Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull

Article abstract of DOI:10.1002/adma.202000364

Visualization of the brain in its native environment is important for understanding common brain diseases. Herein, bright luminogens with remarkable aggregation-induced emission (AIE) characteristics and high quantum yields of up to 42.6% in the solid state are synthesized through facile reaction routes. The synthesized molecule, namely BTF, shows ultrabright far-red/near-infrared emission and can be fabricated into AIE dots by a simple nanoprecipitation procedure. Due to their high brightness, large Stokes shift, good biocompatibility, satisfactory photostability, and large three-photon absorption cross section, the AIE dots can be utilized as efficient fluorescent nanoprobes for in vivo brain vascular imaging through the intact skull by a three-photon fluorescence microscopy imaging technique. This is the first example of using AIE dots for the visualization of the cerebral stroke process through the intact skull of a mouse with high penetration depth and good image contrast. Such good results are anticipated to open up a new venue in the development of efficient emitters with strong nonlinear optical effects for noninvasive bioimaging of living brain.

Full text of DOI:10.1002/adma.202000364

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Facile Synthesis of Efficient Luminogens with AIE Features
for Three-Photon Fluorescence Imaging of the Brain
through the Intact Skull
Wei Qin, Nuernisha Alifu, Jacky W. Y. Lam, Yuhan Cui, Huifang Su, Guodong Liang,*
Jun Qian,* and Ben Zhong Tang*
of its traditional near-infrared (NIR,
Visualization of the brain in its native environment is important for
understanding common brain diseases. Herein, bright luminogens with
remarkable aggregation-induced emission (AIE) characteristics and high
quantum yields of up to 42.6% in the solid state are synthesized through
facile reaction routes. The synthesized molecule, namely BTF, shows
ultrabright far-red/near-infrared emission and can be fabricated into AIE
dots by a simple nanoprecipitation procedure. Due to their high brightness,
large Stokes shift, good biocompatibility, satisfactory photostability, and large
three-photon absorption cross section, the AIE dots can be utilized as efficient
fluorescent nanoprobes for in vivo brain vascular imaging through the intact
skull by a three-photon fluorescence microscopy imaging technique. This is
the first example of using AIE dots for the visualization of the cerebral stroke
process through the intact skull of a mouse with high penetration depth and
good image contrast. Such good results are anticipated to open up a new
venue in the development of efficient emitters with strong nonlinear optical
effects for noninvasive bioimaging of living brain.
7
00–1000 nm) excitation and low ten-
[
4]
dency for photobleaching. Through
optical access to the mouse brain by
common
craniotomy,
researchers
can obtain clear images through 2PF
[5]
microscopy. However, even with the
help of a cranial window, 2PF imaging
is generally limited to image the super-
ficial depth due to light scattering of
[
6]
the excitation beam in biosubstrates.
Moreover, such invasive operation is
far from satisfactory in practical appli-
cation owing to the permanent loss
of integral skull that results in a low
survival rate of animal after operation.
Furthermore, the perturbation of native
environment and inflammation of the
brain tissue are unavoidable destruc-
tive interference, which leads to com-
[
7]
promised imaging quality. Therefore,
the observation of brain in its native
Fluorescence imaging provides a new platform for real-time
tracking of complicated biological process with high sensi-
tivity and fast response.[1] Blood vasculature is the crucial
component of the circulatory system[ and the application of
fluorescent nanoprobes for the visualization of blood vessels
of brain in vivo is important to gain insight into the common
diseases of brain.[ Two-photon fluorescence (2PF) microscopy
is commonly utilized for in vivo deep-tissue imaging because
environment with a noninvasive method is highly desirable for
research on brain disease.
[
8]
To solve the abovementioned problems, three-photon fluo-
rescence (3PF) microscopy imaging is developing rapidly as an
2]
[
9]
effective imaging modality. Unlike conventional 2PF imaging,
3PF imaging technique utilizes higher-order nonlinear localized
excitation at NIR-II region (1000−1700 nm) to enable significant
improvement in penetration depth, spatiotemporal resolution,
3]
Dr. W. Qin, Y. Cui, Prof. G. Liang
PCFM and GDHPPC Labs
School of Materials Science and Engineering
Sun Yat-sen University
Guangzhou 510275, China
E-mail: lgdong@mail.sysu.edu.cn
Dr. N. Alifu, Prof. J. Qian
State Key Laboratory of Modern Optical Instrumentations
Center for Optical and Electromagnetic Research
JORCEP (Sino-Swedish Joint Research Center of photonics)
Zhejiang University
Hangzhou 310058, China
E-mail: qianjun@zju.edu.cn
Dr. N. Alifu
College of Medical Engineering and Technology
Xinjiang Medical University
Urumqi 830011, China
Dr. J. W. Y. Lam, Prof. B. Z. Tang
Department of Chemistry
Hong Kong Branch of Chinese National Engineering Research Centre
for Tissue Restoration and Reconstruction
Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong
E-mail: tangbenz@ust.hk
Dr. H. Su
Department of Orthopaedic Surgery
The First Affiliated Hospital of Zhengzhou University
Zhengzhou, Henan Province 450052, China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adma.202000364.
DOI: 10.1002/adma.202000364
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and signal-to-background ratio (SBR).[9] Taking the full advan-
tage of 3PF microscopy imaging, scientists have made original
works based on commercial fluorescent proteins in imaging the
blood vasculature through the intact skull of mouse with high
sensitivity. This is the first example of using AIE dots for the
visualization of cerebral thrombosis process through the intact
skull of mouse with high penetration depth and good image
contrast. Thus, this is expected to inspire new insights into the
development of efficient NIR emitters with an outstanding 3PF
effect for biomedical applications.
[
10]
structure and function of a mouse brain with high contrast.
Despite these advantages, a major limitation of 3PF imaging
is the lack of organic dyes with large three-photon absorption
[
11]
cross section and high fluorescence quantum efficiency.
To date, various types of three-photon fluorescent bioprobes
BBF and BTF were facilely prepared using inexpensive com-
mercial products according to synthetic pathways in Figure 1
and Scheme S1 (Supporting Information). Their chemical
structures were fully confirmed by nuclear magnetic resonance,
high-resolution mass spectrometry, and single-crystal X-ray dif-
fraction (Figures S1−S6, Supporting Information). BBF can be
facilely prepared by a one-step reaction route (Scheme S1, Sup-
porting Information). Incorporation of diphenylamine (DPA)
derivatives into the BBF structure generates a new adduct
abbreviated as BTF (Figure 1A) with strong electron-donating
triphenylamine (TPA) carrying tert-butyl (t-Bu) groups and
electron-accepting fumaronitrile (FN) moiety. Such a donor–
acceptor (D−A) structure is expected to endow BTF with the
FR/NIR emission and distinct multiphoton absorption. More-
over, BTF contains more freely rotatable phenyl rings and t-Bu
groups to favor the consumption of excited-state energy in the
solution state through active intramolecular motions. Such
motions are restricted in the aggregated state, which enables
[
12]
have been developed, including fluorescent proteins, inor-
ganic quantum dots (QDs),[ and organic dyes. Unfortu-
nately, fluorescent proteins are unstable to laser irradiation and
enzyme degradation, while inorganic QDs suffer from irregular
13]
[14]
[
15]
blinking and potential toxicity in biological environment.
On the other hand, organic fluorophores with tunable absorp-
tion and emission wavelengths can circumvent the abovemen-
tioned problems owing to the satisfactory stability and good
compatibility.[ The conventional design of organic multi-
photon absorbing materials for 3PF microscopy imaging is
mainly based on the introduction of strong electron-donating
16]
[
14]
and electron-accepting moieties in the molecular structures.
However, most organic chromophores are constructed from
hydrophobic skeletons and suffer from aggregation-caused
quenching (ACQ) effect in the aggregated state with weakened
fluorescence efficiency and compromised imaging perfor-
mance.[ To overcome the limitations of ACQ chromophores,
organic luminogens with aggregation-induced emission (AIE)
characteristics have been designed and synthesized for different
purposes.[ The active AIE research in the past decade has gen-
erated a large variety of AIE luminogens (AIEgens), and most
17]
[
25]
the excitons to decay radiatively to facilitate the AIE process.
In addition, the twisted TPA moieties and the bulky t-Bu
groups of BTF hinder the formation of strong π–π stacking
interactions. These collective factors enable BTF to show long
wavelength emission and high quantum efficiency.
18]
[
19]
of them are based on tetraphenylethylene (TPE) skeleton.
However, due to the difficulty in delicate molecular design and
chemical synthesis, few AIEgens with large absorption cross
sections are reported for 3PF bioimaging applications. It is
still challenging to design and synthesize efficient AIE lumino-
gens with simple structures for 3PF bioimaging.
BBF and BTF show good solubility in common organic
solvents, such as toluene (Tol), 1,4-dioxane (DO), dichlo-
romethane (DCM), tetrahydrofuran (THF), and ethyl acetate
(EA), but are not soluble in water. The absorption spectra
of BBF and BTF measured in THF are peaked at 345 and
498 nm, respectively (Figure S7, Supporting Information)
and vary slightly by solvent polarity (Figures S8A and S9A,
Supporting Information). The photoluminescence (PL)
spectra of BBF and BTF in solvents with different polarities
are demonstrated in Figures S8 and S9 (Supporting Infor-
mation). When the solvent polarity is increased from non-
polar Tol to polar EA, the PL maximum of BBF varies in a
small extent (≈4 nm). Under the same condition, the emis-
sion of BTF shifts bathochromically by 20 nm from 629 to
649 nm. This reveals that after introduction of DPA into the
molecular structure of BBF, the emission of BTF is signifi-
cantly red-shifted with positive solvatochromism due to the
[
20]
Stroke is a sudden onset disease of brain. It occurs when the
supply of blood to part of the brain is either interrupted or sud-
denly decreased to normally lead to permanent brain damage
or death.[ Thus, its relevant studies are of great importance
for fundamental research and clinical diagnosis.[ Common
nonfluorescence-based methods for brain imaging are based on
21]
22]
bulky and expensive modern instruments, such as computed
tomography and magnetic resonance imaging.[
23]
However,
these methods possess several disadvantages, such as low spa-
tial resolution and high energy radiation.[ On the other hand,
cerebral thrombosis is generally associated with abnormal
conditions in blood flow. Through the use of 3PF nanoprobes
as hemodynamic indicators in blood flow, the detailed pro-
cess of cerebral thrombosis can be effectively visualized and
monitored.
24]
[
26]
remarkable intramolecular charge transfer (ICT) effect.
To investigate the effects of aggregation and solvent polarity
on the emission process of BBF and BTF, their PL spectra in
With these ideas in mind, we have designed and synthe-
sized non-TPE-based luminogens, namely BBF and BTF. These
molecules possess simple structures and are efficient solid-state
AIE emitters with high quantum yields. BTF with efficient far-
red/near-infrared (FR/NIR) emission can be formulated into
AIE nanodots with a high quantum efficiency of 36.1%, a large
Stokes shift, satisfactory biocompatibility, good photostability,
and large three-photon absorption cross section. They can be
utilized as ultrabright fluorescent probes for 3PF imaging of
THF/water mixtures with different water fractions (f ) were
w
measured. BBF emits a weak blue fluorescence in pure THF.
The PL intensity starts to increase at a high water content of
80% and reaches its maximum value at 95% water content,
which is 20-fold higher than that in pure THF (Figure S10,
Supporting Information). BTF, on the other hand, emits a
weak fluorescence with a maximum at 647 nm in pure THF
(Figure 1C). When a small amount of water (fw ≤ 50%) was
added to the THF solution of BTF, its emission is weakened
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A
B
Facile synthesis
6
5
4
3
2
1
0
fw (vol %)
C
90
D
9
8
7
6
0
0
0
0
80
50
40
30
20
10
0
70
0
60
10-50
550
650
750
850
0
20
40
60
80
100
Wavelength (nm)
Water fraction (vol %)
Figure 1. A) Chemical structure and B) synthetic pathway of BTF. C) PL spectra of BTF in THF/water mixtures with different water fractions (f ). D) Plot
w
of the relative PL intensity (I/I ) versus the composition of the THF/water mixtures of BTF. I = emission intensity in pure THF. Solution concentration =
0
0
−
6
1
0 × 10 ꢀ; Excitation wavelength = 500 nm. Inset: fluorescence photograph of BTF powders taken under 365 nm UV irradiation.
and red-shifted to 650 nm due to the typical twisted
intramolecular charge transfer (TICT) effect.[27] TICT is a
common photophysical phenomenon in organic molecules
constructed from electron donating and accepting units. In
polar environment, these molecules undergo rapid intra-
molecular electron transfer from the donor to the acceptor
moiety, and intramolecular D–A conformation change from
a coplanar structure to a twisted conformation.[ However,
the TICT effect and the AIE property are competitive factors
for determination of the PL intensity. Further increment of
the water content (fw ≥ 60%), BTF molecules form nano-
aggregates due to their hydrophobic effect. Such hydrophobic
environment in the nanoaggregates alleviates the TICT effect
and enables the AIE effect more pronounced as the domi-
nant factor, leading to the increased PL intensity of BTF
mixture.[ The higher the water fraction, the stronger will
be the fluorescence intensity. At fw = 90%, the maximum PL
intensity is attained, which is fivefold of that in pure THF
solution (Figure 1D). Clearly, BTF is AIE-active. The same
conclusion is also drawn from the quantitative measurement.
To obtain further insight into the effect of the molecular
structures on fluorescence emission of BBF and BTF, their
sigle crystals were grown and analyzed by X-ray diffraction.
The crystal data and the collection of parameters were sum-
marized in Tables S2–S4 (Supporting Information). As shown
by the crystal structures in Figure S12 (Supporting Informa-
tion), the cyano groups of BTF adopt a trans-conformation.
The large dihedral angle of 177.05° between the cyano group
and the central double bond reveals that they are intrinsically
planar and well conjugated with good π-electron delocaliza-
tion (Figure 2A). On the other hand, the propeller-shaped
TPA unit is twisted from the central olefin plane. The tor-
sion angle between the central double bond and the adjacent
phenyl rings is measured to be 36.31°. The dihedral angles of
the phenyl rings of TPA are calculated to be 66.54°, 56.02°, and
75.45° (Figure 2A). In addition, the t-Bu groups and adjacent
phenyl rings are also nonplanar with torsion angles from
4.71° to 64.43° (Table S5, Supporting Information). BTF packs
in the triclinic crystal system, from which two representa-
tive dimers are extracted to study the molecular packing. It
is interesting to note that the hydrogen atoms of benzene
rings and t-Bu groups participate in multiple CH⋯N and
CH⋯π interactions in BTF crystals (Figure 2B,C). Multiple
strong CH⋯N interactions are present in crystals with short
hydrogen bonding distances (e.g., 2.957, 3.054, and 3.243 Å in
dimer 1 and 2.782, 3.220, and 3.281 Å in dimer 2). Abundant
CH⋯π interactions exist in adjacent molecules with short
bond lengths (e.g., 3.164, 3.362, and 3.381 Å). Such abundant
27]
28]
The fluorescence quantum efficiency (Φ ) of BBF and BTF
F
in pure THF solution is 0.2% and 2.7%, which dramatically
enhances to 39.8% and 42.6%, respectively, in the solid state
(
Table S1, Supporting Information). The lifetimes of BBF and
BTF powders are measured to be 1.9 and 2.0 ns, respectively,
demonstrating the fluorescence nature of the light emission
rather than long-lived delayed fluorescence or phosphores-
cence (Figure S11, Supporting Information).
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Figure 2. A) Single crystal structure of BTF. B,C) Two dimers extracted from the single-crystal structure of BTF with bond lengths. D) Molecular stacking
structures along the a, b, and c directions in BTF crystals.
interactions between adjacent molecules greatly restrict the
molecular motions and efficiently suppress the nonradiative
relaxation of excitons to enable the molecules to emit intense
FR/NIR emission in crystals. It is noteworthy that the coop-
eration of multiple interactions enables the BTF molecules to
arrange in a slipped parallel manner within the crystals along
the a, b, and c directions with intermolecular distances of 7.582,
Information). A similar slipped parallel molecular arrangement
with intermolecular distances of 7.409, 9.561, and 14.750 Å was
observed (Figure S14, Supporting Information).
To understand the electronic distributions and molecular
geometries of BBF and BTF at the molecular level, density
functional theory calculations were carried out (Figure S15,
Supporting Information). The electron clouds of the highest
occupied molecular orbital (HOMO) and the lowest unoccu-
pied molecular orbital (LUMO) of BBF are dispersed on the
whole molecule. In contrast, the HOMO of BTF is contributed
by orbitals of the FN core and the TPA moieties. The electron
clouds of the LUMO, on the other hand, are concentrated on
the 2,3-diphenylfumaronitrile unit. The remarkable difference
11.424, and 13.796 Å, respectively (Figure 2D). Similar to BTF,
BBF also adopts a twisted conformation in the crystal state
owing to the nonplanarity of the t-Bu groups (Figure S13 and
Tables S6–S8, Supporting Information). It also packs in tri-
clinic crystal system, where multiple CH⋯N and CH⋯π
interactions exist in the crystal lattice (Figure S14, Supporting
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Figure 3. A) Illustration of the formulation process of BTF dots using F127 as the polymer matrix. B) Schematic illustration of 1PF, 3PF, and THG
processes. C) Nonlinear optical property of BTF in aqueous dispersion measured under 1550 nm fs laser excitation. Inset: THG and 3PF images of
BTF aqueous dispersion in capillary glass tube. D) Power dependence relationship of the fluorescence intensity of the BTF dots under 1550 nm fs laser
excitation. E) 3PF intensity of the BTF dots at different time points under continuous laser irradiation at 1550 nm.
in electron distribution of HOMO and LUMO of BTF reveals
an intrinsic ICT feature, which is consistent with the result
from Figure S9 (Supporting Information).
shell. The size of the BTF dots in aqueous solution was meas-
ured by dynamic light scattering (DLS) with a hydrodynamic
diameter of 128 nm (Figure S16A, Supporting Information),
which was further confirmed by scanning electron micro-
scopy (SEM). The BTF dots display an absorption maximum
at 500 nm in aqueous solution, which well fits with the com-
mercial laser excitation at 515 nm. The PL spectrum of the BTF
dots is peaked at the red region (645 nm) and well extends to
the NIR region (800 nm) (Figure S16B, Supporting Informa-
tion). The BTF dots enjoy a larger Stokes shift (>130 nm) than
those of conventional organic dyes (normally <25 nm), which is
The FR/NIR emission of BTF with a high Φ in the aggre-
F
gated state prompts us to study its bioimaging application. To
endow the hydrophobic BTF with good water dispersibility in
aqueous media, amphiphilic polymeric Pluronic F-127 was
applied as biocompatible encapsulation agent to formulate
BTF dots (Figure 3A). In aqueous solution, the lipophilic part
of Pluronic F-127 and AIE molecules form the nanodot core.
While the hydrophilic part extends into water to form outer
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beneficial for high-contrast bioimaging applications due to the
lesser tendency for emission quenching by self-absorption.
of the BTF dots was investigated by monitoring their PL change
upon continuous laser irradiation at 1550 nm (Figure 3E). After
40 min continuous irradiation, the BTF dots maintain more
than 90% of their initial 3PF intensity in PBS and aqueous
suspension. Such good photostability enables long-term bioim-
aging applications.
[
29]
The fluorescence quantum yield (Φ ) of the BTF dots was deter-
F
mined to be 36.1% by using 4-(dicyanomethylene)-2-methyl-6-
(
p-dimethylaminostyryl)-4H-pyran (Φ = 43% in methanol) as
F
standard.
The BTF dots possess a long-term stability shown in
Encouraged by the good 3PF and biocompatibility of the
BTF dots, we first investigated the real-time imaging of mouse
cerebrovascular through a small cranial window using a 3PF
imaging system (Figure S23, Supporting Information). The
representative vasculature images of the mouse brain at pen-
etration depths from 0 to 900 µm are shown in Figure S24
(Supporting Information). The intrinsic THG signal generated
at various penetration depths was also observed in Figure S25
(Supporting Information), which could provide additional
structural information at superficial depths. As shown in
Figure 4A−C, the 3PF and THG signals are well-matched and
this suggests they origin from the same nanoprobe. The combi-
nation of different imaging modes with 3PF and THG enhances
the reliability of deep-tissue imaging. Figure 4D−F shows the
3D high resolution reconstruction vasculature images of the
mouse brain at various penetration depths from 0 to 900 µm.
Careful measurement of the images enables the quantitative
analysis of the full width at half maximum (FWHM) of the indi-
cated tiny capillary, which is equal to 1.68 and 1.80 µm at depths
of 300 and 600 µm, respectively (Figure 4G,H). The SBRs of the
3PF images at depths of 200, 300, 400, and 500 µm are calcu-
lated to be 15.6, 8.9, 5.7, and 5.3, respectively (Figure 4I). The
large SBRs are attributed to the high-order nonlinear optical
effect and the strong 3PF signal of the BTF dots as well as
good penetration capability of the NIR-II excitation light. The
gradual fluorescence loss is attributed to the absorption/scat-
tering of the deep-tissue of mouse brain. Through combination
of images at different penetration depths (Figure 4J), high reso-
lution 3D images are reconstructed from different visual angles
(Figure 4K,L), which provide a general and clear spatial picture
about major blood vasculature networks and details of the tiny
capillaries.
Taking the full advantage of 3PF imaging, the fluorescent
BTF dots are further utilized for practical applications in vis-
ualization of brain blood vessels of mouse with intact skull
in vivo. The noninvasive operation was shown in Figure S26
(Supporting Information), which could avoid the physiolog-
ical alternations and the functional interference of the brain.
Figure 5A−C, on the other hand, shows the 3D high resolu-
tion reconstruction images of the mouse brain vessels, through
which an unambiguous spatial picture of the major vascular
system and the tiny capillary network is provided. The FWHM
of the indicated tiny capillary is calculated to be 0.95 µm
at 200 µm depth, 1.59 µm at 300 µm depth, and 2.08 µm at
400 µm depth (Figure 5D−F). The above results demonstrate
that the AIE dots are promising nanoprobes for in vivo deep-
tissue imaging.
Figure S17 (Supporting Information). Their PL spectra remain
almost unchanged even their suspension solutions in phos-
phate buffer saline (PBS) are stored at room temperature for a
long time. The HeLa cells incubated with BTF dots exhibit no
fluorescence, indicative of little or no endocytosis (Figure S18,
Supporting Information). The cytotoxicity of the BTF dots was
evaluated by their incubation with HeLa cells at different con-
−1
centrations (12.5, 25, 50, and 100 µg mL ) for 24 h (Figure S19,
Supporting Information). The cell viability all remains at above
9
5%, revealing the low cytotoxicity of the dots. As chemical sta-
bility is one of the essential prerequisites for in vivo biological
applications, the BTF dots were dispersed at different pH and
their optical properties were studied as shown in Figure S20
(Supporting Information). No obvious changes in UV and PL
are detected in buffer solutions with different pH. The hydro-
dynamic diameter of the BTF dots in buffer solutions also
maintains at the value of 119−134 nm even the pH is varied
(Figure S21, Supporting Information). All these results suggest
that the BTF dots show high chemical stability, which makes
them promising as fluorescent nanoprobes for in vivo fluores-
cence imaging.
To investigate the nonlinear optical property of the BTF dots,
a measuring system was built as shown in Figure S22 (Sup-
porting Information). Under NIR-II femtosecond (fs) laser
excitation at 1550 nm, three photons of 1550 nm were absorbed
simultaneously in the excitation process followed by the same
radiative decay pathway as that of one-photon process as sug-
gested by the similarity of the obtained PL spectra (Figure 3B).
The bright 3PF of the BTF dots was observed at ≈650 nm
accompanied with a sharp signal peak from the third-harmonic
generation (THG)[ at 517 nm (Figure 3B,C). 3PF and THG
are both high-order nonlinear optical effects. 3PF is approxi-
mate to a fifth-order nonlinear optical effect, while THG is a
third-order nonlinear optical effect. Herein, under 1550 nm
fs laser excitation, the 3PF signal of the BTF dots is much
stronger than their THG signal. Moreover, the 3PF emission
maximum locates in FR/NIR region at 650 nm, which is longer
than that of THG in green region at 517 nm. Therefore, intense
[
33]
30]
3
PF of the BTF dots is more suitable for in vivo bioimaging
due to higher penetration depth and lower light absorption in
biological tissues. The power dependence of 3PF intensity of
the BTF dots on excitation intensity of the 1550 nm fs laser was
plotted. The 3PF intensity of the BTF dots is proportional to the
cubic power of the excitation intensity at 1550 nm (Figure 3D),
which is demonstrative of the main nonlinear optical process of
3
PF. The three-photon absorption cross section (σ ) of the BTF
3
dots was further measured by the three-photon induced fluo-
The blood flow velocity in brain is one of the important
hemodynamic features of brain conditions and can be inves-
tigated using the noninvasive approach through-skull 3PF
imaging. The BTF dots are dispersed in the blood plasma
rather than stained on the red blood cells (RBCs) (Figure S27,
Supporting Information) as indicated by the shadows of RBCs
rescence method. At 1550 nm, the σ of BTF was measured to
3
−
79
6
2
be 2.56 × 10 cm s , which is much higher than that of com-
−
81
6
2 [31]
monly used organic dye Rh6G (6 × 10 cm s ) and some
previously reported dyes.[ The higher σ value of the BTF dots
32]
3
is advantageous for deep-tissue bioimaging. The photostability
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Figure 4. A) THG, B) 3PF, and C) overlay image of the mouse brain vessels at penetration depth of 55 µm. Excitation wavelength: 1550 nm. Scale bar:
00 µm. D−F) In vivo 3D high resolution images of the mouse brain vessels at penetration depth from 0 to 900 µm. G,H) A cross-sectional intensity
1
profile measured along the blue line in (D) and (E). I) Attenuation curve of the fluorescence signal. J) Fluorescence images of the brain vessels at
various penetration depths. Scale bar: 100 µm. K) The side and L) top view of the constructed 3D images of the blood vessels of the mouse brain at
penetration depth from 0 to 900 µm. Scale bar: 100 µm.
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Figure 5. A−C) In vivo 3D high-resolution images of the mouse brain vessels with intact skull at penetration depth from 0 to 400 µm. Excitation wave-
length: 1550 nm. Scale bar: 100 µm. D−F) A cross-sectional intensity profile measured along the yellow line in (A)–(C). G−J) 3PF images of brain blood
vessels of a mouse with intact skull before (G,H) and after (I,J) brain thrombosis. Scale bar: 50 µm. K) A cross-sectional intensity profile measured
along the yellow line in (G) and (I). L) Schematic illustration of the visualization of brain thrombosis process with intact skull based on the AIE dots
through the 3PF microscopy imaging technique.
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flowing with the fluorescent plasma. Through tracking the dis-
tance traveled by the fluorescent spot against time, the average
blood flow velocity (dx/dt) of the indicated capillary was calcu-
lated to be ≈3. 8 mm s− (Figure S28, Supporting Information),
corresponding to a volume blood flow of 1.45 × 10 µL min .
Cerebral thrombosis is a prevalent brain disease and can
induce acute brain injury and even death. Thus, we further uti-
lized the BTF nanodots to monitor the cerebral stroke process
of mouse with intact skull. 3PF imaging of the brain of a mouse
with intact skull was first conducted using AIE dots, through
which the general information on vasculature structure with a
large field of view was obtained (Figure 5G,H) by an infrared
stroke process in mice noninvasively through 3PF microscopy
imaging. These good results will shed light on the development
of efficient solid-state NIR emitters for noninvasive monitoring
disease or dysfunction in brain.
1
−2
−1
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
antireflection low magnification objective. After metabolism for Acknowledgements
several hours, no 3PF signal of BTF dots in the brain blood ves-
W.Q. and N.A. contributed equally to this work. The financial support
sels of mouse was observed (Figure S29, Supporting Informa-
tion). Then a microsurgery was performed on the same mouse
to induce occlusion in the origin of the middle cerebral artery
was partially received from National Natural Science Foundation of
China (21971265, 21374136, 61975172, 61735016, and 21788102), the
Fundamental Research Funds for the Central Universities (18lgpy04 and
17lgjc03), the Zhejiang Provincial Natural Science Foundation of China
(MCA) followed by intravenous injection of the BTF dots. After
(LR17F050001), the Research Grants Council of Hong Kong (C6009-17G),
formation of occlusion of MCA in mouse brain, the indicated
brain blood vessels in the right cerebral hemisphere were invis-
ible in the same location (Figure 5I,J) due to the lack of flowing
blood. Evidently, the process of cerebral thrombosis was clearly
visualized through the intensity change of 3PF signal of the AIE
dots. Same conclusion was drawn through quantitative analysis
of the highlighted lines in the images (Figure 5K). Strong 3PF
signal with a high value of 92.1 from the indicated blood ves-
sels can be effectively monitored in the normal condition of the
brain. After formation of cerebral thrombosis, the 3PF intensity
is dramatically decreased to a low value of 1.1, which is diffi-
cult to distinguish from the background signal. Similar results
induced by occlusion of MCA are also observed at the middle of
cerebral hemisphere of mouse (Figure S30, Supporting Infor-
mation). While the mouse without microsurgery was used as
control under the same 3PF microscopy imaging condition.
No obvious cerebral thrombosis process could be observed
and the Innovation and Technology Commission (ITC-CNERC14SC01).
The authors are grateful to Miss Y. Zhang from Basic Medical Sciences
and Mr. L. Zhu from Interdisciplinary Institute of Neuroscience and
Technology (ZIINT) in Zhejiang University for their help in animal
experiments. The authors are also grateful to Dr. Y. Zhou from School
of Chemistry and Chemical Engineering in Guangxi University for
Nationalities for analysis of single-crystal structure. The procedures
for all the animal experiments were approved by the Institutional
Ethical Committee of Animal Experimentation of Zhejiang University
in China. They were strictly performed in compliance with the rules of
governmental and international guidelines on animal experimentation.
According to requirements for Biosafety and Animal Ethics, all efforts
were made to minimize the number of animals used and their suffering.
Conflict of Interest
The authors declare no conflict of interest.
(Figure S31, Supporting Information). Therefore, the fluores-
cence microscopy images reveal the cause of disease through
the remarkable difference in 3PF intensity of the cerebral
vasculature in complicated central neural system, which signifi-
cantly benefits for understanding the pathogenesis of brain dis-
eases. In recent years, some luminogens have been synthesized
Keywords
aggregation-induced emission, cerebral thrombosis, intact skull, NIR-II
excitation, three-photon fluorescence imaging
Received: January 16, 2020
Revised: March 30, 2020
Published online:
for deep-tissue imaging with good performance by NIR-II laser
excitation.[11b,32,34]
Different from these molecules prepared
by multistep synthesis (normally 5−7 steps) with complicated
chemical structures, the present molecule can be facilely syn-
thesized (2 steps) and possesses a simple chemical structure
and a suitable molecular weight. More importantly, the cerebral
thrombosis process in a living mouse with intact skull was first
visualized noninvasively with high contrast based on ultrabright
AIE dots with remarkable nonlinear optical effect (Figure 5L).
In summary, bright AIE luminogens are facilely synthesized.
The FR-NIR luminogen namely BTF shows a high quantum
efficiency of up to 42.6% in powders. The resulting AIE dots
enjoy high brightness, large Stokes shift, good biocompatibility,
satisfactory photostability, and large three-photon absorption
cross section. They can be utilized as highly efficient fluores-
cent probes for in vivo brain vascular imaging by NIR-II laser
excitation with high penetration depth and image contrast. It is
the first report of using AIE dots for the visualization of cerebral
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