnected
neurons and characterized by crucial emergent properties), studying constituent components
in isolation can provide little insight into many of the most significant mysteries.
Alternatively, converting the brain—or more broadly the entire metazoan (multicellular
animal) organism—into an assembly of reactants anchored onto a new and versatile three-
dimensional (3D) coordinate system has recently emerged as a complementary strategy (
23
,
24
). Coupling individual subsets of chemically defined biomolecules to functional groups,
covalently anchoring or entangling these in turn within a polymer lattice, and then working
with this structure (effectively a 3D assembly of spatially tagged molecular reactants) (
23
,
24
) has already opened the door to a diverse array of novel approaches and discoveries in
biology.
The technique builds in part from (among several other foundations in science and
engineering) the chemistry of hydrogels, which are 3D polymeric networks of connected
hydrophilic components. Gels and polymers have a long history of use in biology, including
for providing physical support of tissues during sectioning and imaging, as well as for a
number of important clinical applications in regenerative medicine and tissue engineering.
But in the basic science of hydrogel-tissue chemistry (
23
,
24
), specific classes of native
biomolecules in tissue are immobilized or covalently anchored (for example, through
individualized interface molecules to gel monomer molecules) and precisely timed
polymerization causing tissue-gel hybrid formation is triggered within all the cells across the
tissue in an ordered and controlled process (Figure 1) to ultimately create an optically and
chemically accessible biomolecular matrix. Indeed, when the biomolecules of interest are
thereby transferred to the polymer lattice, a robust new composite hydrogel-tissue material
results (
23
,
24
), which becomes the substrate for future chemical and optical interrogation
that can be probed and manipulated in new ways. This approach has been diversified (Figure
2) to address needs and opportunities in organisms and tissues across biology (including in
cancer diagnostics, bacterial and HIV infection of mammalian tissues, developmental
biology, parkinsonism, Alzheimer’s disease, multiple sclerosis, autism, drug abuse, and fear/
anxiety disorders). Here, we review the fundamentals of this approach, the rapidly
expanding scope of discoveries that have resulted, and emerging directions and opportunities
for the future.
DEVELOPMENT OF METHODS
Biomolecule functionalization and multistep linkage to a versatile tissue-hydrogel scaffold
(Figure 2) within the cells of vertebrates (mouse, fish, and human) (
15
,
16
,
23
) were
described in an initial version called CLARITY; this method was optimized for application
to the vertebrate nervous system (
15
,
16
,
23
). The hydrogel-tissue hybrid brains were
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transparent (i.e., clarified) and thus permissive of intact whole-organ imaging at high
resolution (
16
). It was noted that the resulting hydrogel-tissue hybrid “expanded” upon lipid
removal in aqueous solution but “did not cause net tissue deformation...[R]emaining
secured in place were fine structural details” (
16
, p. 334) since the expansion could be
reversed with a solution change. Other diverse strategies for reducing opacity of intact tissue
had been explored for years (though with varying degrees of efficacy and versatility) (Figure
3), but transparency was not the only experimental leverage achieved with the hydrogel-
tissue chemistry (HTC) approach; for example, the new hybrids were designed to be
macromolecule permeant—enabling multiple rounds of molecular interrogation of preserved
biomolecules (proteins and nucleic acids) that had been anchored into the new physical
structure (
16
,
23
,
125
).
Single-photon confocal microscopy was initially used to image many-millimeter-thick
blocks of the resulting clarified and fluorescently labeled human brain tissue, zebrafish
central nervous systems, and whole adult mouse brain hemispheres (
16
). Diverse lines of
work eventually emerged from this publication (
23
); as was noted therein, “infused elements
need not be exclusively hydrogel monomers or acrylamide-based, and the properties of
infused elements may be adjusted for varying degrees of clarity, rigidity, macromolecule-
permeability or other functionality” (
16
, pp. 336–37). Also in 2013, a broad diversity of
additional compositions, including those with acrylates or alginates, was described (
25
), and
indeed variations and innovations on the theme rapidly emerged (Figure 4) (reviewed in
23
,
53
).
Also introduced was an electrophoretic tissue clearing (ETC) technique to accelerate lipid
removal (
16
); lipid removal promotes tissue transparency and macromolecular interrogation,
and this process can be carried out nondestructively after hydrogel-tissue hybrid formation
(Figure 1). ETC employs electric field-forced clearance of lipid-containing ionic-detergent
sodium dodecyl sulfate (SDS) micelles (Figure 1). Although helpful, ETC is not absolutely
necessary to remove lipids, and the following year an ETC-independent approach was
reported—passive CLARITY. This variant was initially described by Zhang et al. (
147
) and
was found to be effective for adult central nervous systems and spinal cords. Passive
CLARITY was soon thereafter reported to apply also to brain slices (
104
), and when
combined with CLARITY-optimized light-sheet microscopy (COLM) this variant enabled
imaging of entire adult mouse brains at subcellular resolution within several hours (
131
). At
the same time, another CLARITY variant (PACT) was described (
142
), presenting
modifications to the CLARITY reagents to passively achieve fast clearing of thick samples.
After overnight tissue fixation in 4% paraformaldehyde (PFA), tissues were embedded in a
4% acrylamide hydrogel solution without the 4% PFA and 0.05% bisacrylamide of the
original hydrogel formulation to minimize cross-linking (
133
,
142
). In addition, a relatively
inexpensive refractive index-matching solution, termed RIMS, was introduced (
142
).
The data of both Yang et al. (
142
) and Tomer et al. (
131
) in 2014 showed a moderate degree
of tissue expansion associated with the HTC process, as had been described by Chung et al.
(
16
) and indeed also as had been seen with earlier tissue clearing approaches (Figure 5).
Although this effect had not been amplified to explore potential advantages, over the next
two years, several HTC papers {11 [expansion microscopy (ExM) in 2015], 131 [expansion
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passive CLARITY technique (ePACT) in 2015], and 62 [magnified analysis of the proteome
(MAP) in 2016]} soon enabled much-enhanced swelling of HTC hybrids to improve
resolution of densely packed features. In a method unique for preserving endogenous
fluorescence, ePACT (
133
) uses collagenase to enhance the magnitude of the size change.
Two of the other versions, ExM (
11
,
30
) and MAP (
64
), also embed tissue in a similar
hydrogel network (reviewed in
53
). In these formulations, which prescribe inclusion of
acrylates (R2 in Figure 2) alongside acrylamide to enhance swelling (Figures 2 and 4),
proteolysis can be carried out to facilitate this process but is not required. MAP additionally
allows reversible expansion of the tissue-hydrogel hybrid (Figure 5) and super-resolution
imaging of subcellular structures using high concentrations of acrylamide (30% acrylamide
with 10% acrylate) to promote protein attachment to the hydrogel and prevent intra- and
inter-protein cross-linking (
64
).
A large number of subsequent HTC studies put forward additional enhancements, including
modifications of the ETC process and device (
5
,
59
,
71
,
72
,
117
,
121
), of the hydrogel
monomer and cross-linker levels (
5
,
32
,
63
,
131
,
133
,
142
) and of other parameters while
maintaining the basic hydrogel-tissue chemistry (
18
,
20
,
22
,
32
,
63
,
80
,
84
,
108
,
122
,
140
,
142
,
143
,
145
,
149
). In addition to the acrylamide and/or acrylate-based PFA-coupled
hydrogels noted above (PACT/ePACT, ExM, MAP), other gelation mechanisms have also
been described. The SWITCH approach uses pH changes to synchronize formation of a
glutaraldehyde-crosslinked matrix within tissue before CLARITY-type lipid removal via
SDS, resulting in a heat- and chemical-resistant tissue-hydrogel hybrid that facilitates
multiple rounds of labeling, elution, and relabeling (
94
,
106
). Also described in the study
that introduced PACT was a strategy termed PARS (perfusion-assisted agent release in situ)
for whole-body clearing and labeling using perfusion through the vasculature to deliver
hydrogel, clearing, labeling, and imaging reagents (
133
,
142
). PACT and other passive
CLARITY-based HTC methods were further adapted to tissues otherwise difficult or
impossible to image intact, from the rigid and opaque bone [PACT-deCAL (
133
,
140
) and
Bone CLARITY (
44
)] to the soft and friable clinical samples and embryos (
27
,
51
,
148
).
In addition to small-molecule dyes, cellular stains, and protein labels (e.g., lectin) that can
directly target proteins, DNA, and other biomolecules, tissues cleared using HTC can be
stained using fluorescently tagged whole antibodies as well as smaller antibody formulations
such as FAB (fragment antigen-binding antibody) fragments (
15
,
16
,
131
,
133
). Nanobodies
were effective in staining PACT-cleared tissues (
142
); at 10% the size of full antibodies and
stable over a variety of pH and temperature conditions, nanobodies are particularly
appealing for labeling cleared thick tissues (
133
). The ETC process was accelerated using an
approach called stochastic electrotransport (
59
), and an electrophoretically driven approach
transported antibodies across a few millimeters of cleared tissue in less than an hour,
approximately 800 times faster than via passive diffusion (
75
). PRESTO (pressure-related
efficient and stable transfer of macromolecules into organs) conferred increased antibody
penetration depth and speed, particularly in cleared peripheral organs, by application of
either centrifugal force or convection flow using a syringe pump during sample incubation in
an antibody solution (
71
).
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To broaden the types of macromolecular information obtained, recent studies have
developed methods for visualizing lipids and RNA in HTC samples. Following earlier work
that demonstrated the detection of endogenous mRNA in CLARITY specimens via standard
in situ hybridization protocols (
16
), Yang et al. (
142
) showed that PACT hydrogels
supported the use of single-molecule fluorescence in situ hybridization (smFISH) to detect
individual mRNA transcripts at depth. In optimizing retention of RNA for labeling in cleared
hydrogel-tissue hybrids, a carbodiimide compound [1-ethyl-3–3-dimethyl-aminopropyl
carbodiimide (EDC)] was discovered to be useful for specifically linking RNA nucleotides
directly to the tissue hydrogel (
125
) (Figure 2), and application of the hairpin chain reaction
(HCR) amplification system facilitated multiplexed RNA labeling in these EDC-CLARITY
samples that could be at least 3 mm thick. A 1% acrylamide hydrogel exhibited improved
RNA labeling (for both total RNA and specifically mRNA) when compared to CLARITY
samples (with 4% acrylamide) (
125
). Multiplexed single-molecule HCR was also
demonstrated as an effective in situ hybridization technique in HTC brain slices embedded
and cleared with PACT or ExM (
12
,
27
,
115
). Other methods led to improved visualization
of fluorescent nanoparticles (polyethylene glycol-coated quantum dots) (
116
,
117
), creation
of nonfluorescent (dark) reaction products (horseradish peroxidase colorimetric labeling)
(
122
), and development of lipophilic dyes that were altered to be aldehyde fixable to
proteins to mark membranes even after HTC lipid removal (
52
).
HYDROGEL-TISSUE CHEMISTRY-BASED DISCOVERY IN NEUROSCIENCE
AND THROUGHOUT THE ORGANISM
HTC methods have proven powerful for neuroscience; only a few examples of resulting
discoveries are collected here to illustrate current capabilities and opportunities. First, a large
number of studies have used the HTC approach to identify local and global wiring patterns
of targeted neurons, beginning with the demonstration that a specific class of spinal cord
neuron (NECAB expressing) exhibits midline crossing (
147
), and subsequently with the
mapping of infection distribution for viral vectors microinjected into the lateral amygdala
(LA) to analyze the neural mechanism of cocaine-cue memory engram formation in mice
(
50
). Similarly, in a study analyzing the morphology of raphe-spinal fibers in the spinal cord,
passive CLARITY provided visualization of a unique branching pattern of serotonergic
fibers along the rostrocaudal axis as they extended toward the lateral motor neuron column
(
77
,
78
). Using rabies virus-based circuit mapping, passive CLARITY and COLM provided
unbiased global mapping of all the neurons in the brain that project to dopamine neurons in
the substantia nigra pars compacta, which in turn project to dorsolateral versus dorsomedial
striatum (
73
). Likewise, rabies virus-based methods were used to trace monosynaptic inputs
to projection-defined dopamine neurons via whole-brain CLARITY (in this case also with
ETC and light-sheet imaging) (
90
). Anterograde tracing followed by CLARITY (using both
ETC and passive clearing) provided visualization of synaptic targets of GABAergic
projections from the medial septum (
136
). And in a study analyzing top-down control of
anxiety and fear, passive CLARITY was used to track and map a distinct novel projection
from ventromedial prefrontal cortex to basomedial amygdala (
1
). Integrating passive
CLARITY with light-sheet microscopy and behavior, researchers implemented multiple-
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animal whole-brain activity mapping protocols for HTC alongside a strategy termed
CAPTURE (
143
) for quantifying numbers and projections of behaviorally activated neurons.
PACT was used to study the distribution and morphology of astroglia in thick tissue sections
(
92
) and the 3D distribution of multiple genetically defined neuron types in mouse brains
(
103
). Passive CLARITY on sections of medial prefrontal cortex (mPFC) established the
presence of nonoverlapping corticotropin-releasing factor and corticotropin-releasing factor
receptor-1 circuits relevant to acute stress (
138
) and was used to map brain-wide viral
expression in mice inoculated with western equine encephalitis virus in the foot pad (
101
).
The distribution of microglia within the subventricular zone (a neurogenic region of the
adult central nervous system) was mapped using passive CLARITY (
38
), and in the
periventricular zone of the cerebellum, passive CLARITY was employed to analyze the
organization of astrocytes during development (
43
). Passive CLARITY was used to show
increased dendritic complexity in hippocampal pyramidal neurons of transgenic mice that
exhibit enhanced learning (
114
) and to observe the localization of cells expressing
neuromedin B, a bombesin-like neuropeptide that influences sighing behavior, around the
facial nucleus, including the retrotrapezoid nucleus (a control center for breathing) (
76
). In
transgenic mice using the nicotinic acetylcholine receptor
α
2 subunit (Chrna2) locus to mark
deep-layer V Martinotti cells, passive CLARITY was used to verify labeling, specificity, and
morphology of the targeted cells (
47
). For examining somatostatin-expressing interneurons
in the dentate gyrus, CLARITY allowed demonstration of the axonal projections of a
specific subset to the medial septum (
146
). Subcellular localization of a specific
transcription factor, ESRRA, was analyzed using CLARITY (1% acrylamide with ETC) in
brain sections (200 μm) to help elucidate the protein’s role in cell signaling (
111
). Using
viral vector tracing to label mPFC-projecting neurons in the basolateral amygdala (BLA),
CLARITY provided visualization of the target specificity of those neurons, which aided in
investigation of their role in manipulating fear associations (
60
). To analyze neuronal
organization in the hypothalamus, whole-brain mapping of tyrosine hydroxylase (TH)-
positive neurons and projections was performed with CLARITY followed by
immunostaining and COLM (
109
).
In addition to enabling these basic discoveries, HTC work has also stimulated technical and
engineering advances. Passive CLARITY of electrolytically lesioned slices was used to
correct electrode placement for fast-scan cyclic voltammetry (
120
) and to identify locations
of implanted optical fibers (
89
). Following penetrating brain injury, passive CLARITY
permitted brain-wide visualization of specific peptide accumulation in studies exploring
targeted delivery of diagnostic and therapeutic compounds (
86
). And more broadly, body-
wide biodistribution studies looking at chemicals or biologicals were found to benefit from
HTC; for example, Treweek and coworkers (
134
) and Deverman et al. (
28
) demonstrated
that whole-body PARS (
142
) could facilitate the generation of transduction maps of
systemically delivered genes by adeno-associated viruses, which in turn facilitated
characterization and discovery of new viral variants for targeting the central and peripheral
nervous systems (
8
). HTC-based clearing has also technically enabled quadruple
immunofluorescent staining as well as multiple rounds of labeling to reveal a variety of
richly defined subcellular domains and molecule types in single human cerebellar sections
(
102
).
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Several studies have combined magnetic resonance imaging (MRI) with CLARITY. In
probing the contribution of myelination to measurables from diffusion tensor imaging,
passive CLARITY revealed that myelination correlates strongly with fractional anisotropy
but only partially with radial diffusivity (
9
). The differential contributions of lipids and
proteins to MRI contrast were analyzed using passive CLARITY to remove lipids and
preserve proteins: Cleared tissues showed minimal contrast, increased relaxation times, and
diffusion rates similar to free water, and lipids were thus demonstrated to be the dominant
source of MRI contrast in brain tissue (
74
). In experimental autoimmune encephalomyelitis
(a mouse model of multiple sclerosis), a direct relationship was defined between gray matter
atrophy visualized using MRI and the number of axonal end bulbs in spinal cord visualized
using passive CLARITY (
118
). This type of ground-truth work on clinical biomarkers is of
immense and rapidly increasing value, particularly given the epidemiology of
neurodegenerative diseases.
Disease model work in general has progressed rapidly with HTC. In a mouse model for
Parkinson’s disease, passive CLARITY revealed fragmented nigrostriatal axons (
97
). In
addition to related studies in rat models (
80
,
119
), direct human-disease HTC applications
have also advanced rapidly. The effectiveness of CLARITY on postmortem human brain
tissue was demonstrated using 500-μm thick tissue blocks from clinical autism samples that
had been stored in formalin for over six years, revealing 3D morphologies not readily
accessible using traditional sectioning (
16
). Similarly, passive CLARITY has been used to
examine the 3D architecture of amyloid and tau aggregates in 500-μm thick banked tissue
from Alzheimer’s disease patients (
3
), and passive CLARITY has been used on 3-mm thick
blocks of fresh or formalin-fixed tissue from Parkinson’s disease patients to reveal Lewy
body inclusions nearly 1 mm deep in the tissue (
80
).
NONNEURAL TISSUES
Although originally conceived for studying the brain (
23
,
24
), the HTC approach can be
extended to a wide variety of other organs and tissue types, including spinal cord, lung,
heart, intestine, spleen, kidney, muscle, testis, pancreas, liver, skin, and bone (
32
,
44
,
71
,
72
,
100
,
140
,
142
). Its usefulness for imaging infection was demonstrated using PACT in mice
infected with fluorescent
Mycobacterium tuberculosis,
which enabled visualization of 3D
spatial distribution of bacteria throughout intact lungs (
20
). A modified PACT, MiPACT (for
microbial identification after PACT) was designed to label bacterial rRNA (via HCR) for
analysis of spatial organization and metabolic activity of bacteria in amorphous sputum
samples from cystic fibrosis patients (
27
). Also in lung, localization of nestin-expressing
cells was observed throughout the vasculature (not the airway system) of tissue cleared via
PACT, which motivated and guided investigation of the role of these cells in development of
pulmonary hypertension (
110
). In a mouse model of lung adenocarcinoma, applying
CLARITY to whole-lung tumors (clearing with two days of ETC) provided a comprehensive
demonstration of significant differences in the cellular density and morphology of tumor
cells with and without depletion of regulatory T cells (
54
). In pancreatic tissue, an evaluation
of p53 loss of heterozygosity in tumor progression was enabled by HTC (
95
).
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In liver, 3D positioning within the portal system (relative to the canals of Hering) was
demonstrated using passive CLARITY for periportal hepatocytes, which undergo
proliferation following injury (
37
). After application of passive CLARITY to rat kidneys,
superresolution-STED microscopy revealed 3D positioning information at the nanometer
scale (
137
). HTC on mouse and human gut tissue was achieved using passive CLARITY and
immunostaining to visualize structures in the enteric nervous system, vasculature, smooth
muscle layers, and epithelium, while also demonstrating compatibility with classical
pathological stains such as hematoxylin-eosin and Heidenhain’s Azan (
96
). Early systemic
viral spread of human immunodeficiency virus 1 (HIV-1) in humanized mice was analyzed
from gut-associated lymphoid tissues using PACT (
58
), and HTC (with ETC) was found
useful for studying even dense and fibrous mouse hind-limb skeletal muscle tissue (
91
). In
virgin and lactating mouse mammary glands, epithelial and tumor cells were made visible
using PACT (
82
), whereas with passive CLARITY on intact mouse ovaries, the architecture
and growth of ovarian follicles and their relationship to vasculature was analyzed throughout
the mouse reproductive life (
35
,
83
). Embryonic and neoplastic tissue analysis has been
similarly optimized (
48
,
88
,
132
), and fast clearing was achieved by HTC in liver tissue (
69
)
as well as in the growth plates of distal limbs (
17
).
In hatched chickens, adult
Xenopus,
and adult zebrafish, the comparative organization of
HTC-stabilized cerebrospinal fluid-contacting cells revealed similarities pointing to a
common bony vertebrate ancestor (
141
). Legs from chicken embryos were analyzed using
passive CLARITY to reveal embryonic development of hallux positioning in the avian
grasping foot (
6
). Passive CLARITY was also applied to the mouse nasal septum to
visualize the morphology of horizontal basal cells in the olfactory epithelium following
lesion of the olfactory bulb (
112
). The effect of subcutaneous injection of poly(methacrylic
acid-co-methyl methacrylate) beads on vascularization was observed using passive
CLARITY in mouse skin tissue (
79
). A dual-illumination-side light-sheet microscope
optimized for imaging cardiac tissue over 1 cm
3
in volume, combined with HTC, enabled
researchers to measure ventricular dimensions, track the lineage of cardiac cells, and view
the spatial distribution of cardiac-specific proteins within intact hearts (
29
). CLARITY also
has been employed in intact mouse hearts as well as human heart tissue up to several
millimeters thick (
42
,
62
).
Host-pathogen interactions were studied using passive CLARITY and PACT to
comprehensively examine morphology of necrotic granulomas from adult zebrafish infected
with
Mycobacterium marinum
(
19
,
20
). PACT and CUBIC (
123
) were found well suited for
imaging the intact zebrafish testis at cellular resolution (
39
). Passive CLARITY was applied
to transgenic
Xenopus
tadpoles to locate and quantify thyroid hormone signaling disruption
by contaminants introduced during brain development (
36
). Applying passive CLARITY to
the intact liver of lamprey undergoing metamorphosis provided visualization of the process
of biliary degeneration, a process that occurs in human infants with biliary atresia via a
mechanism that is still unknown (
14
), and passive CLARITY/COLM imaging in the
lamprey was used to visualize the spatial organization of neuronal inputs and outputs in the
optic tectum with the Neurobiotin tracer (
55
).
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Addressing challenges beyond soft tissue, Bone CLARITY (
44
) was developed and applied
along with a CLARITY-optimized light-sheet microscope to quantify marrow cells from
cleared adult intact mouse bones, revealing differences in fluorescent stem cell count and
distribution after bone-forming agent administration (
44
). HTC approaches have been
applied to multicellular plants as well via plant-enzyme-assisted (PEA)-CLARITY, an
adaptation to perform optical clearing and antibody interrogation on plant tissues. Using cell
wall-degrading enzymes to increase permeability and starch-hydrolyzing enzymes to
improve transparency following passive clearing, PEA-CLARITY enabled visualization of
fluorescent signals from expressed proteins as well as antibody staining in whole, intact
tobacco and
Arabidopsis
leaves (
98
). The PEA-CLARITY protocol was later applied to
study the 3D architecture of the
Medicago truncatula
root nodules (
128
).
OUTLOOK
The proven application domain of HTC in biology and medicine is rapidly expanding and
has already resulted in numerous basic science discoveries and opportunities for clinical
medicine (e.g.,
24
,
51
,
143
). However, the novelty of the preparation and its resulting data
streams have created challenges. Here, we consider the current rate-limiting steps as well as
opportunities for the future.
Early on, one of the clearest applications of the HTC approach was enabling high-resolution
optical access to large intact tissues, organs, and organisms. Although this major goal was
achieved, collecting high-resolution volumetric image data from large samples created new
issues. For example, the transparency of the hydrogel-tissue hybrid allowed confocal or two-
photon imaging over large volumes, but these slow point-scanning techniques led to
bottlenecks in image acquisition (e.g., the collection of high-resolution structural data sets
for an adult mouse brain required several days of imaging). Data collection on this timescale
is associated with problems ranging from photobleaching to simple microscope
overoccupancy, but rapid development of advanced light-sheet imaging, which offers orders-
of-magnitude improvement in speed (
29
,
41
,
44
,
107
,
115
,
130
,
131
,
143
), addressed this
acquisition problem. Subsequent HTC-focused work included stochastic electrotransport
(
59
); super-resolution-STED microscopy (
137
); adaptive optics (
105
); HTC sample handling
chambers (
44
,
92
,
93
,
135
); custom ETC and staining chambers (
59
,
71
); and microfluidic
chip-based embedding, clearing, and labeling (
13
).
The initial expansion found associated with HTC methods (
16
,
131
,
142
) was counteracted
with size-normalization/contraction strategies during the refractive index-matching step to
allow high-resolution objectives with limited working distance to access more of the brain
(
16
). This strategy also had the effect of reducing the data set size, an important
consideration for tractability. However, these considerations have become progressively less
important with the advent of new hardware, including customized long-working-distance
and high-resolution CLARITY objectives (
87
,
131
) as well as distributed computing
strategies.
Many studies have employed automated analysis pipelines for manipulating large CLARITY
data sets; commercial 3D rendering software programs, such as Imaris or Arivis, can
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automate manually intensive data processing steps such as cell counting. Automation
becomes even more valuable when analyzing thicker tissue sections or whole organs (
44
,
92
,
143
), but the utility of automated analysis extends beyond the domain of cell body
recognition and counting. To quantify neural projection patterns, an automated method has
been developed to compute 3D structure tensors from CLARITY images, and input of the
tensors into diffusion tractography software yielded reconstruction of calculated streamlines
mapped onto fibers from the CLARITY images (
143
). With this approach, connectivity
between a seed region and specific downstream targets could be visualized and
quantitatively evaluated by counting streamlines (
143
). In addition, alignment of
autofluorescence images from multiple sample organs can be used to create a common
reference space. When autofluorescence is combined with segmentation algorithms for
automated cell detection, a transformation of the acquired signal from each sample onto this
reference space can be used to compare the regional distribution of labeled cells across brain
samples and allow registration to public atlases, such as the Allen Brain Institute’s Mouse
Reference Atlas (
90
,
107
,
143
). Automatic annotation of CLARITY brain images (
67
) has
been enabled by registering CLARITY brain images to the Allen atlas using a method called
Mask-LDDMM. TeraFly is a free, open-source software tool designed specifically for 3D
integrated visualization and annotation of massive, terabyte-sized image data sets like those
acquired using the COLM system (
7
), and a manual segmentation tool (ManSegTool) for
segmenting 3D neuronal data sets was demonstrated to enable neuroscientists to extract
neurons from cerebellum slices cleared and imaged using passive CLARITY (
85
). For
automatic annotation and standardization of brainwide data sets, WholeBrain is a free, open-
source software that provides connectivity and activity-based mapping and quantification of
multidimensional data, using a scale-invariant anatomical mouse brain atlas, which allows
comparison of results across experiments and imaging platforms (
40
). Concurrently, an
interactive Web-based framework, Openbrainmap (
http://openbrainmap.org
), was developed
for data visualization and sharing between laboratories (
40
).
Tissue clarification is only one of many application domains of HTC methods, although it is
arguably the most developed. Beyond tissue transparency, two studies have applied the
hydrogel tissue-embedding step of CLARITY to stabilize mouse embryos or adult mouse
brain tissue for micro-computed tomography (micro-CT) imaging using contrast agents that
typically shrink tissue (
2
,
139
). CLARITY was also used to reveal the 3D structure of
patterned microtissues (
129
). And in stem cell-derived organoids, passive CLARITY
followed by immunostaining was used to model and explore effects of cocaine exposure on
the human fetal brain (
70
).
A final emerging domain of substantial interest, and an initial motivation for HTC (
26
), is
the development of hydrogel-tissue hybrids with diverse types of functionalization, which
would enable experiments extending far beyond static structural and molecular analysis. For
example, creation of active constructs based on polymers with electrically conductive
properties could allow new forms of interrogation of biological systems, and diverse
additional forms of HTC and variants are in the process of emerging. Rooted in fundamental
chemistry, the broad concept of envisioning (and remaking) metazoan animals and tissues as
metareactants—that is, positionally intact and chemically versatile scaffolds of molecular
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reactants—may continue to open up new and unanticipated domains of investigation and
discovery across diverse fields of biology.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
We thank Prof. Kwanghun Chung, Prof. Zhenan Bao, Dr. Ritchie Chen, Dr. Xiao Wang, Dr. Emily Sylwestrak, and
members of our laboratories for helpful comments on the manuscript. K.D. is supported by the National Institutes
of Health (NIH) R01DA03537701, R01MH075957, and R01MH086373, as well as by the Defense Advanced
Research Projects Agency and Army Research Laboratory NeuroFAST program (Cooperative Agreement
W911NF-1420013). V.G. is supported by the NIH via the New Innovator Award DP2NS087949 and the
Presidential Early Career Award for Science and Engineers, OT2OD023848–01, and R01AG047664; V.G. is also a
Heritage Medical Research Institute Investigator and director of the Center for Molecular and Cellular
Neuroscience in the Chen Institute at Caltech.
LITERATURE CITED
1. Adhikari A, Lerner TN, Finkelstein J, Pak S, Jennings JH, et al. 2015 Basomedial amygdala
mediates top-down control of anxiety and fear. Nature 527:179–85 [PubMed: 26536109]
2. Anderson R, Maga AM. 2015 A novel procedure for rapid imaging of adult mouse brains with
microCT using iodine-based contrast. PLOS ONE 10:e0142974 [PubMed: 26571123]
3. Ando K, Laborde Q, Lazar A, Godefroy D, Youssef I, et al. 2014 Inside Alzheimer brain with
CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D. Acta Neuropathol. 128:457–59
[PubMed: 25069432]
4. Aoyagi Y, Kawakami R, Osanai H, Hibi T, Nemoto T. 2015 A rapid optical clearing protocol using
2,2
′
-thiodiethanol for microscopic observation of fixed mouse brain. PLOS ONE 10:e0116280
[PubMed: 25633541]
5. Bastrup J, Larsen PH. 2017 Optimized CLARITY technique detects reduced parvalbumin density in
a genetic model of schizophrenia. J. Neurosci. Methods 283:23–32 [PubMed: 28342832]
6. Botelho JF, Smith-Paredes D, Soto-Acuna S, Nunez-Leon D, Palma V, Vargas AO. 2017 Greater
growth of proximal metatarsals in bird embryos and the evolution of hallux position in the grasping
foot. J. Exp. Zool. B Mol. Dev. Evol 328:106–18 [PubMed: 27649924]
7. Bria A, Iannello G, Onofri L, Peng H. 2016 TeraFly: real-time three-dimensional visualization and
annotation of terabytes of multidimensional volumetric images. Nat. Methods 13:192–94 [PubMed:
26914202]
8. Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, et al. 2017 Engineered AAVs for efficient
noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci 20:1172–79
[PubMed: 28671695]
9. Chang EH, Argyelan M, Aggarwal M, Chandon TS, Karlsgodt KH, et al. 2017 The role of
myelinationin measures of white matter integrity: combination of diffusion tensor imaging and two-
photon microscopy of CLARITY intact brains. Neuroimage 147:253–61 [PubMed: 27986605]
10. Chang JB, Chen F, Yoon YG, Jung EE, Babcock H, et al. 2017 Iterative expansion microscopy.
Nat. Methods 14:593–99 [PubMed: 28417997]
11. Chen F, Tillberg PW, Boyden ES. 2015 Expansion microscopy. Science 347:543 [PubMed:
25592419]
12. Chen F, Wassie AT, Cote AJ, Sinha A, Alon S, et al. 2016 Nanoscale imaging of RNA with
expansion microscopy. Nat. Methods 13:679–84 [PubMed: 27376770]
13. Chen YY, Silva PN, Syed AM, Sindhwani S, Rocheleau JV, Chan WC. 2016 Clarifying intact 3D
tissues on a microfluidic chip for high-throughput structural analysis. PNAS 113:14915–20
[PubMed: 27956625]
Gradinaru et al.
Page 11
Annu Rev Biophys
. Author manuscript; available in PMC 2019 February 02.
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author Manuscript
14. Chung-Davidson YW, Davidson PJ, Scott AM, Walaszczyk EJ, Brant CO, et al. 2014 A new
clarification method to visualize biliary degeneration during liver metamorphosis in Sea Lamprey
(Petromyzon marinus). J. Vis. Exp 6:88
15. Chung K, Deisseroth K. 2013 CLARITY for mapping the nervous system. Nat. Methods 10:508–
13 [PubMed: 23722210]
16. Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, et al. 2013 Structural and
molecular interrogation of intact biological systems. Nature 497:332–37 [PubMed: 23575631]
17. Collette JC, Choubey L, Smith KM. 2017 Glial and stem cell expression of murine Fibroblast
Growth Factor Receptor 1 in the embryonic and perinatal nervous system. PeerJ 5:e3519
[PubMed: 28674667]
18. Costantini I, Ghobril JP, Di Giovanna AP, Allegra Mascaro AL, Silvestri L, et al. 2015 A versatile
clearing agent for multi-modal brain imaging. Sci. Rep 5:9808 [PubMed: 25950610]
19. Cronan MR, Beerman RW, Rosenberg AF, Saelens JW, Johnson MG, et al. 2016 Macrophage
epithelial reprogramming underlies mycobacterial granuloma formation and promotes infection.
Immunity 45:861–76 [PubMed: 27760340]
20. Cronan MR, Rosenberg AF, Oehlers SH, Saelens JW, Sisk DM, et al. 2015 CLARITY and PACT-
based imaging of adult zebrafish and mouse for whole-animal analysis of infections. Dis. Model.
Mech 8:1643–50 [PubMed: 26449262]
21. Cui Y, Wang X, Ren W, Liu J, Irudayaraj J. 2016 Optical clearing delivers ultrasensitive
hyperspectral dark-field imaging for single-cell evaluation. ACS Nano 10:3132–43 [PubMed:
26895095]
22. d’Esposito A, Nikitichev D, Desjardins A, Walker-Samuel S, Lythgoe MF. 2015 Quantification of
light attenuation in optically cleared mouse brains. J. Biomed. Opt 20:80503 [PubMed: 26277988]
23. Deisseroth K 2016 A look inside the brain. Sci. Am 315:30–37
24. Deisseroth K 2017 Optical and chemical discoveries recognized for impact on biology and
psychiatry. EMBO Rep. 18:859–60 [PubMed: 28566521]
25. Deisseroth KA, Chung K. 2015 Methods and compositions for preparing biological specimens for
microscopic analysis.
www.google.com/patents/US20150144490
. Filing date: March 13, 2013. US
Patent Appl. No. US20150144490
26. Deisseroth KA, Gradinaru V. 2014 Functional targeted brain endoskeletonization.
www.google.com/patents/US20140030192
. Filing date: Jan 26, 2012. US Patent Appl. No.
US20140030192
27. DePas WH, Starwalt-Lee R, Van Sambeek L, Ravindra Kumar S, Gradinaru V, Newman DK. 2016
Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis
sputum via hydrogel embedding, clearing, and rRNA labeling. mBio 7:5e00796–16
28. Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, et al. 2016 Cre-dependent selection
yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotech 34:204–9
29. Ding Y, Lee J, Ma J, Sung K, Yokota T, et al. 2017 Light-sheet fluorescence imaging to localize
cardiac lineage and protein distribution. Sci. Rep 7:42209 [PubMed: 28165052]
30. Dodt H-U. 2015 The superresolved brain. Science 347:474–75 [PubMed: 25635071]
31. Dodt H-U, Leischner U, Schierloh A, Jahrling N, Mauch CP, et al. 2007 Ultramicroscopy: three-
dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4:331–36
[PubMed: 17384643]
32. Epp JR, Niibori Y, Liz Hsiang HL, Mercaldo V, Deisseroth K, et al. 2015 Optimization of
CLARITY for clearing whole-brain and other intact organs. eNeuro 2:1–15
33. Erturk A, Becker K, Jährling N, Mauch CP, Hojer CD, et al. 2012 Three-dimensional imaging of
solvent-cleared organs using 3DISCO. Nat. Protoc 7:1983–95 [PubMed: 23060243]
34. Feldman MY. 1973 Reactions of nucleic acids and nucleoproteins with formaldehyde. Prog. Nucl.
Acid Res. Mol. Biol 13:1–49
35. Feng Y, Cui P, Lu X, Hsueh B, Möller Billig F, et al. 2017 CLARITY reveals dynamics of ovarian
follicular architecture and vasculature in three-dimensions. Sci. Rep 7:44810 [PubMed: 28333125]
Gradinaru et al.
Page 12
Annu Rev Biophys
. Author manuscript; available in PMC 2019 February 02.
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author Manuscript
36. Fini JB, Mughal BB, Le Mevel S, Leemans M, Lettmann M, et al. 2017 Human amniotic fluid
contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos.
Sci. Rep 7:43786 [PubMed: 28266608]
37. Font-Burgada J, Shalapour S, Ramaswamy S, Hsueh B, Rossell D, et al. 2015 Hybrid periportal
hepatocytes regenerate the injured liver without giving rise to cancer. Cell 162:766–79 [PubMed:
26276631]
38. Fourgeaud L, Traves PG, Tufail Y, Leal-Bailey H, Lew ED, et al. 2016 TAM receptors regulate
multiple features of microglial physiology. Nature 532:240–44 [PubMed: 27049947]
39. Fretaud M, Riviere L, Job E, Gay S, Lareyre JJ, et al. 2017 High-resolution 3D imaging of whole
organ after clearing: taking a new look at the zebrafish testis. Sci. Rep 7:43012 [PubMed:
28211501]
40. Fürth D, Vaissière T, Tzortzi O, Xuan Y, Märtin A, et al. 2017 An interactive framework for whole-
brain maps at cellular resolution. Nat. Neurosci 21:139–49 [PubMed: 29203898]
41. Glaser AK, Reder NP, Chen Y, McCarty EF, Yin C, et al. 2017 Light-sheet microscopy for slide-
free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng 1:0084 [PubMed:
29750130]
42. Gloschat CR, Koppel AC, Aras KK, Brennan JA, Holzem KM, Efimov IR. 2016 Arrhythmogenic
and metabolic remodelling of failing human heart. J. Physiol 594:3963–80 [PubMed: 27019074]
43. Gonzalez-Gonzalez MA, Gomez-Gonzalez GB, Becerra-Gonzalez M, Martinez-Torres A. 2017
Identification of novel cellular clusters define a specialized area in the cerebellar periventricular
zone. Sci. Rep 7:40768 [PubMed: 28106069]
44. Greenbaum A, Chan KY, Dobreva T, Brown D, Balani DH, et al. 2017 Bone CLARITY: clearing,
imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci. Transl.
Med 9:387
45. Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, et al. 2015 ScaleS: an optical clearing
palette for biological imaging. Nat. Neurosci 18:1518–29 [PubMed: 26368944]
46. Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, et al. 2011 Scale: a chemical approach
for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci 14:1481–
88 [PubMed: 21878933]
47. Hilscher MM, Leao RN, Edwards SJ, Leao KE, Kullander K. 2017 Chrna2-Martinotti cells
synchronize layer 5 type A pyramidal cells via rebound excitation. PLOS Biol. 15:e2001392
[PubMed: 28182735]
48. Hirashima T, Adachi T. 2015 Procedures for the quantification of whole-tissue
immunofluorescence images obtained at single-cell resolution during murine tubular organ
development. PLOS ONE 10:e0135343 [PubMed: 26258587]
49. Hou B, Zhang D, Zhao S, Wei M, Yang Z, et al. 2015 Scalable and DiI-compatible optical
clearance of the mammalian brain. Front. Neuroanat 9:19 [PubMed: 25759641]
50. Hsiang HL, Epp JR, van den Oever MC, Yan C, Rashid AJ, et al. 2014 Manipulating a “cocaine
engram” in mice. J. Neurosci 34:14115–27 [PubMed: 25319707]
51. Hsueh B, Burns VM, Pauerstein P, Holzem K, Ye L, et al. 2017 Pathways to clinical CLARITY:
volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease. Sci.
Rep 7:5899 [PubMed: 28724969]
52. Jensen KHR, Berg RW. 2016 CLARITY-compatible lipophilic dyes for electrode marking and
neuronal tracing. Sci. Rep. 6:32674 [PubMed: 27597115]
53. Jensen KHR, Berg RW. 2017 Advances and perspectives in tissue clearing using CLARITY. J.
Chem. Neuroanat 86:19–34 [PubMed: 28728966]
54. Joshi NS, Akama-Garren EH, Lu Y, Lee DY, Chang GP, et al. 2015 Regulatory T cells in tumor-
associated tertiary lymphoid structures suppress anti-tumor T cell responses. Immunity 43:579–90
[PubMed: 26341400]
55. Kardamakis AA, Pérez-Fernández J, Grillner S. 2016 Spatiotemporal interplay between
multisensory excitation and recruited inhibition in the lamprey optic tectum. eLife 5:e16472
[PubMed: 27635636]
56. Ke MT, Fujimoto S, Imai T. 2013 SeeDB: a simple and morphology-preserving optical clearing
agent for neuronal circuit reconstruction. Nat. Neurosci 16:1154–61 [PubMed: 23792946]
Gradinaru et al.
Page 13
Annu Rev Biophys
. Author manuscript; available in PMC 2019 February 02.
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author Manuscript
57. Kellner M, Heidrich M, Lorbeer RA, Antonopoulos GC, Knudsen L, et al. 2016 A combined
method for correlative 3D imaging of biological samples from macro to nano scale. Sci. Rep
6:35606 [PubMed: 27759114]
58. Kieffer C, Ladinsky MS, Ninh A, Galimidi RP, Bjorkman PJ. 2017 Longitudinal imaging of HIV-1
spread in humanized mice with parallel 3D immunofluorescence and electron tomography. eLife
6:e23282 [PubMed: 28198699]
59. Kim S-Y, Cho JH, Murray E, Bakh N, Choi H, et al. 2015 Stochastic electrotransport selectively
enhances the transport of highly electromobile molecules. PNAS 112:E6274–83 [PubMed:
26578787]
60. Klavir O, Prigge M, Sarel A, Paz R, Yizhar O. 2017 Manipulating fear associations via optogenetic
modulation of amygdala inputs to prefrontal cortex. Nat. Neurosci 20:836–44 [PubMed:
28288126]
61. Klingberg A, Hasenberg A, Ludwig-Portugall I, Medyukhina A, Mann L, et al. 2017 Fully
automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using
lightsheet microscopy. J. Am. Soc. Nephrol 28:452–59 [PubMed: 27487796]
62. Kolesova H, Capek M, Radochova B, Janacek J, Sedmera D.2016 Comparison of different tissue
clearing methods and 3D imaging techniques for visualization ofGFP-expressing mouse embryos
and embryonic hearts. Histochem. Cell Biol 146:141–52 [PubMed: 27145961]
63. Krolewski DM, Kumar V, Martin B, Tomer R, Deisseroth K, et al. 2018 Quantitative validation of
immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations. Brain
Struct. Funct 223:987–99 [PubMed: 29243106]
64. Ku T, Swaney J, Park JY, Albanese A, Murray E, et al. 2016 Multiplexed and scalable super-
resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat.
Biotechnol 34:973–81 [PubMed: 27454740]
65. Kubota SI, Takahashi K, Nishida J, Morishita Y, Ehata S, et al. 2017 Whole-body profiling of
cancer metastasis with single-cell resolution. Cell Rep. 20:236–50 [PubMed: 28683317]
66. Kurihara D, Mizuta Y, Sato Y, Higashiyama T. 2015 ClearSee: a rapid optical clearing reagent for
whole-plant fluorescence imaging. Development 142:4168–79 [PubMed: 26493404]
67. Kutten KS, Vogelstein JT, Charon N, Ye L, Deisseroth K, Miller MI. 2016 Deformably registering
and annotating whole CLARITY brains to an atlas via masked LDDMM. Presented at Proc. SPIE
Opt., Photonics, Digit. Technol. for Imaging Appl. IV, Brussels, Belg.
68. Lai HM, Liu AKL, Ng W-L, DeFelice J, Lee WS, et al. 2016 Rationalisation and validation of an
acrylamide-free procedure in three-dimensional histological imaging. PLOS ONE 11:e0158628
[PubMed: 27359336]
69. Lai M, Li X, Li J, Hu Y, Czajkowsky DM, Shao Z. 2017 Improved clearing of lipid droplet-rich
tissues for three-dimensional structural elucidation. Acta Biochim. Biophys. Sin 49:465–67
[PubMed: 28338831]
70. Lee CT, Chen J, Kindberg AA, Bendriem RM, Spivak CE, et al. 2017 CYP3A5 mediates effects of
cocaine on human neocorticogenesis: studies using an in vitro 3D self-organized hPSC model with
a single cortex-like unit. Neuropsychopharmacology 42:774–84 [PubMed: 27534267]
71. Lee E, Choi J, Jo Y, Kim JY, Jang YJ, et al. 2016 ACT-PRESTO: rapid and consistent tissue
clearing and labeling method for 3-dimensional (3D) imaging. Sci. Rep 6:18631 [PubMed:
26750588]
72. Lee H, Park JH, Seo I, Park SH, Kim S.2014 Improved application of the electrophoretic tissue
clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung,
and intestine. BMC Dev. Biol 14:48 [PubMed: 25528649]
73. Lerner TN, Shilyansky C, Davidson TJ, Evans KE, Beier KT, et al. 2015 Intact-brain analyses
reveal distinct information carried by SNc dopamine subcircuits. Cell 162:635–47 [PubMed:
26232229]
74. Leuze C, Aswendt M, Ferenczi E, Liu CW, Hsueh B, et al. 2017 The separate effects of lipids and
proteins on brain MRI contrast revealed through tissue clearing. Neuroimage 156:412–22
[PubMed: 28411157]
75. Li J, Czajkowsky DM, Li X, Shao Z. 2015 Fast immuno-labeling by electrophoretically driven
infiltration for intact tissue imaging. Sci. Rep 5:10640 [PubMed: 26013317]
Gradinaru et al.
Page 14
Annu Rev Biophys
. Author manuscript; available in PMC 2019 February 02.
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author Manuscript
76. Li P, Janczewski WA, Yackle K, Kam K, Pagliardini S, et al. 2016 The peptidergic control circuit
for sighing. Nature 530:293–97 [PubMed: 26855425]
77. Liang H, Schofield E, Paxinos G. 2016 Imaging serotonergic fibers in the mouse spinal cord using
the CLARITY/CUBIC technique. J. Vis. Exp 108:53673
78. Liang H, Wang S, Francis R, Whan R, Watson C, Paxinos G. 2015 Distribution of raphespinal
fibers in the mouse spinal cord. Mol. Pain 11:42 [PubMed: 26173454]
79. Lisovsky A, Zhang DK, Sefton MV. 2016 Effect of methacrylic acid beads on the sonic hedgehog
signaling pathway and macrophage polarization in a subcutaneous injection mouse model.
Biomaterials 98:203–14 [PubMed: 27264502]
80. Liu AKL, Hurry MED, Ng OTW, DeFelice J, Lai HM, et al. 2016 Bringing CLARITY to the
human brain: visualization of Lewy pathology in three dimensions. Neuropathol. Appl. Neurobiol
42:573–87 [PubMed: 26526972]
81. Liu AKL, Lai HM, Chang RCC, Gentleman SM. 2017 Free of acrylamide sodium dodecyl sulphate
(SDS)-based tissue clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional
visualization of human brain tissues. Neuropathol. Appl. Neurobiol 43:346–51 [PubMed:
27627784]
82. Lloyd-Lewis B, Davis FM, Harris OB, Hitchcock JR, Lourenco FC, et al. 2016 Imaging the
mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence
methods. Breast Cancer Res. 18:127 [PubMed: 27964754]
83. Lu X, Guo S, Cheng Y, Kim J-h, Feng Y, Feng Y. 2017 Stimulation of ovarian follicle growth after
AMPK inhibition. Reproduction 153:683–94 [PubMed: 28250241]
84. Magliaro C, Callara AL, Mattei G, Morcinelli M, Viaggi C, et al. 2016 Clarifying CLARITY:
quantitative optimization of the diffusion based delipidation protocol for genetically labeled tissue.
Front. Neurosci 10:179 [PubMed: 27199642]
85. Magliaro C, Callara AL, Vanello N, Ahluwalia A. 2017 A manual segmentation tool for
threedimensional neuron datasets. Front. Neuroinform 11:36 [PubMed: 28620293]
86. Mann AP, Scodeller P, Hussain S, Joo J, Kwon E, et al. 2016 A peptide for targeted, systemic
delivery of imaging and therapeutic compounds into acute brain injuries. Nat. Commun 7:11980
[PubMed: 27351915]
87. Marx V 2014 Microscopy: seeing through tissue. Nat. Methods 11:1209–14 [PubMed: 25423017]
88. Mayrhofer M, Gourain V, Reischl M, Affaticati P, Jenett A, et al. 2017A novel brain tumour model
in zebrafish reveals the role of YAP activation in MAPK- and PI3K-induced malignant growth.
Dis. Model. Mech 10:15–28 [PubMed: 27935819]
89. Menegas W, Babayan BM, Uchida N, Watabe-Uchida M. 2017 Opposite initialization to novel
cues in dopamine signaling in ventral and posterior striatum in mice. eLife 6:e21886 [PubMed:
28054919]
90. Menegas W, Bergan JF, Ogawa SK, Isogai Y, Umadevi Venkataraju K, et al. 2015 Dopamine
neurons projecting to the posterior striatum form an anatomically distinct subclass. eLife 4:e10032
[PubMed: 26322384]
91. Milgroom A, Ralston E. 2016 Clearing skeletal muscle with CLARITY for light microscopy
imaging. Cell Biol. Int 40:478–83 [PubMed: 26732743]
92. Miller SJ, Rothstein JD. 2016 Astroglia in thick tissue with super resolution and cellular
reconstruction. PLOSONE 11:e0160391
93. Miller SJ, Rothstein JD. 2017 3D printer generated tissue iMolds for cleared tissue using single-
and multi-photon microscopy for deep tissue evaluation. Biol. Proced. Online 19:7 [PubMed:
28690429]
94. Murray E, Cho JH, Goodwin D, Ku T, Swaney J, et al. 2015 Simple, scalable proteomic imaging
for high-dimensional profiling of intact systems. Cell 163:1500–14 [PubMed: 26638076]
95. Muzumdar MD, Dorans KJ, Chung KM, Robbins R, Tammela T, et al. 2016 Clonal dynamics
following p53 loss of heterozygosity in Kras-driven cancers. Nat. Commun 7:12685 [PubMed:
27585860]
96. Neckel PH, Mattheus U, Hirt B, Just L, Mack AF. 2016 Large-scale tissue clearing (PACT):
technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.
Sci. Rep 6:34331 [PubMed: 27680942]
Gradinaru et al.
Page 15
Annu Rev Biophys
. Author manuscript; available in PMC 2019 February 02.
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author Manuscript
97. Nordstroma U, Beauvais G, Ghosh A, Pulikkaparambil Sasidharan BC, Lundblad M, et al. 2015
Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous
mouse model of Parkinson’s disease. Neurobiol. Dis 73:70–82 [PubMed: 25281317]
98. Palmer WM, Martin AP, Flynn JR, Reed SL, White RG, et al. 2015 PEA-CLARITY: 3D molecular
imaging of whole plant organs. Sci. Rep 5:13492 [PubMed: 26328508]
99. Pan C, Cai R, Quacquarelli FP, Ghasemigharagoz A, Lourbopoulos A, et al. 2016 Shrinkage-
mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13:859–67
[PubMed: 27548807]
100. Pan M, Reid MA, Lowman XH, Kulkarni RP, Tran TQ, et al. 2016 Regional glutamine deficiency
in tumours promotes dedifferentiation through inhibition of histone demethylation. Nat. Cell Biol
18:1090–101 [PubMed: 27617932]
101. Phillips AT, Rico AB, Stauft CB, Hammond SL, Aboellail TA, et al. 2016 Entry sites of
Venezuelan and western equine encephalitis viruses in the mouse central nervous system
following peripheral infection. J. Virol 90:5785–96 [PubMed: 27053560]
102. Phillips J, Laude A, Lightowlers R, Morris CM, Turnbull DM, Lax NZ. 2016 Development of
passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum:
understanding mechanisms of neurodegeneration in mitochondrial disease. Sci. Rep 6:26013
[PubMed: 27181107]
103. Plummer NW, Evsyukova IY, Robertson SD, de Marchena J, Tucker CJ, Jensen P. 2015
Expanding the power of recombinase-based labeling to uncover cellular diversity. Development
142:4385–93 [PubMed: 26586220]
104. Poguzhelskaya E, Artamonov D, Bolshakova A, Vlasova O, Bezprozvanny I. 2014 Simplified
method to perform CLARITY imaging. Mol. Neurodegener 9:19 [PubMed: 24885504]
105. Reinig MR, Novak SW, Tao X, Bentolila LA, Roberts DG, et al. 2016 Enhancing image quality in
cleared tissue with adaptive optics. J. Biomed. Opt 21:121508 [PubMed: 27735018]
106. Ren J, Choi H, Chung K, Bouma BE. 2017 Label-free volumetric optical imaging of intact
murine brains. Sci. Rep 7:46306 [PubMed: 28401897]
107. Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M.2014 iDISCO: a simple, rapid
method to immunolabel large tissue samples for volume imaging. Cell 159:896–910 [PubMed:
25417164]
108. Roberts DG, Johnsonbaugh HB, Spence RD, MacKenzie-Graham A. 2016 Optical clearing of the
mouse central nervous system using passive CLARITY. J. Vis. Exp 112:e54025
109. Romanov RA, Zeisel A, Bakker J, Girach F, Hellysaz A, et al. 2017 Molecular interrogation of
hypothalamic organization reveals distinct dopamine neuronal subtypes. Nat. Neurosci 20:176–
88 [PubMed: 27991900]
110. Saboor F, Reckmann AN, Tomczyk CU, Peters DM, Weissmann N, et al. 2016 Nestin-expressing
vascular wall cells drive development of pulmonary hypertension. Eur. Respir. J 47:876–88
[PubMed: 26699726]
111. Saul MC, Seward CH, Troy JM, Zhang H, Sloofman LG, et al. 2017 Transcriptional regulatory
dynamics drive coordinated metabolic and neural response to social challenge in mice. Genome
Res. 27:959–72 [PubMed: 28356321]
112. Schnittke N, Herrick DB, Lin B, Peterson J, Coleman JH, et al. 2015 Transcription factor p63
controls the reserve status but not the stemness of horizontal basal cells in the olfactory
epithelium. PNAS 112:E5068–77 [PubMed: 26305958]
113. Schwarz MK, Scherbarth A, Sprengel R, Engelhardt J, Theer P, Giese G. 2015 Fluorescent-
protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLOS ONE
10:e0124650 [PubMed: 25993380]
114. Serita T, Fukushima H, Kida S. 2017 Constitutive activation of CREB in mice enhances temporal
association learning and increases hippocampal CA1 neuronal spine density and complexity. Sci.
Rep 7:42528 [PubMed: 28195219]
115. Shah S, Lübeck E, Schwarzkopf M, He TF, Greenbaum A, et al. 2016 Single-molecule RNA
detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.
Development 143:2862–67 [PubMed: 27342713]
Gradinaru et al.
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