Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Surgery

Full text of DOI:10.1016/j.chempr.2019.07.015

Article
Boosting Fluorescence-Photoacoustic-Raman
Properties in One Fluorophore for Precise
Cancer Surgery
Ji Qi, Jun Li, Ruihua Liu, ...,
Dingbin Liu, Dan Ding, Ben
Zhong Tang
dingd@nankai.edu.cn (D.D.)
tangbenz@ust.hk (B.Z.T.)
HIGHLIGHTS
Fluorescence, photoacoustic, and
Raman properties can be boosted
in one molecule
Preoperative fluorescence/PA
imaging deciphers
comprehensive tumor information
Intraoperative fluorescence/
Raman imaging helps to delineate
tiny residual tumors
The one-for-all organic agent
provides a unique platform for
precise cancer surgery
A one-for-all organic agent for integrated triple-modality imaging-guided cancer
surgery has been developed, in which the fluorescence, photoacoustic (PA), and
Raman properties could be precisely tuned and boosted by tuning the molecular
structure and intramolecular motions. By taking advantage of the merits of each
mode, the organic nanoagent helps to decipher tumor information at different
surgical stages and improve cancer surgery outcomes significantly. The
preoperative fluorescence and PA imaging provide comprehensive information
about tumors, while intraoperative fluorescence and Raman imaging accurately
delineate tiny residual tumors.
Qi et al., Chem 5, 1–21
October 10, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2019.07.015

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Article
Boosting Fluorescence-Photoacoustic-Raman
Properties in One Fluorophore
for Precise Cancer Surgery
Ji Qi,1,5 Jun Li,2,5 Ruihua Liu,2 Qiang Li,3 Haoke Zhang,1 Jacky W.Y. Lam,1 Ryan T.K. Kwok,1
1,4,6,
Dingbin Liu,3 Dan Ding,2, and Ben Zhong Tang
*
*
SUMMARY
The Bigger Picture
High-performance molecular
agent enables maximal diagnosis
and treatment outcomes (e.g.,
cancer surgery). The ideal image-
guided cancer surgery calls for
diverse imaging methods at
different stages of the complex
cancer operation, yet there is no
individual imaging technique
meeting all the requirements
throughout the entire surgery
process. Here, we report a kind of
one-for-all organic agent, in which
the fluorescence, photoacoustic,
and Raman properties can be
simultaneously boosted by tuning
the molecular structure and
intramolecular motion, for triple-
modality imaging-guided precise
cancer surgery. The preoperative
fluorescence and photoacoustic
imaging help to decipher
Multi-modality organic imaging agents hold great potential for comprehensive
disease diagnosis and treatment by taking advantage of each mode, yet the
development is less than satisfied. Here, we develop the one-for-all agent
with boosted fluorescence, photoacoustic (PA), and Raman properties by tuning
the molecular structure and intramolecular motion for triple-modality imaging-
guided cancer surgery. Compared with the other analogs, compound OTPA-
TQ3 can generate the highest fluorescence, PA, and Raman (2,215 cmÀ1) signals,
which presents the first organic molecule integrating with these optical imaging
modalities. In vivo experiments with the OTPA-TQ3-based nanoagent help to
decipher tumor information at different surgical stages and improve cancer sur-
gery outcomes. The pre-operative fluorescence and PA imaging are capable of
providing comprehensive tumor information, while the intraoperative fluores-
cence and Raman imaging delineate tumor margins in a sensitive high-contrast
manner. This one-for-all organic molecular agent allows for accurate cancer im-
aging and resection, rendering great promise for integrated multi-modality im-
aging applications.
INTRODUCTION
Complete removal of tumor tissues is decisively important for prolonging the pa-
tients’ lifetime and even thoroughly curing cancers.1,2 Image-guided cancer surgery
that aims to employ molecular imaging techniques to help the surgeon catch and re-
move all the tumor nodules thus arise at the historic moment and has been used clin-
ically in recent years.3–5 Ideal image-guided cancer surgery calls for diverse imaging
methods at the different stages of cancer operation.6,7 Before surgery, the basic in-
formation such as the size, number, and location of the tumors inside the body has to
be confirmed, which requires the imaging technique possessing excellent spatial
resolution and high sensitivity.8,9 Nevertheless, during surgery, the surgeon faces
several conundrums such as identification of tiny tumors (e.g., < 1 mm) and the
margin between normal and tumorous tissues, as well as judgment of whether res-
idue tumors exist after main tumor resection.10,11 Hence, the imaging technique
with superb sensitivity and prominent signal-to-background ratio is highly desirable
intraoperatively. Unfortunately, there is no individual imaging technique meeting all
the requirements throughout the entire surgery process. Taking versatile optical im-
aging techniques for examples, fluorescence imaging possesses excellent sensi-
tivity, but the penetration capability and spatial resolution are limited.12,13 Photoa-
coustic (PA) imaging can provide large penetration depth beyond the optical
diffusion limit meanwhile maintaining high spatial resolution yet the sensitivity is
comprehensive tumor
information, while intraoperative
fluorescence and Raman imaging
delineate the tumor margins
accurately. This work highlights a
new strategy to develop multi-
functional organic agents with
one-for-all signature for
comprehensive cancer surgery,
rendering great promise for
clinical translation.
Chem 5, 1–21, October 10, 2019 ª 2019 Elsevier Inc.
1

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
not that satisfied.14–17 Besides, Raman imaging is a complementary optical imaging
technique, featuring a cell-silent region (1,800–2,800 cmÀ1), which permits high-
contrast imaging with zero interference of biological background and thus holds
great potential for precise intraoperative inspection of residual tumors.18–20 As a
consequence, the combined advantages of fluorescence, PA, and Raman imaging
modalities decidedly promote the cancer surgical outcomes, which calls for highly
efficient fluorescence-PA-Raman triple-modality imaging agents.
At present, the most commonly used strategy for preparing multi-modality imaging
agents is to combine various components into one platform (all-in-one strategy) to
make use of their respective functions.21–25 Although effective, this method is hin-
dered by the complicated composition, reduced reproducibility and uncertain
pharmacokinetics, hence less accessible for clinical translation.26,27 Alternatively,
one-for-all organic agents with multiple imaging capacities in one molecule have
received more attention due to the lower complicity, simpler preparation, defined
structure, and far better reproducibility than the all-in-one agents.28,29 To our
knowledge, however, one-for-all organic agents with simultaneous fluorescence,
PA, and Raman imaging capabilities have been scarcely reported since it is consid-
erably hard to develop a molecular guideline to enable and boost every optical im-
aging efficacy at the same time. All the three imaging modalities stem from external
light excitation, e.g., fluorescence and PA are associated with the radiative and
nonradiative decay pathways from the excited state to the ground state, respec-
tively, while Raman signal originates from the relaxation of virtual energy state
(Scheme S1). The radiative and nonradiative pathways are expected to be greatly
impacted by the molecular motions (e.g., rotation, vibration, and twisting) that
could consume the excited-state energy, and Raman signal is a kind of molecular
vibration and rotation.18,30 These processes are competitive to each other, so it
is really difficult to simultaneously boost them in one organic molecule. Since the
aforementioned three optical imaging capacities and the corresponding photo-
physical processes are closely related to intramolecular motions, we wonder
whether organic molecules with rotation and vibration units can serve as a high-per-
1Department of Chemistry, The Hong Kong
Branch of Chinese National Engineering
forming fluorescence-PA-Raman triple-modality imaging agent, which has never
been explored before.
Research Center for Tissue Restoration and
Reconstruction, Institute for Advanced Study, and
Department of Chemical and Biological
Engineering, The Hong Kong University of
Science and Technology, Clear Water Bay,
Kowloon, Hong Kong, China
Molecular motions that play a pivotal role in determining many fundamental
physical and chemical processes hold stupendous potential for advancing the
biomedical field, as controllability and utilization of dynamic molecular motions
can lead to functional or smart materials with accurately tunable properties,
benefiting to precision medicine and personalized theranostics.31–33 For example,
our recent studies about the aggregation-induced emission (AIE) luminogens
clearly demonstrate that intramolecular motions contribute greatly to the photo-
physical energy dissipation pathways and that restriction of intramolecular mo-
tions significantly promotes fluorescence in the aggregates.34–36 However, so
far, there have been few reports on the design of multi-functional bioagents
for precision medicine by fully taking advantage of active intramolecular motion
after light absorption, as it is indeed challenging to command and unify micro-
cosmic molecular dynamic behaviors to determine macroscopic biomedical
function and optimize the efficacy. This motivates us to develop advanced optical
bioprobes with biomedical effectiveness not achievable by currently available
ones.
2State Key Laboratory of Medicinal Chemical
Biology, Key Laboratory of Bioactive Materials,
Ministry of Education, and College of Life
Sciences, Nankai University, Tianjin 300071, China
3College of Chemistry, Research Center for
Analytical Sciences, State Key Laboratory of
Medicinal Chemical Biology, and Tianjin Key
Laboratory of Molecular Recognition and
Biosensing, Nankai University, Tianjin 300071,
China
4Center for Aggregation-Induced Emission,
SCUT-HKUST Joint Research Institute, State Key
Laboratory of Luminescent Materials and
Devices, South China University of Technology,
Guangzhou 510640, China
5These authors contributed equally
6Lead Contact
*Correspondence: dingd@nankai.edu.cn (D.D.),
tangbenz@ust.hk (B.Z.T.)
In this contribution, we report for the first time that boosted fluorescence, PA,
and Raman properties can be integrated into one organic fluorophore, in which
https://doi.org/10.1016/j.chempr.2019.07.015
2
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Scheme 1. Schematic Illustration of the One-for-All Organic Agent for Image-Guided Surgery
The one-for-all agent can be used for comprehensive preoperative fluorescence and PA imaging of
tumors, and intraoperative fluorescence and Raman imaging of tiny residual tumors to boost cancer
surgery outcomes. ex, excitation light.
these optical imaging properties are significantly impacted by the molecular
structure and intramolecular motion. We start with the design and synthesis of
a series of near-infrared (NIR)-absorbing organic fluorophores (named OTPA-
TQ1-3) with different substituted units (phenyl, phenyl-alkyne, and phenyl-
alkyne-phenyl, respectively, Scheme S2), followed by significant enhancement
of the water solubility of these hydrophobic molecules via formulation into well-
dispersed nanoparticles (NPs). Compared with the other two analogs, compound
OTPA-TQ3 with the large phenyl-alkyne-phenyl substitutes can generate the
highest fluorescence, PA, and Raman (in cell-silent region) signals, which presents
the first organic molecule integrating with these optical imaging modalities.
Lastly, the feasibility of such one-for-all multi-functional OTPA-TQ3 NPs in intri-
cate biomedical application such as image-guided cancer surgery was investi-
gated. In vivo study reveals that precise cancer surgical treatment can be
achieved under the guidance of OTPA-TQ3 NPs permitting preoperative NIR
fluorescence and PA imaging as well as intraoperative fluorescence and
Raman imaging (Scheme 1). This study suggests that the one-for-all organic
agent with optimized imaging performance holds great promise for practical
applications.
RESULTS
Synthesis, Characterization, and Photophysical Properties
The push-pull or donor-acceptor (D-A) approach, in which the electron-donating
and electron-withdrawing moieties are alternatively arranged along the conjugated
structure, is effective to reduce the bandgap.37,38 In this work, we employ alkoxy-
substituted triphenylamine (OTPA) as the donor and thiadiazoloquinoxaline (TQ)
as the acceptor to construct the NIR chromophores. The strong D-A interaction en-
sures efficient intramolecular charge transfer (ICT), which is beneficial to realizing
small electronic band gap and thus NIR absorption and emission. The octyloxy sub-
stitutes in triphenylamine unit could increase the electron-donating nature, as well as
endow the resultant compounds with good solubility and processability. Moreover,
the long side chains are designed to retain some room between the conjugated
backbones, which is favorable for intramolecular motions in aggregated state.39,40
A series of analogs with different substituted groups (i.e., phenyl, phenyl-alkyne,
and phenyl-alkyne-phenyl) in TQ core have been synthesized (Figure 1A) to investi-
gate their influence on the optical imaging properties in terms of fluorescence, PA,
Chem 5, 1–21, October 10, 2019
3

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 1. Structure, Calculation, and Photophysical Properties of OTPA-TQs
(A and B) Shown in (A) are chemical structures and in (B) are optimized molecular geometries of OTPA-TQ1-3 in the ground state.
(C) Absorption spectra of OTPA-TQ1-3 in THF (20 mM).
(D) PL spectra of OTPA-TQ3 (20 mM) and (E) aAIE value versus water fraction (fw) in THF-water mixtures. aAIE is defined as the ratio of the PL intensities of
the compounds in THF-water mixtures and pure THF (fw = 0).
and Raman. The synthetic route to OTPA-TQ molecules is presented in Scheme S2.
Key synthesis steps include Stille cross-coupling reaction between the tributyltin-
substituted OTPA (5) and the dibromo-molecule (6) to produce the dinitro-com-
pound (7) as a dark purple solid, followed by the iron-catalyst nitro reduction and
subsequent cyclization with benzils to obtain the final compounds. The benzil deriv-
atives (9–11) with different substitutes were prepared in advance. The intermediates
and final compounds are characterized by 1H NMR, 13C NMR, and high-resolution
mass spectrum (HRMS). Detailed synthesis processes and characterizations are
shown in the Supplemental Information (Figures S1–S22).
To gain in-depth understanding about the molecular geometry, we conducted den-
sity functional theory (DFT) calculations with Gauss 09 program at B3LYP/6-31G(d)
4
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
level. In order to simplify the calculations, we abbreviated the substituted aliphatic
groups as methoxy units. The optimized molecular geometries of the compounds in
ground state are presented in Figure 1B. They possess similar geometry with largely
twisted intramolecular rotors, which would dissipate the excited-state energy as the
free high-frequency rotation in solution and be beneficial for realizing the AIE effect
and bright emission in aggregation and solid state. As shown in Figures S23–S25, the
wave function of highest occupied molecular orbital (HOMO) distributes along both
OTPA and TQ units, whereas the lowest unoccupied molecular orbital (LUMO) is
mostly localized on electron-deficient TQ core, indicating D-A characteristic and effi-
cient ICT.41 Further study on other molecular orbitals (HOMO-1, HOMO-2, HOMO-
3, and HOMO-4 and LUMO+1, LUMO +2, LUMO +3, and LUMO +4, Figures S26–
S28) suggests that the two phenyl-alkyne-phenyl groups in OTPA-TQ3 enable better
conjugation and electronic transition.
The absorption spectra of compounds OTPA-TQ1-3 in tetrahydrofuran (THF) show
strong absorption in the range of 600–750 nm (Figure 1C), matching well with the
excitation light sources of commercially available fluorescence and PA imaging
systems. The maximal absorption coefficients increase in the order of OTPA-
TQ2 < OTPA-TQ1 < OTPA-TQ3, which is in the same trend as the calculated oscil-
lator strengths (Table S1). The higher absorption coefficient of OTPA-TQ3 would be
favorable for efficient light excitation. The photoluminescence (PL) maxima of com-
pounds OTPA-TQ1-3 in THF solution are in sequence at 894, 911, and 910 nm,
respectively (Figure S29; Table S1). The large Stokes shifts of about 200 nm effica-
ciously avoid the overlap between excitation and emission spectra, allowing for effi-
cient utilization of the fluorescent light. We then studied their fluorescence property
by adding water (poor solvent) into THF (good solvent) solution. For all three com-
pounds, the PL intensities slightly decrease when gradually increasing water fraction
from 0% to 30% (Figures 1D, 1E, and S30) due to the solvent polarity effect and
therefore the transformation to twisted intramolecular charge transfer (TICT) state.42
The emission intensities largely intensify when further increasing water fraction to
95%, representing typical AIE signature. Interestingly, the aAIE value (defined as
the ratio of maximal PL intensity in aggregate state and solution state) of OTPA-
TQ3 in THF-water mixtures with 95% water fraction is higher than the other two
derivatives (Table S1), attributing to that the large twisted rotors (phenyl-alkyne-
phenyl) on OTPA-TQ3 result in reduced intermolecular interactions such as
p-p stacking in the aggregated state.43 This result reveals that the size of intramo-
lecular rotors would significantly influence the fluorescence property.
Preparation, Characterization, and Fluorescence Properties of the NPs
To render the hydrophobic compounds with good in vivo biocompatibility, OTPA-
TQs were formulated into stable and small-nanosized NPs through nanoprecipita-
tion method using an amphiphilic lipid-PEG2000 co-polymer as the encapsulation
matrix (Figure 2A). During this process, the hydrophobic organic molecule randomly
assembles in the core, and the amphiphilic surfactant forms the shell to result in wa-
ter-soluble NPs.15 The size and morphology of OTPA-TQ1-3 NPs, respectively, were
characterized by transmission electron microscopy (TEM) and dynamic light scat-
tering (DLS) measurements. As depicted in Figures 2B and S31, the DLS data sug-
gest that the hydrodynamic diameters of OTPA-TQ1-3 NPs are 141, 142, and
144 nm, respectively, and the TEM images indicate that all the three NPs possess
approximately spherical morphology with similar average diameters of
110–120 nm (Table S2). The smaller sizes obtained from TEM measurement is prob-
ably due to the shrinkage of the hydration layer in the dried TEM samples.37 The ob-
tained NPs are a kind of green solution with good transparency, which is similar as
Chem 5, 1–21, October 10, 2019
5

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 2. Characterization and Photophysical Properties of the NPs
(A) Schematic illustration of the nanoprecipitation process.
(B) Representative DLS result and TEM image of OTPA-TQ3 NPs.
(C and D) Shown in (C) is the absorption and in (D) PL spectra of the NPs (20 mM).
(E) PLE mapping of OTPA-TQ3 NPs in aqueous dispersion (20 mM).
the molecules in THF (Figure S32). The absorption maxima of OTPA-TQ1-3 NPs are
centered at 684, 701, and 705 nm, respectively (Figure 2C; Table S1), which are red-
shifted slightly as compared with those in solution states. On the contrary, the PL
spectra of OTPA-TQ1-3 NPs are centered at 880, 897, and 895 nm, respectively (Fig-
ure 2D; Table S1). The hypsochromic shifts of NPs emission spectra for about 15 nm
when compared with those in THF solutions may be attributed to that the fluoro-
phoric molecules in NPs are surrounded by the same molecules with less polarity
than THF. The PL spectra of the compounds in toluene (Figure S29B) exhibit hypso-
chromic shifts of about 20 nm as compared to that of THF solution, which is similar as
the NPs form and confirms the influence of polarity effect.
By using indocyanine green (ICG) as the reference (with a nominal photolumines-
cence quantum yield [PLQY] of 13% in dimethyl sulfoxide [DMSO]),44 the PLQYs of
OTPA-TQ1-3 NPs are measured to be 2.5%, 1.8%, and 2.7%, respectively. These
values are comparable to the recent reported organic emitters with similar emission
range, and much higher than the widely used carbon nanotubes (ꢀ0.4%).45,46 The
changes of PLQYs are in the same trend as the aAIE values (Table S1), suggesting
that AIE characteristic is vital for realizing highly luminescent organic NPs. To better
understand the AIE characteristics and photophysical processes of the molecules,
we measured the PL lifetime in solution and NPs forms (Figure S33; Table S3),
and the corresponding radiative and nonradiative decay rates are presented in
Table S4. Of note, the radiative decay rates increase slightly from solution to NPs
6
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
state, while the nonradiative decay rates decrease significantly. In solution, the mol-
ecules undergo active intramolecular motions, which would consume the excited-
state energy and result in dominated nonradiative relaxation. In contrast, the molec-
ular environment in the aggregate state imposes higher structural rigidity, which
blocks the nonradiative decay channel of the excited state and the radiative one
opens, giving rise to increased PL efficiency.34,47 The highest fluorescent brightness
and aAIE value of OTPA-TQ3 NPs should be attributed to the most twisted molecular
structure, which could reduce the intermolecular interactions within NPs. PL excita-
tion (PLE) mapping was also performed to gain deep understanding about the exci-
tation-emission relationships, and PLE maps of the organic NPs (Figures 2E, S34, and
S35) manifest eligible excitation and emission in NIR region.
Photoacoustic Properties of the Compounds and NPs
We next studied the structure-PA property relationships of OTPA-TQ molecules and
the influence of rotor size in PA signal output. Firstly, glycerol was employed to
elevate solution viscosity on the intramolecular motions of the compounds, which
has been confirmed by the increased PLQYs in viscous environments (Table S5).
Noteworthy, OTPA-TQ3 exhibits the largest brightness enhancement, probably
attribute to the prominent AIE signature. As shown in Figure 3A, the PA intensities
of all three molecules decrease upon increasing the glycerol fraction in N,N-dime-
thylformamide (DMF)-glycerol mixtures, revealing the key role of intramolecular mo-
tions for PA signal output.48 It is also noted from Figure 3A that the PA amplitude of
OTPA-TQ3 profoundly declines of ꢀ65% from pure DMF to 90% glycerol fraction,
which is more pronounced than that of the other two derivatives (40% for OTPA-
TQ1 and 50% for OTPA-TQ2). This result suggests that the excited-state intramolec-
ular motions of larger molecular rotors contributes greater on PA generation.
The PA spectra of OTPA-TQ1-3 NPs were recorded by measuring the PA intensity at
different wavelengths from 680 to 950 nm. As shown in Figure 3B, the PA spectra of
OTPA-TQ1-3 NPs match well with the absorption profiles (Figure 2C), indicating that
the PA signals are produced from the NIR absorption of the molecules. Under the
irradiation of 680 nm NIR pulsed laser, for each OTPA-TQ molecule (50 mM), its
NPs state (ꢀ140 nm by DLS) shows around 2.5-fold higher PA intensity than its
bare aggregate (without lipid-PEG2000) state in THF/water mixture with 95% water
fraction (the aggregates possess broad size distribution of >400 nm measured by
DLS). As the amphiphilic co-polymer lipid-PEG2000 acting as the surfactant can
essentially improve the water solubility of the hydrophobic molecules and provides
much larger specific surface area, which permits more effective excited-state intra-
molecular motions in aqueous media, and this result further validates that there is
a positive correlation between excited-state intramolecular motion and PA signal
output,29,48 in good accordance with that of Figure 3A.
When comparing the PA amplitudes among the three OTPA-TQ NPs, as depicted in
Figure 3C, OTPA-TQ3 NPs exhibit the highest PA intensity, which is about 1.4-fold
higher than the other two counterparts NPs. This could be ascribed to that the large
phenyl-alkyne-phenyl rotors lead to stronger intramolecular motions43 and thus
generate stronger PA signal, agreeing well with Figure 3A. The higher absorption
coefficient of OTPA-TQ3 might also account for the pronounced fluorescence and
PA performance. The PA amplitude of OTPA-TQ NPs was also compared with the
well-known high-performing PA imaging agents including semiconducting polymer
NPs (SPNs) (Figure S36) and methylene blue (MB).15,49 Since MB, SPNs, and OTPA-
TQ1-3 NPs have similar absorption at about 680 nm (Figure S37), rational compari-
son in the same concentration (50 mM) was allowed using a 680 nm pulsed laser. As
Chem 5, 1–21, October 10, 2019
7

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 3. PA Properties of the Compounds and NPs
(A) Relative PA intensity of OTPA-TQ molecules (50 mM) in DMF-glycerol mixtures with different
glycerol fractions at 680 nm.
(B) PA spectra of OTPA-TQ1-3 NPs (50 mM).
(C) PA intensity of MB, semiconducting polymer NPs (SPNs), and OTPA-TQ1-3 NPs with the same
concentration (50 mM) at 680 nm. The molar concentration of SPNs is based on the repeating unit.
Data are presented as the means G SD (n = 3).
(D) PA amplitudes of OTPA-TQ3 NPs as a function of concentration. Data are presented as the
means G SD (n = 3).
shown in Figure 3C, under the same experimental condition, the PA amplitude of
OTPA-TQ3 NPs is ꢀ1.7-fold and ꢀ2.4-fold higher than that of SPNs and MB, respec-
tively. Furthermore, the PA intensity of OTPA-TQ3 NPs shows a good linear relation-
ship with the molar concentration of OTPA-TQ3 (Figure 3D), indicating the potential
for quantitative analysis.
Raman Properties of the Compounds and NPs
The alkyne group is introduced into OTPA-TQ2 and OTPA-TQ3 molecules as it has
been well accepted to produce Raman signature in the cell-silent region.50,51 Fig-
ure 4A displays the Raman spectra of the OTPA-TQ1-3 NPs. Noteworthy, OTPA-
TQ3 NPs other than the other two NPs possess rather strong Raman signal at
2,215 cmÀ1, which is in the cell-silent region and refers to the typical carbon-carbon
triple bond stretching and vibration signature of alkyne groups. The strong Raman
signal of OTPA-TQ3 is probably ascribed to the large conjugation of the phenyl-
alkyne-phenyl units as confirmed by DFT calculation (Figure S28),52,53 warranting
it an efficient biomarker for highly specific Raman imaging with negligible
8
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 4. Raman Properties of the Compounds and NPs
(A) Raman spectra of OTPA-TQ1-3 NPs (100 mM, excitation: 532 nm).
(B) Raman intensity of OTPA-TQ3 (50 mM, excitation: 532 nm) in pure THF, THF-water mixture with
10% THF fraction, and encapsulated NPs.
background. Furthermore, the influence of intramolecular motions on Raman prop-
erty was studied by investigating the Raman spectra of OTPA-TQ3 in the forms of
free molecule (in THF solution), large aggregate (in THF-water mixture with 90% wa-
ter fraction) and NPs. As depicted in Figure 4B, the OTPA-TQ3 NPs show much
stronger Raman intensity at 2,215 cmÀ1 than the large aggregates, whereas the
free molecule has the highest Raman signal, which is in accord with previous reports
that molecular motion brings about Raman scattering.54,55 As the polymer surfactant
lipid-PEG2000 favors improved solubility of OTPA-TQ3 in water, benefiting to pro-
moted intramolecular motions, this result implies that the Raman intensity from
phenyl-alkyne-phenyl in the unconfined free-motion form would be stronger than
the restricted aggregation. From the basic theory of Raman scattering, after light ab-
sorption, the molecules are excited to a virtual energy state.56 In our case, the wave-
length of the incident light (532 nm) could also lead to the electronic transition of
OTPA-TQ3 molecule. Therefore, the result in Figure 4B suggests that intramolecular
motions in the aforementioned high energy state would significantly enhance the
Raman signal from phenyl-alkyne-phenyl.
The interplay of fluorescence, PA and Raman properties, and the corresponding
photophysical processes in different states are depicted in Scheme S3. By gradually
activating the intramolecular motions (e.g., from large aggregate to NPs state and
further to THF solution), the PA and Raman properties are enhanced, while the fluo-
rescence is influenced adversely. The twisted 3D molecular structure and pro-
nounced AIE effect enable high NIR fluorescent brightness in aqueous media. The
strong excited-state intramolecular motions and high absorption coefficient of
OTPA-TQ3 are responsible for the strong PA generation capability. Moreover, the
conjugated phenyl-alkyne-phenyl units and intramolecular motions warrant OTPA-
TQ3 strong Raman signal in the cell-silent region. Accordingly, the best imaging per-
formance of OTPA-TQ3 can be attributed to both the large conjugated substitutes
and intramolecular motions.
Stability of the NPs
Photostability is of critical importance for optical imaging agents; hence, we inves-
tigated the photobleaching resistance capacities of OTPA-TQ1-3 NPs under light
irradiation and the clinically used MB was used as a control. After exposure to
Chem 5, 1–21, October 10, 2019
9

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 5. Stability of the NPs
(A) Photostability of the NPs and MB under continuous light (650 nm, 200 mW cmÀ2) irradiation. A and A0 are the maximal PL intensity of OTPA-TQs NPs
and MB without and with light irradiation.
(B) Plot of I/I0 versus RONS (ClOÀ and ,OH, 1 mM) treatment. I and I0 are the maximal PL intensity of OTPA-TQs NPs and ICG (black, OTPA-TQ1 NPs;
blue, OTPA-TQ2 NPs; red, OTPA-TQ3 NPs; green, ICG) in PBS solutions in the presence and absence of RONS, respectively. Data are presented as the
means G SD (n = 3).
(C) Plots of photoacoustic intensity of OTPA-TQ3 NPs in a phantom (50 mM) against number of laser pulses at 700 nm (2.4 3 104 pulses; 17.5 mJ cmÀ2
laser and 10 Hz pulse repetition rate).
continuous red light (650 nm, 200 mW cmÀ2) irradiation for 60 min, the absorption
and emission properties of all three NPs remain constant, whereas MB dye with
similar absorption maximum is easily photobleaching, as evidenced by the PL inten-
sity decreasing to ꢀ55% of the original value (Figure 5A). Additionally, reactive ox-
ygen and nitrogen species (RONS) are a kind of important signaling molecules
closely related to body health, which are also known to overexpress in many
diseased regions, e.g., cancer, inflammation, and cardiovascular diseases.57,58 As
a consequence, stable optical probes that are resistant to RONS are momentous
for in vivo disease detection. As presented in Figure 5B, all the OTPA-TQ1-3 NPs
show excellent resistance to various RONS. In contrast, the FDA-approved ICG is
severely destroyed in the presence of ClOÀ and ,OH, which is likely due to the
degradation of alternatively arranged single-double bonds. We further studied
the probe stability under PA experiment condition. After exposure to 2.4 3 104
pulses (17.5 mJ cmÀ2 laser and 10 Hz pulse repetition rate), nearly no PA signal
loss is observed (Figure 5C), indicating good photostability and suitability for PA im-
aging. Noteworthy, the NPs also show good colloidal stability, as no precipitation is
observed after storage at room temperature for 2 weeks, and the average diameters
nearly do not change as well (Figure S38). Taken together, these results reveal that
the organic nanoagents possess superb stability with various treatments, which are
suitable for the long-term in vivo applications.
Preoperative Fluorescence and PA Imaging of Tumors
The aforementioned results have reasonably demonstrated that OTPA-TQ3 NPs
possess the optimized fluorescence, PA, and Raman signals in aqueous environ-
ment. Thus, the OTPA-TQ3 NPs were used for the following in vivo utilization to
investigate whether such an optical agent with excellent fluorescence-PA-Raman
properties could be beneficial to precise image-guided cancer surgery. As preoper-
ative imaging requires both good spatial resolution and high sensitivity to reveal the
size, number, and location of tumors in vivo, both NIR fluorescence imaging and
PA imaging were performed before surgery with 4T1 subcutaneous tumor-bearing
mice by intravenous administration of OTPA-TQ3 NPs. After NPs injection, the
10
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 6. Preoperative Fluorescence and PA Imaging of Tumor-Bearing Mice In Vivo
(A) Representative fluorescence images of tumor-bearing mice after intravenous injection of OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3) at
different time points as indicated.
(B) Fluorescence intensity of the tumor site as a function of post-injection time. Data are presented as the means G SD (n = 3 mice).
(C) Fluorescence intensities of slices of tumor and main organs (heart, liver, spleen, lung, and kidneys) resected from the tumor-bearing mice at 24 h
post-injection. All the tissues were sliced with the same thickness of 1 mm for fluorescence imaging and quantitative analysis in order to better eliminate
the influence of tissue penetration depth. Data are presented as the means G SD (n = 3 mice).
(D and E) Shown in (D) are representative PA images and in (E) the corresponding PA intensity of tumor site after intravenous administration of OTPA-
TQ3 NPs at different time points as indicated. Data are presented as the means G SD (n = 3 mice).
tumor-bearing mice were concurrently scanned by IVIS imaging system and small-
animal opt-acoustic tomography system (MOST) at designated time intervals. The
time-dependent in vivo non-invasive NIR fluorescence images are shown in Fig-
ure 6A, and the corresponding fluorescence intensity-time relationship in tumor is
depicted in Figure 6B, which reveal that the NIR fluorescence signal at tumor site be-
comes intense gradually as the time elapses stemming from the passive enhanced
permeability and retention (EPR) effect.59 The outstanding EPR effect of OTPA-
TQ3 NPs should be benefited from their appropriate size and surface chemistry. Be-
sides tumor, the reticuloendothelial system (RES) organs including liver and spleen
Chem 5, 1–21, October 10, 2019
11

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
where the NPs are prone to accumulate are also lighted up (Figures 6C and S39).60 At
24 h post-injection, the fluorescence signal at tumor site reaches the maximum and
the tumor signal-to-background (surrounding normal skin autofluorescence) ratio is
as high as ꢀ9.2, which is considered to be very high according to the literatures.61
On the other hand, the mice were also imaged by PA instrument within 24 h study
duration. As shown in Figure 6D, the PA signal in tumor gradually amplifies over
time and arrives at the peak at 24 h post-injection, coinciding well with the trend
of time-dependent fluorescence imaging results (Figures 6A and 6B). Noteworthy,
the average PA signal in tumor at 24 h is ꢀ7.0 times higher than the background
(0 h) before NPs injection (Figure 6E), giving better performance than many high-
performing PA imaging agents.37,62 Both preoperative NIR fluorescence imaging
and PA imaging indicate that the OTPA-TQ3 NPs can detect the tumors in vivo in
an extremely high-contrast manner at 24 h post-injection, providing the surgeon
with important information on surgical plan.
Intraoperative Fluorescence and Raman Imaging of Tiny Residual Tumors
Next, tumor resection surgery was conducted by a surgeon from Tianjin First Central
Hospital (Tianjin, China) based on the information by preoperative imaging. In the
clinic, the most challenging issues for the surgeon during surgery are to evaluate
whether there are residual tumors left behind post-major tumor excision as well as
to differentiate the boundaries between normal and tumor tissues.2,63 Thereby,
the combination of fluorescence and Raman imaging holding the integrated advan-
tages of fast and real-time mode, excellent sensitivity and high signal-to-back-
ground ratio are desirable during cancer operation. In our case, the first surgery
(S1) was performed by the surgeon with his experience after the OTPA-TQ3 NPs
were intravenously injected into 4T1 tumor-bearing mice for 24 h, which was fol-
lowed by NIR fluorescence and Raman imaging at the same time. In case that the tu-
mors are totally removed by the surgeon, as confirmed by hematoxylin and eosin
(H&E) histological analyses, there are no fluorescence and Raman signals at the sur-
gical incision sites. In other cases that there are tiny tumors left behind, a certain de-
gree of fluorescence signal can be observed (Figure 7A). Although OTPA-TQ3 NPs
possess rather high NIR fluorescence, the volume of residue tumors is tiny, leading
to only small amount of NPs in them. Besides, the normal tissue autofluorescence
compromises the signal-to-background ratio as well. Thus, the surgeon cannot accu-
rately assess whether the weak fluorescent areas are indeed tumors. Even so, intra-
operative fluorescence imaging is quite necessary, as it is fast, sensitive, and real
time and can rapidly point out the suspicious areas of residual tumors.
To pursue precise surgical treatment, Raman imaging with microscopic resolution was
conducted in the suspicious areas with faint fluorescence. As illustrated in Figure 7B,
the OTPA-TQ3 NPs with strong Raman signal (2,215 cmÀ1) in the cell-silent region
can sensitively visualize the residual tumors and their boundaries to normal tissues by
Raman imaging with ‘‘yes-or-no’’ signature. Such excellent effectiveness of residual tu-
mor detection during surgery should be attributed to both the high Raman signal of
OTPA-TQ3 NPs and the innate zero-background nature of Raman imaging in cell-silent
region. It is noted that 94% of the tested tiny areas with Raman signal are confirmed as
tumors when consulting the H&E histological analysis (Figure 7C). As Raman imaging is
much faster than histological analysis, it holds great promise for intraoperative residual
tumor inspection.6,64 Noteworthy, the intraoperative fluorescence-Raman imaging with
OTPA-TQ3 NPs can clearly delineate tiny residual tumors after S1 with diameters of
about 450 mm (Figure S40). After demonstrating the existence of residual tumors, the
surgeon could perform the second surgery (S2) to remove the residual tumors until there
are no fluorescence and Raman signals (Figures 7D–7F).
12
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Figure 7. Intraoperative Fluorescence and Raman Imaging of Tiny Residual Tumors
(A) Representative fluorescence images of the OTPA-TQ3 NPs-treated tumor-bearing mice before and after S1 treatment.
(B and C) Shown in (B) is Raman imaging and in (C) H&E-stained tissues of the operative incision site after S1.
(D) Representative fluorescence images of the OTPA-TQ3 NPs-treated tumor-bearing mice before and after S2 treatment.
(E and F) Shown in (E) is Raman imaging and in (F) are H&E-stained tissues of the operative incision site after S2.
(G) Survival curves for the tumor-bearing mice after various treatments as indicated (n = 10 mice per group).
(H and I) Representative H&E-stained images of the lung slices from the dead mice are shown in (H) Control, and in (I) are S1 groups. The black dashed
lines circle the tumor areas.
(J) Representative H&E-stained images of the lung slices from the mice in S2 group that survived 60 days.
The survival rates of mice after S1 (with fluorescence and Raman signals at the sur-
gical incision sites) and S2 (without any signals), respectively, were monitored with
the mice received no surgical treatment as the control. As shown in Figure 7G, the
mice without surgical treatment and only undergoing S1 all died within 40 days
post-surgery. In contrast, the 10 mice in S2 group survived 40 days, however,
2 mice were then dead on day 43 and 47 after surgery, respectively. The other
8 mice survived during 60-day study duration. Given that it has been well accepted
that 4T1 tumor model has aggressive metastatic potential,65 the influence of lung
metastasis on survival rates was assessed. In this experiment, the lung organ of
each dead mouse in the 3 groups was excised, sliced, and stained with H&E for his-
tological analysis. As displayed in Figures 7H and 7I, it is found that almost all the
dead mice in the Control and S1 groups suffer from lung metastases, as confirmed
by the H&E staining. Moreover, the primary tumors from mice in these two groups
also grew bigger as the time elapsed. Therefore, the mice in Control and S1
Chem 5, 1–21, October 10, 2019
13

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
cohorts died probably attributed to both the lung metastases and primary tumor
growing.
On the other hand, for S2 group, since there was no primary tumor recurrence observed
forthe2deadmice, thelungmetastaseswereexamined. AsshowninFigureS41, obvious
lung metastases are observed, which should be the main cause of death for these 2 mice.
Furthermore,theother8survivingmiceinS2groupweresacrificedattheendofthe study
(on day 60 after surgery), and the histologicalanalysesrevealneither primary tumor recur-
rence nor obvious lung metastases (Figure 7J). We then investigated the possible reason
for the less lung metastases in S2group. Since the surgeries (both S1 and S2) were carried
out on day 9 after the 4T1 cancer cells were injected subcutaneously into the mouse axil-
lary space, the lung tissues were examined with H&E on day 9 post-cancer cell inocula-
tion. As shown in Figure S42A, the H&E-stained lung slices show negligible tumor metas-
tasesonthisday,implyingthatthelungmetastasesmaynotsignificantlyoccuratthistime
point. It is also found that distinct lung metastases can be observed on day 21 after 4T1
cancer cell inoculation (Figure S42B). Therefore, the complete tumor resection was per-
formed at the relatively early stage of cancer, which would greatly reduce the risk of sub-
sequent lung metastases, leading to 8 of 10 mice in S2 group surviving 60 days.
Biocompatibility is critically important for molecular probes, so we studied the
biosafety and excretion of OTPA-TQ3 NPs. The cellular viability was evaluated by
co-culturing the NPs with different cell lines (4T1 cancer cells, NIH 3T3 cells, and De-
troit 551 human fibroblast cells, respectively). As depicted in Figure S43, all the cells
treated with a high concentration of NPs (30 mM) display good viability of higher than
90%, suggesting low cytotoxicity. The hepatic and renal function analyses (Fig-
ure S44) and blood routine examination (Figure S45) of the OTPA-TQ3 NPs-treated
mice also show no noticeable abnormalities, indicating the low in vivo toxicity. To
evaluate how the NPs are cleared from the body, after intravenous administration
of OTPA-TQ3 NPs into the healthy mice, the feces and urine of the mice were then
collected and imaged at designated time intervals. As shown in Figure S46, signifi-
cant fluorescence signal from the OTPA-TQ3 NPs can be observed in the collected
feces within 7 days after injection, demonstrating that the NPs are mainly excreted
from the body through biliary pathway, i.e., from liver to bile duct, intestine, and
finally to feces.37,66 On the other hand, there is negligible fluorescence signal in
the urine, indicating that the NPs would not be cleared via renal pathway. Taken
together, these results indicate that the NPs have good biocompatibility and no
obvious side effect is observed. Systematic investigation may be required to study
the long-term fate and biocompatibility of the organic nanoprobes in the future.
To study the influence of agent dose on the imaging performance, different concen-
trations (0, 81, 162, 325, and 650 mM) of OTPA-TQ3 NPs with the same volume of
200 mL were intravenously injected into the tumor-bearing mice, and the PA, fluores-
cence and Raman imaging were investigated. To make a rational comparison, the
imaging was carried out in the same condition as indicated in the experimental pro-
cedures. Generally, the signal intensity intensifies with increased concentration (Fig-
ures S47 and S48). For preoperative PA imaging, the high concentration of 650 mM
can provide detailed information about tumor size in a high-contrast manner, while
other lower concentrations are not able to illuminate the tumor site clearly. The
intraoperative fluorescence imaging could only illuminate residual tumors with
the high-concentration nanoagent, possibly because there is tissue autofluores-
cence and a low NPs concentration in the tiny residual tumor nodules, while Raman
imaging can provide a better signal in a relatively low nanoagent concentration,
probably because of the zero-background signature in the cell-silent region. As a
14
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
consequence, a concentration of 650 mM could provide the most comprehensive in-
formation about tumor at different surgical stages, and no obvious side effect was
observed, representing the optimal experimental condition for image-guided tumor
surgery. We hope that these comparisons would provide useful guidance for other
organic molecular probes.
DISCUSSION
We have developed a kind of one-for-all molecular agent for comprehensive image-
guided surgery application. The fluorescence, PA, and Raman properties in one organic
molecule are greatly impacted by the molecular structure and intramolecular motion, as
they have an effect on the photophysical properties, and the corresponding optical im-
aging performance. As compared with the other two compounds, OTPA-TQ3 with the
largest intramolecular rotation units has the most twisted 3D molecular structure and the
strongest AIE effect, benefiting to the highest NIR fluorescent brightness in aqueous
media. Moreover, the strong excited-state intramolecular motions and high absorption
coefficient endow OTPA-TQ3 with the strongest PA signal generation capability.
Furthermore, the phenyl-alkyne-phenyl units warrant OTPA-TQ3 NPs strong Raman
signal at 2215 cmÀ1 in the cell-silent region, and the intramolecular motions in high en-
ergy state after light excitation are demonstrated to significantly enhance the Raman
signal. Taking advantages of the high sensitivity of fluorescence imaging and good
spatial resolution and penetration depth of PA technique, the intravenously adminis-
trated OTPA-TQ3 NPs enable tumor detection preoperatively and then guidance of sur-
gical plan. Further intraoperative imaging during cancer surgery manifests that the
OTPA-TQ3 NPs can help the surgeon accurately remove all of the tiny residual tumors
by virtue of the fast, real-time, and sensitive fluorescence imaging and high-contrast
Raman imaging with zero background, which greatly prolong the lifetimes of mice
post-surgery. Moreover, the organic nanoprobe shows good biocompatibility, and no
side effect was observed from both the in vitro and in vivo tests. This work represents
the first example of boosting fluorescence-PA-Raman properties in one organic fluoro-
phore, which allows for accurate cancer imaging and resection, rendering great promise
for integrated multi-modality imaging applications.
There are still several aspects that can be considered for meeting the clinical trans-
lation of multi-modality optical imaging agent. First, the probes with longer excita-
tion and emission wavelengths would be better, for example, to increase the absorp-
tion and emission wavelength of the probes to the recently developed second NIR
window would enable larger penetration depth, and higher spatial resolution. Sec-
ond, other post-surgery treatment (e.g., immunotherapy) may be needed to
combine with the multi-modality image-guided precision surgery, especially for pa-
tients with metastases, to inhibit both the primary tumors and metastatic tumors
effectively. Third, new instrumentation that integrating the complex measurements
and data analyses in one platform could definitely facilitate in situ monitoring and
improve the performance of multi-modality imaging, benefiting for clinical use.
EXPERIMENTAL PROCEDURES
Materials and Characterizations
All the chemicals and reagents were purchased from chemical sources and were
used as received. The nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker AV 400 spectrometer. HRMS were measured with a GCT premier
CAB048 mass spectrometer in matrix assisted laser desorption ionization-time of
flight (MALDI-TOF) mode. The theoretical calculation was carried out at the level
of B3LYP/6-31G* using DFT method with the Gaussian 09 program package (the
Chem 5, 1–21, October 10, 2019
15

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Cartesian coordinates of OTPA-TQ1-3; see Tables S6–S8). The ultraviolet-visible
(UV-vis) absorption spectra were performed using a Shimadzu 2550 UV-vis scanning
spectrophotometer. The steady-state PL spectra were conducted on a Horiba Fluo-
rolog-3 spectrofluorometer. Transient PL at room temperature was measured using
an Edinburgh FLS1000 fluorescence spectrophotometer with a photodetector of
PMT-980. Raman spectra were acquired by a confocal Raman microspectroscopy
with a 532 nm excitation (Renishaw). DLS was measured on a 90 plus particle size
analyzer. TEM images were obtained from a JEM-2010F transmission electron mi-
croscope with an accelerating voltage of 200 kV.
Preparation of the NPs
1 mg of the organic compound and 2 mg of amphiphilic lipid-PEG (DSPE-PEG2000
)
were dissolved in 1 mL of THF. The obtained THF solution was poured into 9 mL
of deionized water under sonication with a microtip probe sonicator (XL2000, Miso-
nix Incorporated, NY). The mixture was then sonicated for another 1 min and
violently stirred in fume hood overnight at room temperature to evaporate residue
THF, and the obtained NPs solution was used directly.
Photoacoustic Properties
The PA properties were studied by using a multi-spectral optoacoustic tomography
(iTheraMedical, Germany), which was equipped with
a wavelength-tunable
(680–980 nm) optical parametric oscillator pumped by a Nd:YAG laser with excita-
tion pulses of 7 ns duration at a repetition rate of 10 Hz. The light from the fiber
covered an area of 4 cm2 with a maximum incident pulse energy of approximately
70 mJ (100 mJ, 70% fiber coupling efficiency). This generated an optical fluence
of 17.5 mJ cmÀ2, which was well within the safe exposures according to the American
National Standard for Safe Use of Lasers. The PA intensity was measured by finely
analyzing the regions of interest of acquired images. PA spectra of the NPs solutions
were obtained by recording the PA signals at different wavelengths from 680 nm to
950 nm (10 nm wavelength for each slice). The relationship between PA intensity and
NPs concentration was established by using various concentrations of NPs solutions
(10, 25, 50, 100, and 200 mM). The comparison of PA intensity of different agents
(50 mM) was conducted by the excitation of 680 nm pulsed laser. The probe stability
during PA experiment was evaluated by scanning OTPA-TQ3 NPs in a phantom
(50 mM) with 2.4 3 104 of laser pulses at 700 nm (17.5 mJ cmÀ2 laser and 10 Hz pulse
repetition rate).
Cytotoxicity Study
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used
to evaluate the cytotoxicity of the NPs in different cell lines. 4T1 breast cancer cells,
NIH 3T3 cells and Detroit 551 human fibroblast cells were respectively harvested in a
logarithmic growth phase and seeded in 96-well plates (5000 cells per well with
100 mL suspension) for 24 h and grew to ꢀ80% confluence. Then the culture medium
was replaced with 100 mL of fresh culture medium containing OTPA-TQ3 NPs with
various concentrations (the concentrations based on OTPA-TQ3 are: 0 mM,
1.5 mM, 3 mM, 6 mM, 15 mM, and 30 mM), separately. After incubating for 24 h, the
culture medium was removed and the wells were washed three times with PBS,
and 100 mL of MTT dissolved in serum-free culture medium (0.5 mg/mL) was added
into each well. After 4 h, the MTT solution was removed cautiously and 100 mL of
DMSO was added into the wells, followed by gently shaking for 10 min. Then, the
absorbance of MTT was measured by a Bio-Rad 680 microplate reader at 490 nm
to evaluate the viability of cells inside.
16
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Animal Experiments
All animal studies were conducted under the guidelines set by Tianjin Committee of
Use and Care of Laboratory Animals, and the overall project protocols were
approved by the Animal Ethics Committee of Nankai University.
Tumor-Bearing Mice
6-Week-old female BALB/c mice were obtained from the Laboratory Animal Center
of the Academy of Military Medical Sciences (Beijing, China). To establish the xeno-
graft 4T1 tumor-bearing mouse model, 4T1 breast cancer cells (1 3 106) suspended
in 30 mL of RPMI-1640 medium were injected subcutaneously into the right axillary
space of the BALB/c mouse. After about 9 days, mice with tumor volumes of about
80–120 mm3 were used subsequently.
Preoperative Fluorescence and Photoacoustic Imaging
The xenograft 4T1 tumor-bearing female mice were used for the following experi-
ments (n = 3 mice for quantitative analyses of all imaging modalities). The tumor-
bearing mice were anesthetized using 2% isoflurane in oxygen, and OTPA-TQ3
NPs (200 mL, 650 mM based on OTPA-TQ3) were intravenously injected into the tu-
mor-bearing mice using a microsyringe. Then in vivo NIR fluorescence and PA imag-
ing were concurrently carried out to provide comprehensive information about the
tumor. For fluorescence imaging, it was performed with the Maestro EX fluorescence
imaging system (CRi, Inc.) with excitation at 704 nm and signal collection in the spec-
tral region of 740–950 nm. For PA imaging, it was performed with the same mice as
fluorescence imaging on a commercial small-animal opt-acoustic tomography sys-
tem (MOST, iTheraMedical, Germany). The PA data were acquired at 700 nm excita-
tion (17.5 mJ cmÀ2 laser and 10 Hz pulse repetition rate), after which the images were
reconstructed using the model-based algorithm supplied within the ViewMSOT soft-
ware suite (V3.6, iThera Medical). The fluorescence and PA images were recorded at
designated time intervals after injection.
Tumor Resection and Intraoperative Fluorescence-Raman Imaging
Based on the information provided by preoperative fluorescence and PA imaging at
24 h post-injection, the tumors were resected (n = 20 mice). Briefly, the tumor-
bearing mice were anesthetized using 2% isoflurane in oxygen. With the experience
of a surgeon, the tumor tissues were aseptically prepped and sterile instruments
were employed to excise the tumors (S1). The operative incision sites were followed
by NIR fluorescence imaging to detect if there were residual tumors left behind. The
fluorescence imaging was performed similar to the aforementioned in vivo case
(Maestro EX fluorescence imaging system with excitation at 704 nm and signal
collection in the spectral region of 740–950 nm). The tissues at the operative incision
sites were subsequently dissected and sliced, and the frozen sections were used for
Raman microscopy and H&E staining. For Raman microscopy, frozen sections were
placed on quartz slides (Ted Pella, Inc.) and air-dried. A 503 or 123 objective lens
was used, and Raman spectral maps and correlating white light images were ac-
quired using the Renishaw Streamline function (excitation at 532 nm, the irradiated
power was ꢀ2 mW and the exposure time for each line was 1 s). The Raman spectra
were analyzed by least squares analysis using Wire 2.0 software (Renishaw). At the
same time, H&E staining was performed to confirm whether there were residual tu-
mors in the operative sites left behind after surgery, and the obtained slices were
examined by a digital microscope (Leica QWin). After demonstrating the existence
of residual tumors after the first surgery (S1), the second surgery (S2) was performed
to remove the tumors until there were no fluorescence and Raman signals, as well as
confirmed by H&E staining.
Chem 5, 1–21, October 10, 2019
17

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Histological Analysis
After S1 and the intraoperative imaging with fluorescence and Raman techniques,
tissues at the operative sites were collected and fixed in 4% paraformaldehyde at
4ꢁC overnight. Then the samples were embedded in paraffin, sliced at a thickness
of 5 mm, and the slices were stained with H&E, and imaged by an optical microscopy
(Leica QWin). After S2, the surgical sites were also examined by H&E staining in a
similar manner.
Survival Rate Study
The xenograft 4T1 tumor-bearing mice were randomly selected and then divided
into three groups (n = 10 mice per group), which were named ‘‘Control,’’ ‘‘S1 with
FL-RA signals,’’ and ‘‘S2 without any signals,’’ respectively. For the ‘‘Control’’ group,
the mice were intravenously injected with OTPA-TQ3 NPs (200 mL, 650 mM based on
OTPA-TQ3). For the ‘‘S1 with FL-RA signals’’ group, after the intravenous injection of
OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3) for 24 h, the tumor-bearing
mice were imaged with fluorescence and PA imaging, and tumor resection surgery
was conducted by a surgeon with his experience (S1), but there were still fluores-
cence and Raman signals at the operative sites. For the ‘‘S2 without any signals’’
group, after the intravenous injection of OTPA-TQ3 NPs (200 mL, 650 mM based
on OTPA-TQ3) for 24 h, the tumor-bearing mice were imaged with fluorescence
and PA imaging, and tumor resection surgery was conducted by a surgeon with
his experience (S1). With the information provided by the intraoperative fluores-
cence and Raman imaging, the second surgery (S2) was performed, after which no
fluorescence and Raman signals could be detected. The survival rates were moni-
tored within 60-day study duration.
Serum Biochemistry Assay and Complete Blood Count
Healthy BALB/c mice were randomly assigned to two groups (n = 4 per group). On
day 0, mice in one group were intravenously injected with 200 mL of OTPA-TQ3 NPs
(650 mM based on OTPA-TQ3), and mice in another group was not treated for con-
trol. On day 7, all mice were sacrificed, and their blood was collected from the arter-
iae ophthalmica. The blood samples were utilized for blood routine examination and
hepatic and renal function analyses, respectively.
Statistical Analysis
Statistical analysis was performed by GraphPad Prism. Quantitative data were ex-
pressed as means G standard deviation (SD). One-way ANOVA and unpaired Stu-
dent’s t test were utilized for statistical analyses. p Value < 0.05 was considered sta-
tistically significant.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2019.07.015.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation of China (51622305,
21788102, and 51873092), the National Basic Research Program of China
(2015CB856503), the Research Grants Council of Hong Kong (C6009-17G,
C2014-15G, A-HKUST605/16, 16308016, and 2018YFE0190200), the Innovation
and Technology Commission (ITC-CNERC14SC01 and ITS/254/17), the Funda-
mental Research Funds for the Central Universities, Nankai University, and the
18
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
Science and Technology Plan of Shenzhen (JCYJ20160229205601482 and
JCY20170818113602462).
AUTHOR CONTRIBUTIONS
D.D. and B.Z.T. conceived and designed the study. J.Q. synthesized and character-
ized the compounds. J.Q. and J.L. performed the NPs preparation and in vitro ex-
periments. J.L., R.L., and Q.L. performed the in vivo experiments. H.Z. provided
technical assistance with the theoretical calculation. J.Q., J.L., J.W.Y.L., R.T.K.K.,
D.L., D.D., and B.Z.T. analyzed the data and participated in the discussion. J.Q.,
J.L., D.D., and B.Z.T. contributed to the writing of this paper.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: December 4, 2018
Revised: January 28, 2019
Accepted: July 17, 2019
Published: August 8, 2019
REFERENCES AND NOTES
1. Lukianova-Hleb, E.Y., Kim, Y.-S.,
Chang, J., et al. (2018). Light-triggered
retention and cascaded therapy of albumin-
based theranostic nanomedicines to alleviate
tumor adaptive treatment tolerance. Adv.
Funct. Mater. 28, 1707291.
16. Yang, B., Chen, Y., and Shi, J. (2018). Material
chemistry of two-dimensional inorganic
nanosheets in cancer theranostics. Chem 4,
1284–1313.
Belatsarkouski, I., Gillenwater, A.M., O’Neill,
B.E., and Lapotko, D.O. (2016). Intraoperative
diagnostics and elimination of residual
microtumours with plasmonic nanobubbles.
Nat. Nanotechnol. 11, 525–532.
17. Kim, T., Zhang, Q., Li, J., Zhang, L., and Jokerst,
J.V. (2018). A gold/silver hybrid nanoparticle for
treatment and photoacoustic imaging of
9. Fan, Q., Cheng, K., Yang, Z., Zhang, R., Yang,
M., Hu, X., Ma, X., Bu, L., Lu, X., Xiong, X., et al.
(2015). Perylene-diimide-based nanoparticles
as highly efficient photoacoustic agents for
deep brain tumor imaging in living mice. Adv.
Mater. 27, 843–847.
2. Wang, P., Fan, Y., Lu, L., Liu, L., Fan, L., Zhao,
M., Xie, Y., Xu, C., and Zhang, F. (2018). NIR-II
nanoprobes in-vivo assembly to improve
image-guided surgery for metastatic ovarian
cancer. Nat. Commun. 9, 2898.
bacterial infection. ACS Nano 12, 5615–5625.
18. Wei, L., Hu, F., Chen, Z., Shen, Y., Zhang, L., and
Min, W. (2016). Live-cell bioorthogonal
chemical imaging: stimulated Raman
scattering microscopy of vibrational probes.
Acc. Chem. Res. 49, 1494–1502.
3. Vahrmeijer, A.L., Hutteman, M., van der Vorst,
J.R., van de Velde, C.J.H., and Frangioni, J.V.
(2013). Image-guided cancer surgery using
near-infrared fluorescence. Nat. Rev. Clin.
Oncol. 10, 507–518.
10. Bissell, M.J., and Hines, W.C. (2011). Why don’t
we get more cancer? A proposed role of the
microenvironment in restraining cancer
progression. Nat. Med. 17, 320–329.
19. Song, Z.L., Chen, Z., Bian, X., Zhou, L.Y., Ding,
D., Liang, H., Zou, Y.X., Wang, S.S., Chen, L.,
Yang, C., et al. (2014). Alkyne-functionalized
superstable graphitic silver nanoparticles for
Raman imaging. J. Am. Chem. Soc. 136, 13558–
13561.
11. Jokerst, J.V., Cole, A.J., Van de Sompel, D., and
Gambhir, S.S. (2012). Gold nanorods for
ovarian cancer detection with photoacoustic
imaging and resection guidance via Raman
imaging in living mice. ACS Nano 6, 10366–
10377.
4. Antaris, A.L., Chen, H., Cheng, K., Sun, Y.,
Hong, G., Qu, C., Diao, S., Deng, Z., Hu, X.,
Zhang, B., et al. (2016). A small-molecule dye
for NIR-II imaging. Nat. Mater. 15, 235–242.
20. Li, S., Chen, T., Wang, Y., Liu, L., Lv, F., Li, Z.,
Huang, Y., Schanze, K.S., and Wang, S. (2017).
Conjugated polymer with intrinsic alkyne units
for synergistically enhanced Raman imaging in
living cells. Angew. Chem. Int. Ed. Engl. 56,
13455–13458.
5. Gu, X., Zhang, X., Ma, H., Jia, S., Zhang, P.,
Zhao, Y., Liu, Q., Wang, J., Zheng, X., Lam,
J.W.Y., et al. (2018). Corannulene-incorporated
AIE nanodots with highly suppressed
nonradiative decay for boosted cancer
phototheranostics in vivo. Adv. Mater. 30,
e1801065.
12. Miao, Q., Xie, C., Zhen, X., Lyu, Y., Duan, H., Liu,
X., Jokerst, J.V., and Pu, K. (2017). Molecular
afterglow imaging with bright, biodegradable
polymer nanoparticles. Nat. Biotechnol. 35,
1102–1110.
21. Li, M., Luo, Z., and Zhao, Y. (2018). Self-
assembled hybrid nanostructures: versatile
multifunctional nanoplatforms for cancer
diagnosis and therapy. Chem. Mater. 30,
25–53.
6. Kircher, M.F., de la Zerda, A., Jokerst, J.V.,
Zavaleta, C.L., Kempen, P.J., Mittra, E., Pitter,
K., Huang, R., Campos, C., Habte, F., et al.
(2012). A brain tumor molecular imaging
strategy using a new triple-modality MRI-
photoacoustic-Raman nanoparticle. Nat. Med.
18, 829–834.
13. Xu, Z., Huang, X., Han, X., Wu, D., Zhang, B.,
Tan, Y., Cao, M., Liu, S.H., Yin, J., and Yoon, J.
(2018). A visible and near-infrared, dual-
channel fluorescence-on probe for selectively
tracking mitochondrial glutathione. Chem 4,
1609–1628.
22. Goel, S., Ferreira, C.A., Chen, F., Ellison, P.A.,
Siamof, C.M., Barnhart, T.E., and Cai, W. (2018).
Activatable hybrid nanotheranostics for
tetramodal imaging and synergistic
14. Wang, L.V., and Hu, S. (2012). Photoacoustic
tomography: in vivo imaging from organelles
to organs. Science 335, 1458–1462.
7. Qi, J., Chen, C., Zhang, X., Hu, X., Ji, S., Kwok,
R.T.K., Lam, J.W.Y., Ding, D., and Tang, B.Z.
(2018). Light-driven transformable optical
agent with adaptive functions for boosting
cancer surgery outcomes. Nat. Commun. 9,
1848.
photothermal/photodynamic therapy. Adv.
Mater. 30, 29266476.
23. Dong, Z., Feng, L., Hao, Y., Chen, M., Gao, M.,
Chao, Y., Zhao, H., Zhu, W., Liu, J., Liang, C.,
et al. (2018). Synthesis of hollow biomineralized
CaCO3Àpolydopamine nanoparticles for
multimodal imaging-guided cancer
15. Pu, K., Shuhendler, A.J., Jokerst, J.V., Mei, J.,
Gambhir, S.S., Bao, Z., and Rao, J. (2014).
Semiconducting polymer nanoparticles as
photoacoustic molecular imaging probes in
living mice. Nat. Nanotechnol. 9, 233–239.
8. Chen, J., Liu, L., Motevalli, S.M., Wu, X., Yang,
X.-H., Li, X., Han, L., Magrini, A., Guo, W.,
photodynamic therapy with reduced skin
Chem 5, 1–21, October 10, 2019
19

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
photosensitivity. J. Am. Chem. Soc. 140, 2165–
2178.
37. Qi, J., Fang, Y., Kwok, R.T.K., Zhang, X., Hu, X.,
Lam, J.W.Y., Ding, D., and Tang, B.Z. (2017).
Highly stable organic small molecular
nanoparticles as an advanced and
biocompatible phototheranostic agent
of tumor in living mice. ACS Nano 11, 7177–
7188.
temperature phosphorescence. Chem 1,
592–602.
48. Cai, X., Liu, J., Liew, W.H., Duan, Y., Geng, J.,
Thakor, N., Yao, K., Liao, L.-D., and Liu, B.
(2017). Organic molecules with propeller
structures for efficient photoacoustic
24. He, X., Zhao, Z., Xiong, L.H., Gao, P.F., Peng,
C., Li, R.S., Xiong, Y., Li, Z., Sung, H.H.-Y.,
Williams, I.D., et al. (2018). Redox-active
AIEgen-derived plasmonic and fluorescent
core@shell nanoparticles for multimodality
bioimaging. J. Am. Chem. Soc. 140, 6904–
6911.
imaging and photothermal ablation of
cancer cells. Mater. Chem. Front. 1, 1556–1562.
38. Cheng, K., Chen, H., Jenkins, C.H., Zhang, G.,
Zhao, W., Zhang, Z., Han, F., Fung, J., Yang, M.,
Jiang, Y., et al. (2017). Synthesis,
49. Weber, J., Beard, P.C., and Bohndiek, S.E.
(2016). Contrast agents for molecular
photoacoustic imaging. Nat. Methods 13,
639–650.
25. Ai, X., Ho, C.J.H., Aw, J., Attia, A.B.E., Mu, J.,
Wang, Y., Wang, X., Wang, Y., Liu, X., Chen, H.,
et al. (2016). In vivo covalent cross-linking of
photon-converted rare-earth nanostructures
for tumor localization and theranostics. Nat.
Commun. 7, 10432.
characterization, and biomedical applications
of a targeted dual-modal near-infrared-II
fluorescence and photoacoustic
imaging nanoprobe. ACS Nano 11, 12276–
12291.
50. Lin, L., Tian, X., Hong, S., Dai, P., You, Q.,
Wang, R., Feng, L., Xie, C., Tian, Z.Q., and
Chen, X. (2013). A bioorthogonal Raman
reporter strategy for SERS detection of glycans
on live cells. Angew. Chem. Int. Ed. Engl. 52,
7266–7271.
39. Ma, J., Liu, Z., Wang, Z., Yang, Y., Zhang, G.,
Zhang, X., and Zhang, D. (2017). Charge
mobility enhancement for
26. Fan, W., Yung, B., Huang, P., and Chen, X.
(2017). Nanotechnology for multimodal
synergistic cancer therapy. Chem. Rev. 117,
13566–13638.
diketopyrrolopyrrole-based conjugated
polymers by partial replacement of branching
alkyl chains with linear ones. Mater. Chem.
Front. 1, 2547–2553.
51. Wei, L., Hu, F., Shen, Y., Chen, Z., Yu, Y., Lin,
C.C., Wang, M.C., and Min, W. (2014). Live-cell
imaging of alkyne-tagged small biomolecules
by stimulated Raman scattering. Nat. Methods
11, 410–412.
27. Ni, D., Ferreira, C.A., Barnhart, T.E., Quach, V.,
Yu, B., Jiang, D., Wei, W., Liu, H., Engle, J.W.,
Hu, P., et al. (2018). Magnetic targeting of
nanotheranostics enhances cerenkov
40. Zhao, Z., Chen, C., Wu, W., Wang, F., Du, L.,
Zhang, X., Xiong, Y., He, X., Cai, Y., Kwok,
R.T.K., et al. (2019). Highly efficient
photothermal nanoagent achieved by
harvesting energy via excited-state
intramolecular motion within nanoparticles.
Nat. Commun. 10, 768.
radiation-induced photodynamic therapy.
J. Am. Chem. Soc. 140, 14971–14979.
52. Yamakoshi, H., Dodo, K., Palonpon, A., Ando,
J., Fujita, K., Kawata, S., and Sodeoka, M.
(2012). Alkyne-tag Raman imaging for
visualization of mobile small molecules
in live cells. J. Am. Chem. Soc. 134, 20681–
20689.
28. Lovell, J.F., Jin, C.S., Huynh, E., Jin, H., Kim, C.,
Rubinstein, J.L., Chan, W.C.W., Cao, W., Wang,
L.V., and Zheng, G. (2011). Porphysome
nanovesicles generated by porphyrin bilayers
for use as multimodal biophotonic contrast
agents. Nat. Mater. 10, 324–332.
41. Cai, Y., Liang, P., Tang, Q., Yang, X., Si, W.,
Huang, W., Zhang, Q., and Dong, X. (2017).
Diketopyrrolopyrrole-triphenylamine organic
nanoparticles as multifunctional reagents for
photoacoustic imaging-guided
53. Li, X., Jiang, M., Lam, J.W.Y., Tang, B.Z., and
Qu, J.Y. (2017). Mitochondrial imaging with
combined fluorescence and stimulated Raman
scattering microscopy using a probe of
the aggregation-induced emission
characteristic. J. Am. Chem. Soc. 139, 17022–
17030.
29. Feng, G., and Liu, B. (2016). Multifunctional
AIEgens for future theranostics. Small 12, 6528–
6535.
photodynamic/photothermal synergistic
tumor therapy. ACS Nano 11, 1054–1063.
30. Ng, K.K., and Zheng, G. (2015). Molecular
interactions in organic nanoparticles for
phototheranostic applications. Chem. Rev.
115, 11012–11042.
42. Qi, J., Sun, C., Li, D., Zhang, H., Yu, W.,
Zebibula, A., Lam, J.W.Y., Xi, W., Zhu, L., Cai, F.,
et al. (2018). Aggregation-induced emission
luminogen with near-infrared-II excitation and
near-infrared-I emission for ultradeep intravital
two-photon microscopy. ACS Nano 12, 7936–
7945.
54. Jansen, TlC., Snijders, J.G., and Duppen, K.
(2001). Interactioinduced effects in the
nonlinear Raman response of liquid CS2: a
finite field nonequilibrium molecular
dynamics approach. J. Chem. Phys. 114,
10910–10921.
31. Qian, H., Cousins, M.E., Horak, E.H., Wakefield,
A., Liptak, M.D., and Aprahamian, I. (2017).
Suppression of Kasha’s rule as a mechanism
for fluorescent molecular rotors and
aggregation-induced emission. Nat. Chem. 9,
83–87.
43. Zong, L., Zhang, H., Li, Y., Gong, Y., Li, D.,
Wang, J., Wang, Z., Xie, Y., Han, M., Peng, Q.,
et al. (2018). Tunable aggregation-induced
emission nanoparticles by varying isolation
groups in perylene diimide derivatives
and application in three-photon
55. Zheng, M., and Du, W. (2006). Phase behavior,
conformations, thermodynamic properties,
and molecular motion of multicomponent
paraffin waxes: a Raman spectroscopy study.
Vib. Spectrosc. 40, 219–224.
32. Xie, S., Manuguri, S., Proietti, G., Romson, J.,
Fu, Y., Inge, A.K., Wu, B., Zhang, Y., Hall, D.,
¨
Ramstrom, O., et al. (2017). Design and
¨
fluorescence bioimaging. ACS Nano 12,
9532–9540.
56. Troccoli, M., Belyanin, A., Capasso, F.,
Cubukcu, E., Sivco, D.L., and Cho, A.Y. (2005).
Raman injection laser. Nature 433, 845–848.
synthesis of theranostic antibiotic nanodrugs
that display enhanced antibacterial activity and
luminescence. Proc. Natl. Acad. Sci. USA 114,
8464–8469.
44. Qi, J., Sun, C., Zebibula, A., Zhang, H., Kwok,
R.T.K., Zhao, X., Xi, W., Lam, J.W.Y., Qian, J.,
and Tang, B.Z. (2018). Real-time and high-
resolution bioimaging with bright
57. Dickinson, B.C., and Chang, C.J. (2011).
Chemistry and biology of reactive oxygen
species in signaling or stress responses. Nat.
Chem. Biol. 7, 504–511.
33. Zhang, J., Fu, Y., Han, H.H., Zang, Y., Li, J., He,
X.P., Feringa, B.L., and Tian, H. (2017). Remote
light-controlled intracellular target recognition
by photochromic fluorescent glycoprobes.
Nat. Commun. 8, 987.
aggregation-induced emission dots in
short-wave infrared region. Adv. Mater. 30,
e1706856.
58. Mao, D., Wu, W., Ji, S., Chen, C., Hu, F., Kong,
D., Ding, D., and Liu, B. (2017).
45. Hong, G., Antaris, A.L., and Dai, H. (2017).
Near-infrared fluorophores for biomedical
imaging. Nat. Biomed. Eng. 1, 0010.
Chemiluminescence-guided cancer therapy
using a chemiexcited photosensitizer. Chem 3,
991–1007.
34. Mei, J., Leung, N.L.C., Kwok, R.T.K., Lam,
J.W.Y., and Tang, B.Z. (2015). Aggregation-
induced emission: together we
46. Wan, H., Yue, J., Zhu, S., Uno, T., Zhang, X.,
Yang, Q., Yu, K., Hong, G., Wang, J., Li, L., et al.
(2018). A bright organic NIR-II nanofluorophore
for three-dimensional imaging into biological
tissues. Nat. Commun. 9, 1171.
59. Qian, C., Yu, J., Chen, Y., Hu, Q., Xiao, X., Sun,
W., Wang, C., Feng, P., Shen, Q.D., and Gu, Z.
(2016). Light-activated hypoxia-responsive
nanocarriers for enhanced anticancer therapy.
Adv. Mater. 28, 3313–3320.
shine, united we soar! Chem. Rev. 115,
11718–11940.
35. Ding, D., Li, K., Liu, B., and Tang, B.Z. (2013).
Bioprobes based on AIE fluorogens. Acc.
Chem. Res. 46, 2441–2453.
47. Zhao, W., He, Z., Lam, J.Y., Peng, Q., Ma, H.,
Shuai, Z., Bai, G., Hao, J., and Tang, B.Z. (2016).
Rational molecular design for achieving
60. Sun, Q., Zhou, Z., Qiu, N., and Shen, Y. (2017).
Rational design of cancer nanomedicine:
nanoproperty integration and synchronization.
Adv. Mater. 29, 28234430.
36. Qian, J., and Tang, B.Z. (2017). AIE luminogens
for bioimaging and theranostics: from
organelles to animals. Chem 3, 56–91.
persistent and efficient pure organic room-
20
Chem 5, 1–21, October 10, 2019

Please cite this article in press as: Qi et al., Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Sur-
gery, Chem (2019), https://doi.org/10.1016/j.chempr.2019.07.015
61. Liu, J., Chen, C., Ji, S., Liu, Q., Ding, D., Zhao,
D., and Liu, B. (2017). Long wavelength
excitable near-infrared fluorescent
63. Pirro, V., Alfaro, C.M., Jarmusch, A.K., Hattab,
E.M., Cohen-Gadol, A.A., and Cooks, R.G.
(2017). Intraoperative assessment of tumor
margins during glioma resection by
desorption electrospray ionization-mass
spectrometry. Proc. Natl. Acad. Sci. USA 114,
6700–6705.
65. Sagiv-Barfi, I., Kohrt, H.E., Czerwinski, D.K.,
Ng, P.P., Chang, B.Y., and Levy, R. (2015).
Therapeutic antitumor immunity by
checkpoint blockade is enhanced
by ibrutinib, an inhibitor of both BTK and
ITK. Proc. Natl. Acad. Sci. USA 112, E966–
E972.
nanoparticles with aggregation-induced
emission characteristics for image-guided
tumor resection. Chem. Sci. 8, 2782–2789.
62. Pu, K., Mei, J., Jokerst, J.V., Hong, G., Antaris,
A.L., Chattopadhyay, N., Shuhendler, A.J.,
Kurosawa, T., Zhou, Y., Gambhir, S.S., et al.
(2015). Diketopyrrolopyrrole-based
64. Jermyn, M., Mok, K., Mercier, J., Desroches,
J., Pichette, J., Saint-Arnaud, K., Bernstein,
L., Guiot, M.C., Petrecca, K., and Leblond, F.
(2015). Intraoperative brain cancer
66. Liu, Z., Davis, C., Cai, W., He, L., Chen, X., and
Dai, H. (2008). Circulation and long-term fate of
functionalized, biocompatible single-walled
carbon nanotubes in mice probed by Raman
spectroscopy. Proc. Natl. Acad. Sci. USA 105,
1410–1415.
semiconducting polymer nanoparticles for
in vivo photoacoustic imaging. Adv. Mater. 27,
5184–5190.
detection with Raman spectroscopy in
humans. Sci. Transl. Med. 7, 274ra19.
Chem 5, 1–21, October 10, 2019
21