Mini-Symposium
Whole-Brain Analysis of Cells and Circuits by Tissue
Clearing and Light-Sheet Microscopy
Tomoyuki Mano,
1,2
*
X
Alexandre Albanese,
4
*
X
Hans-Ulrich Dodt,
8,9
X
Ali Erturk,
10,11,12
X
Viviana Gradinaru,
13
Jennifer B. Treweek,
13,14
Atsushi Miyawaki,
15,16
Kwanghun Chung,
4,5,6,7,17
and
X
Hiroki R. Ueda
2,3,18
1
Department of Information Physics and Computing, Graduate School of Information Science and Technology,
2
International Research Center for
Neurointelligence, UTIAS,
3
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan,
4
Institute for Medical Engineering and Science,
5
Picower Institute for Learning and Memory,
6
Department of Chemical Engineering,
7
Department of Brain
and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139,
8
Department of Bioelectronics, Vienna University
of Technology, FKE, 1040 Vienna, Austria,
9
Center for Brain Research, Medical University of Vienna, 1090 Vienna, Austria,
10
Institute for Stroke and
Dementia Research, Klinikum der Universita
̈t Mu
̈nchen, Ludwig Maximilians University of Munich, 80539 Munich, Germany,
11
Graduate School of
Systemic Neurosciences, 80539 Munich, Germany,
12
Munich Cluster for Systems Neurology, 81377 Munich, Germany,
13
Division of Biology and Biological
Engineering, California Institute of Technology, Pasadena, California, 91125,
14
Department of Biomedical Engineering, University of Southern California,
Los Angeles, California 90089,
15
Laboratory for Cell Function and Dynamics, Center for Brain Science,
16
Biotechnological Optics Research Team, Center for
Advanced Photonics, RIKEN, Wako-City, 351-0198 Saitama, Japan,
17
Broad Institute of Harvard University and MIT, Cambridge, Massachusetts 02142,
and
18
Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 565-0871 Suita, Japan
In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for
high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-
Symposium presented at the Society for Neuroscience’s 2018 annual meeting.
Introduction
Microscopic analysis of tissues reveals that intricate organization
of cells underlies biological function. Tissues are not translucent
against visible light, so it is impossible to image far beyond the
surface. Thus, conventional tissue imaging applies a microtome
to cut samples into thin sections before staining with dyes and
antibodies to visualize cells. Inferring 3D structure from thin
sections is often problematic, and requires minimal sample dis-
tortion and precision alignment of serial sections.
The analysis of large 3D volumes is necessary for mapping the
connections of far-reaching neurons inside the brain and deter-
mining the nature of cellular interactions underlying proper
function and behavior. In recent years, several techniques have
emerged to achieve optical transparency and enable high-
resolution microscopy of thick tissue sections and whole organs.
These techniques use different strategies to reduce light scattering
in tissues and improve image sharpness.
Scattering occurs in tissues due to light’s heterogeneous inter-
action with different molecules, subcellular structures, mem-
branes, and cell populations inside the tissue. For example, the
interface between a cell’s lipid membrane and the cytoplasm
causes a significant drop in refractive index (RI). Heterogeneity at
the scale of molecules, cells, and tissues contribute to light scat-
tering and requires homogenization via tissue clearing to increase
overall light penetration (
Tainaka et al., 2016
;
Treweek and Gra-
dinaru, 2016
). Combining tissue clearing with light-sheet fluo-
rescence microscopy (LSFM) has paved the road for current
whole-brain imaging by eliminating out-of-focus excitation
(hence reduced background level and greatly preventing photo-
bleaching), and by accelerating image acquisition.
Tissue clearing originated in the early 20th century,
when
Werner Spalteholz experimented with high RI organic sol-
vents (
Spalteholz, 1914
). Organic solvent-based clearing ho-
mogenizes a tissue’s RI by the removal of highly scattering
lipids and the displacement of water by high RI solvents.
Building on this approach, Dodt’s group revitalized organic
solvent-based tissue clearing for the modern era of neurosci-
ence by coupling it with a new optical imaging method that
Received Aug. 31, 2018; revised Sept. 27, 2018; accepted Sept. 27, 2018.
The Dodt laboratory thanks Dr. Saiedeh Saghafi and Dr. Klaus Becker for aspheric optics-based LSFM work, and
the Austrian science funding agency FWF and the German Hertie foundation for support; the Ertu
̈rk laboratory
thanks Ruiyao Cai for uDISCO figure and Synergy Excellence Cluster Munich (SyNergy), Fritz Thyssen Stiftung, and
DFG for support; the Miyawaki laboratory thanks Dr. Hiroshi Hama for Sca
l
eS figure and Grant-in-Aid for Scientific
Research on Priority Areas (JSPS KAKENHI), the Human Frontier Science Program, and the Brain Mapping by Inte-
grated Neurotechnologies for Disease Studies (Brain/MINDS) for support; the Ueda laboratory thanks Tatsuya Mu-
rakami for offering picture materials for CUBIC figure, and Brain/MINDS (AMED/MEXT), the Basic Science and
Platform Technology Program for Innovative Biological Medicine (AMED/MEXT)and Grant-in-Aid for Scientific Re-
search (S) (JSPS KAKENHI) for support; the Chung laboratory thanks The Packard Award, the McKnight Foundation
andtheNIH(1-DP2-ES027992)for
support;theGradinarulaboratorythanksSripriyaRavindraKumar,GerardM.
Coughlin, Rosemary Challis, and Collin Challis for CREATE and VAST images, Min Jee Jang for HCR images, and
J. Ryan Cho for GCaMP imaging, and the NIH BRAIN Initiative and NIH Director’s Office and NSF Neuronex for
support.
The authors declare no competing financial interests.
*T.M. and A.A. contributed equally to this work.
Correspondence should be addressed to either of the following: Dr. Hiroki R. Ueda, Department of Systems
Pharmacology, Graduate School of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan, E-mail:
uedah-
tky@umin.ac.jp
; or Kwanghun Chung, Massachusetts Institute of Technology, Cambridge, MA 02139, E-mail:
khchung@mit.edu
.
https://doi.org/10.1523/JNEUROSCI.1677-18.2018
Copyright © 2018 the authors 0270-6474/18/389330-08$15.00/0
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38(44):9330 –9337
improves the resolution and field-of-view using LSFM (
Fig. 1
;
Dodt et al., 2007
;
Ertu
̈rk et al., 2012
;
Saghafi et al., 2014
). This
technique allows rapid imaging of larger volumes than with
conventional point scanning microscopy. Erturk’s group de-
veloped uDISCO method (
Fig. 2
) to image whole-mouse
bodies (
Pan et al., 2016
) and recently vDISCO method, a
whole-mouse immunolabeling method to amplify the signal
two orders of magnitude for detecting single cells in cleared
mouse bodies through intact bones and muscles (
Cai et al.,
2018
). vDISCO enabled construction of the first whole-mouse
neuronal connectivity map, study brain trauma effects in pe-
ripheral nerves, and image meningeal vessels and their cellular
contest through intact skull.
Some techniques have replaced organic solvents with aqueous
solutions to improve preservation of endogenous reporter pro-
teins and simplicity of experimental handling, since a urea-based
clearing reagent ScaleA2 was developed (
Hama et al., 2011
). To
date, various mixtures and protocols have emerged that use aque-
ous chemicals for whole-tissue clearing. Miyawaki’s group has
additionally developed a urea/sorbitol-based clearing method
called Sca
l
eS (
Hama et al., 2015
) to achieve mild clarification
where synaptic ultrastructure, observed by electron microscopy,
is well preserved. Sca
l
eS was capable of imaging A
plaques in
rodent models of Alzheimer’s disease and human clinical samples
(
Fig. 3
). Ueda’s group has developed a clearing method called
CUBIC (clear, unobstructed brain/body imaging cocktails and
Figure 1.
The optical resolution of light-sheet microscopy is determined by both detection objective NA and thickness of the illuminating laser sheet. In conv
entional light-sheet
microscopy, there is a fundamental tradeoff between light-sheet thickness and the field-of-view; when a thin laser sheet is illuminated (hence a hig
her axial resolution), the effective
imaging area (determined by Rayleigh length) must decrease rapidly. To overcome this tradeoff, one approach is to use a beam that is generated by a sequ
ence of aspheric lenses (
Saghafi
et al., 2014
).
A
, The optical design of this light-sheet generator, along with the measurements of the beam profile along the
x
-
y
plane for confirmation of the extended Rayleigh range.
Each optical element in the design are as follows: (a) first aspheric condenser lens, (b) Powell lens, (c) second aspheric condenser lens, (d) first ac
hromatic cylindrical lens, and (e) second
achromatic cylindrical lens.
B
, A Scheme of the ultramicroscopy setup: (1) 488 nm laser; (2) light-sheet generator optics, duplicate arms are placed on the left and right side of the
specimen container; (3) specimen container with quartz windows; (4) detection arm with an exchangeable objective; and (5) scientific grade CCD came
ra. The objective for imaging is
dipped into the clearing solution from above so that the objectives can be exchanged easily for change of magnification.
C–E
, Using a custom-made ultramicroscope equipped with the
novel light-sheet generator, a cleared fruit-fly brain was imaged and reconstructed in 3D. A virtual cross-section through the head is presented in
D
. om, Ommatidia; m, medulla; lo,
lobula. A virtual cross-section through the thorax is presented in
E
. oe, Esophagus; pv, proventriculus (cardia).
F
,
G
, The hippocampus area of Thy1-EGFP-M mouse brain was dissected and
cleared, and imaged with ultramicroscopy. Close-up with 10
magnification (
F
) and 20
magnification (
G
) are shown. Scale bars:
F
,
G
, 100
m.
Mano, Albanese et al.
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Tissue Clearing and Light-Sheet Microscopy
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• 9331
computational analysis;
Susaki et al., 2014
,
2015
;
Tainaka et al.,
2014
;
Kubota et al., 2017
) and has recently reported controlled
expansion of cleared mouse brains using CUBIC-X (
Fig. 4
;
Mu-
rakami et al., 2018
). Physical expansion of a sample (or swelling)
enables imaging of subcellular structures across the entire mouse
brain. Using LSFM, authors generated a point-based mouse brain
atlas (called CUBIC-Atlas) with single-cell annotation, and
tracked postnatal subregion development in mice.
Another important innovation in tissue imaging and phe-
notyping is the development of technologies that engineer
tissue physicochemical properties, tissue-molecular interac-
tion, and molecular transport within intact tissue. Chung’s
group has developed a suite of technologies that engineer tis-
sue properties to maximally preserve tissue information using
synthetic polymer (e.g., CLARITY, MAP) or crosslinker-based
tissue reinforcement strategies (e.g., SWITCH,
Fig. 5
;
Chung
Figure2.
uDISCOenablesinvestigationoflong-rangeneuronalconnectionsspanningtheentiremousebodyandentireCNSinunprecedenteddetail(
Panetal.,2016
).
A
–
D
,TheentireCNS(whole
brain and spinal cord) of a Thy1-GFP-M mouse was cleared by uDISCO and imaged by LSFM. Because of the superior preservation of the fluorescent protein s
ignals achieved by uDISCO, detailed
neuronal structures are clearly visible. Zoom-in view of the brain hemisphere and spinal cord within boxed region in
A
are shown in
B
and
D
, respectively. Zoom-in view of
B
is shown in
C
, where
individual pyramidal cell bodies and dendrites are clearly resolved. Scale bars:
A
,10mm;
B
, 1 mm;
C
, 100
m;
D
, 1 mm.
E
–
J
, AAV2-Syn-GFP was transduced in the right motor cortex of mice, and
AAV2-Syn-RFP in the left motor cortex. The brain was then cleared by uDISCO and imaged with LSFM. Fluorescence expression of the virally delivered pro
teins was detectable in the brain and
throughout the intact spinal cord (
F
–
J
). Details of neuronal extensions of boxed regions in
F
are shown in
H
(thalamus and midbrain),
I
(cervical), and
J
(thoracic spinal cord regions). Arrowheads
in
G
indicate decussation of the descending motor axons. Scale bars:
F
, 5 mm;
G
, 2 mm;
H
–
J
, 1 mm.
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Mano, Albanese et al.
•
Tissue Clearing and Light-Sheet Microscopy
et al., 2013
;
Murray et al., 2015
;
Ku et al., 2016
). They have also
developed MAP (magnified analysis of proteome) that
expands entire brains fourfold linearly while preserving 3D
proteome for super-resolution molecular imaging. When the
transport of reagents into a large volume is diffusion-limited,
chemical reactions are slow and uneven. To achieve uniform
tissue preservation and staining, the Chung group has devel-
oped SWITCH to control a wide range of chemical reaction
kinetics. In addition, they have developed stochastic electro-
transport (
Kim et al., 2015
) that accelerates lipid removal and
tissue staining to substantially improve the throughput of in-
tact tissue phenotyping approaches. Together, these technol-
ogies enable rapid, integrated, multiscale phenotyping of
neuronal circuits.
Gradinaru’s group has developed hydrogel-tissue chemistry
(HTC)-based strategies to stabilize endogenous fluorescent
proteins and tissue biomolecules during successive rounds of
clearing, RNA labeling
via
single-molecule fluorescence
in situ
hybridization or hybridization chain reaction (HCR), immuno-
histochemistry, and imaging at depth (
Fig. 6
;
Yang et al., 2014
;
Treweek et al., 2015
;
Shah et al., 2016
;
Greenbaum et al., 2017a
,
b
).
Recent work on viral vector engineering has led to the generation of
novel AAV capsids with enhanced tropism for specific cell types, and
with the ability to cross the blood–brain barrier when delivered sys-
temically (
Deverman et al., 2016
;
Chan et al., 2017
). These vectors
permit noninvasive gene transfer and tunable multicolor labeling of
discrete cell populations across the CNS and PNS.
This sampling of current work demonstrates how the breath-
taking complexity of the human brain and brains of other
preclinical models continues to inspire technological advance-
ments in whole-tissue clearing, fluorescent labeling, microscope
design, and analysis of large information-rich datasets. These
Figure 3.
Three-dimensional immunohistochemistry enables analysis of clinical samples, where transgenic or viral labeling strategies cannot be used. Sca
l
eS and AbSca
l
e protocols ensure
reliable, reproducible, and homogeneous penetration of antibodies while conserving tissue ultrastructure (
Hama et al., 2015
).
A
–
C
, A 2-mm-thick brain slice from an Alzheimer’s disease (AD) model
mousewasstainedwithAlexaFluor488-6E10(green)andAlexaFluor546-Iba1(red)usingtheAbSca
l
eprotocol,tovisualizethedistributionofA
plaquesandmicroglia.High-magnificationimages
allow quantification of the distance between A
plaques and microglia in resting or activated states (
B
). Two representative A
plaques and microglia are shown, along with the distribution of
activatedandrestingmicrogliadistances(
C
).Scalebars:
A
–
C
,50
m.
D
,A3DblockofcerebralcortexfromADmodelmousewasstainedwithAlexaFluor488-6E10(green)andTexasRedlectin(red),
to reveal the distribution of A
plaques and blood vessels. Scale bar, 50
m.
E
–
J
, A single plaque was tracked at multiple scales by successive imaging with light (LM) and electron microscopy (EM).
A 2-mm-thick slice from AD model mouse brain was cleared and stained by AbScale (
E
,
F
). After restoration and re-fixation, 50
m sections were made, followed by DAB staining with secondary
antibody (
H
,
I
). Finally, 70-nm-thick ultrathin sections were imaged with TEM. The Sca
l
eS protocol performs excellently in preserving the ultrafine structures at nanometer scale. Scale bars:
F
,
G
,1
mm;
H
,
I
,50
m;
J
,2
m.
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Tissue Clearing and Light-Sheet Microscopy
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Figure 4.
To acquire a fundamental understanding of tissue clearing by hydrophilic reagents, Ueda’s group strategically screened
1600 chemicals, and identified key chemical properties that
contribute to the clearing. These include salt-free amine with high octanol/water partition-coefficient (logP) for delipidation,
N-
alkylimidazole for decoloring, aromatic amide for RI matching, and
protonation of phosphate ion for decalcification (
Tainaka et al., 2018
). By combining these insights, it is now possible to rationally design a new family of CUBIC protocols that are optimized, for
example,forlargeprimateandhumantissues.Further,Ueda’sgroupdevelopedtheCUBIC-Xprotocol,afluorescent-protein-compatible,whole-org
anclearingandhomogeneousexpansionmethod
(
Murakami et al., 2018
), which can be combined with high-resolution LSFM to enable subcellular resolution imaging and digital identification of all cell nuclei in the adul
t mouse brain.
A
,
CUBIC-X-treated whole mouse brain (2.2-fold linear expansion) was stained with propidium iodide, and was imaged with high-resolution LSFM (NA
0.6). Whole-brain 3D rendering (center) and
zoom-in images of representative brain regions (side panels with two-step magnification) are shown. Each individual cell nuclei (and even its conde
nsation pattern in some cells) were clearly
resolved across all brain regions, which were automatically detected by GPU-based high-speed algorithm. This allowed construction of the CUBIC-At
las, a mouse brain atlas comprising an ensemble
of
72 million cells. Scale bars: 50
m (after normalizing the sample expansion).
B
, Using a new CUBIC protocol optimized for preserving fluorescent protein (FP) signals (
Tainaka et al., 2018
), a
virtual-multiplex imaging of whole mouse brain was demonstrated. Seven individual brains with genetically encoded FPs were registered onto the ref
erence brain, which revealed the brain-wide
distribution of unique cell types; pyramidal neurons (Thy1-YFP), astrocytes (Mlc1-YFP), oligodendrocytes (Plp-YFP), dopaminergic neurons (Th-
EGFP), serotonin receptor 5B (Htr5b-YFP), and
dopamine receptor D1 (Drd1-mVenus). Scale bars: macro view, 1500
m; zoom-in view, 500
m.
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Tissue Clearing and Light-Sheet Microscopy
Figure 5.
A
, CLARITY-processed mouse brains were stained with SYTO 16 (green), tomato lectin (red), and anti-histone H3 antibodies (white). Compared with stan
dard diffusion (top row), Stochastic
electro-transport (SE) (bottom row;
Kim et al., 2015
) accelerates tissue
labeling with fluorescent markers by applying a rotational electric field. SE achieves complete and uniform staining of an adult mouse
brain in a single day. Scale bar, 1 mm.
B
, SWITCH (
Murray et al., 2015
) is a method that enables uniform tissue processing for large-scale tissues. Molecule transport is decoupled from its interaction with the
sample. For example, DiD lipid dye under standard diffusion produces incomplete staining mostly saturated at the tissue surface (left). In SWITCH, t
he reactivity of fixatives, dyes, and antibodies is suppressed in
SWITCH-OFF buffer to allow the probes to readily diffuse deep into the tissue. For DiD, OFF buffer contains a low concentration of SDS detergent to bloc
k lipid binding until the tissue is saturated. Then, transfer
to “ON buffer” DiD dye binds to lipids (right). SWICTH technique produces rapid and uniform tissue staining compared with standard diffusion. Scale b
ar, 200
m.
C
, Tissue processing in the Chung laboratory
enable multiple rounds of antibody staining. Here, we show a fully coregistered image showing various cell types in human visual cortex.
D
, MAP (
Ku et al., 2016
) physically expands tissues to achieve a fourfold
increase in image resolution. MAP is applicable to intact organs, such as mouse brain. Scale bar, 1 cm.
E
, Left, A 3D render of a cortical sample stained with neurofilament medium unit (NF-M; blue) and GABAB
receptor subunit-1 (GABA
B
R1; red) antibodies; inset, synaptic neurofilaments colocalized with GABAergenic postsynaptic proteins. Middle, a maximum intensity projection
image of calbindin (CB; magenta)
showing dendritic spines. Right, synaptic structures in MAP samples resolved with a presynaptic (bassoon) and postsynaptic marker (homer1) in the c
ortex. Yellow box shows a GFP-positive neuron; white box
highlights the elliptical structures of presynaptic and postsynaptic proteins distributed at a synaptic junction. The intensity of markers along t
he synapse axis confirms separation of presynaptic and postsynaptic
markers. Scale bars:
white, 50
m; gray, 10
m; yellow, 1
m.
F
, SWITCH glutaraldehyde-reinforced human brain tissue shows a coregistered overlay of 9 of 22 different rounds of immunostaining.
Scale bar, 300
m.
G
, Information-rich datasets obtained from multi-round staining can be used for quantitative analysis of 3D volumes.
H
, As a proof-of-concept soma diameter and distance to
nearest blood vessel are compared for six different cell types (*
p
0.05, ***
p
0.001; ANOVA).
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• 9335
Figure 6.
Advancing modern neuroscience endeavors is the development of techniques for modifying chemically-distinct and/or genetically-specified cells
and circuits for high-resolution
imaging at depth. Among these techniques are as follows: (
A
) HTC-based biomolecule stabilization and tissue-clearing (images adapted from
Yang et al., 2014
;
Treweek et al., 2015
;
Treweek and
Gradinaru, 2016
;
Greenbaum et al., 2017a
;
Gradinaru et al., 2018
), (
B
) viral vector engineering [left, Cre recombinase-based AAV targeted evolution (CREATE); right, vector-assisted spectral tracing
(VAST);
Deverman et al., 2016
;
Chan et al., 2017
;
Bedbrook et al., 2018
;
Challis et al., 2018
], and (
C
,
E
) methodologies for cell-profiling
in vivo
and
in situ
. Recent advances in modern microscopy [e.g.,
LSFM and ultramicroscopy (
Dodt et al., 2007
); CLARITY-optimized light-sheet microscopy (
Tomer et al., 2014
); two-photon microscopy and microendoscopy (
Jung et al., 2004
;
Barretto et al., 2011
;
Marshalletal.,2016
)],allowresearcherstovisualizedeeptissuestructuresinreal-time(
C
),andtoimagelargetissuevolumesrapidlyandwithsubcellularresolution[
D
,
E
;HCR(
Choietal.,2014
;
Shah
et al., 2016
;
Greenbaum et al., 2017b
)]. Complementing these technologies are improved strategies for targeting individual cell populations and neuronal circuits, such as through the
use of
genetically engineered animal models (e.g., rodent fluorescent reporter and Cre driver lines;
D
,
E
) or through the systemic delivery (e.g., via retro-orbital (RO) injection) of viral vectors with unique
cell tropism (
Deverman et al., 2016
;
Chan et al., 2017
);
B
, Top left, For CNS targeting, AAV-PHP.eB was used to package single-stranded (ss) rAAV genomes expressing nuclear localized (NLS)
fluorescent reporters (XFP) from cell type-specific promoters for neurons (hSyn1, green), oligodendrocytes (MBP, red), or astrocytes (GFAP, blue
), resulting in brain-wide gene expression upon RO
delivery;bottomleft,forPNStargeting,AAV-PHP.Swasusedtopackagess-rAAVgenomesexpressingXFPsfromeitherneuron-specific(hSyn1)ortyro
sinehydroxylase(rTH)-specificpromoters,with
RO injection of ssAAVPHP.S:rTH-GFP and ssAAV-PHP.S:hSyn1-tdTomato-f (farnesylated) resulting in gene expression throughout TH
-containing cell bodies (green) and nerve bundles (red) of the
myentericandsubmucosalplexusoftheduodenum;imagesadaptedfrom
Challisetal.,2018
)and/ormulticolorlabelingcapabilities(
Chanetal.,2017
;e.g.,VAST;
B
,right;
E
,left),thelatterofwhich
promotes cell-sorting and long-range projection mapping endeavors (
Bedbrook et al., 2018
).
D
,
E
, Likewise, methodologies for labeling RNA transcripts and protein epitopes have been adapted for
use alongside tissue-clearing protocols (
E
;
Shah et al., 2016
;
Greenbaum et al., 2017b
). Here, HCR is uniquely suited to imaging RNA point-labels at depth with up to single-molecule precision (
D
),
and to resisting label loss during immunohistochemistry (
E
) through its use of fluorescence signal amplification and direct
in situ
hybridization-based readout (
Choi et al., 2014
).
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Tissue Clearing and Light-Sheet Microscopy
technological breakthroughs are paving the road to whole-brain
single-cell atlases and connectomes (maps of connected neurons)
for future neuroscientists.
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