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Accepted Article

Title: Molecular Engineering of NIR-II Fluorophores for Improved

Biomedical Detection

Authors: Fan Zhang and Zuhai Lei

This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.

To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202007040

Link to VoR: https://doi.org/10.1002/anie.202007040

 Angewandte Chemie International Edition 10.1002/anie.202007040

REVIEW

Molecular Engineering of NIR-II Fluorophores for Improved

Biomedical Detection
Zuhai Leia, b, Fan Zhang* a

Keywords：
Fluorophores, NIR-II,

Accepted Manuscript
Bioimaging, Brightness
Fluorescence
biosensing

1
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Angewandte Chemie International Edition
REVIEW
Abstract: The development of fluorophores for the second near-
infrared window (NIR-II, 1000 - 1700 nm) represents a newly
emerging, significant and vibrant field in analytic chemistry, chemical
biology and biomedical engineering. The wavelength, brightness, and
stability are three crucial factors that determine the performance of a
NIR-II fluorophore. Up to now, significant progress has been made in
the development of NIR-II fluorescence molecular probes, including
the synthesis of D-A-D and D--A fluorophores with improved NIR-II
fluorescence imaging performance and the construction of the off-on
probe and ratiometric probe via energy transfer or molecular structure
modification. In this review, we summarize the most recent advances
in molecular engineering design strategies of NIR-II fluorophores and
probes, then highlight a selection of bioimaging and biosensing
applications. We also provide perspectives on potential challenges
and opportunities in this emerging field.
1. Introduction
Fluorescence imaging is one of the remarkable imaging
modalities for improving biomedical detection and image-guided
surgery in both fundamental research and clinical applications.
Compared with the traditional imaging modalities such as X-ray
computed tomography (CT), nuclear magnetic resonance
imaging (NMRI) and positron emission tomography (PET),
fluorescence imaging has advantages such as high
spatiotemporal resolution, real-time detection, non-invasiveness
and low cost.[1] With such a highly sensitive imaging technique,
the molecular or cellular level activities can be well visualized,
which are desirable for us to understand the biological function or
disease. Up to now, fluorescence imaging has been widely used
in immunofluorescence assay, sensing, super-resolution imaging,
single-molecule imaging, 3D imaging and image-guided
surgery.[2] For the past few decades, fluorescence imaging mainly
locates in the visible to NIR-I region (650 - 950 nm).[3] The short
wavelength (< 1000 nm), especially the visible spectrum, results
in poor photon penetration depth, which remains the primary
barricade for in vivo biomedical fluorescence imaging application.
Compared with the visible and NIR-I light, second near-infrared
(NIR-II, 1000 - 1700 nm) light can penetrate more deeply into
biological tissues as less light is scattered at this long-wavelength
window.[4] Unsurprisingly, newly emerging fluorescence imaging
in the NIR-II region has attracted growing attention due to deeper
penetration (approximately 5–20 mm) of biological tissues,
diminished background autofluorescence, and improved signal-
to-noise ratio.[5] Furthermore, recent study has shown that NIR-II
imaging has advantages over NIR-I imaging in clinical
applications.[6]
NIR-II molecular fluorophore is the cornerstone of NIR-II
fluorescence imaging. Recent years have witnessed a fast-
increasing shift to the use of NIR-II fluorophores for in vivo
imaging. Despite the tremendous and urgent need, high-
performance NIR-II fluorophores are still limited. The quantum
yield () of a NIR-II fluorophore is still much lower than the visible
fluorophore due to the small HOMO-LUMO gap and poor
structural rigidity, which results in weak NIR-II fluorescence
brightness.[7] Besides, the conjugative backbone of a long-
wavelength fluorophore is prone to be attacked by reactive
biological species such as ONOO-, cysteine.[8] Therefore, there is
an ongoing effort toward the development of longer-wavelength
(> 1000 nm), bright, stable and biocompatible NIR-II fluorophores.
2
This article is protected by copyright. All rights reserved.
10.1002/anie.202007040
Up to now, several reviews focused on NIR-II fluorescence
bioimaging have been reported.[7, 9] Herein, we review the state-
of-the-art development of NIR-II fluorophores, with particular
attention on high-performance fluorophore and probe design
principles from the molecular engineering point of view. We first
introduce the crucial guidelines of high-performance NIR-II
fluorophore molecular engineering strategies, then highlight a
selection of bioimaging and biosensing performance based on the
fluorophore design, such as dynamic bioimaging and multiplexed
detection. Finally, the current challenges and opportunities of
molecular engineering design principles of NIR-II fluorescent
probes are discussed. We believe this review will help the
Accepted Manuscript
researchers to systematically understand the chemical principles
behind the NIR-II fluorophore for improved bioimaging and
biosensing applications.
2. Molecular engineering strategies for NIR-II
fluorophores development
The pioneering work of NIR-II fluorescence bioimaging was
reported in 2009, and high-resolution images of intravital tumor
vessels beneath thick skin were presented with the ingeniously
prepared single-walled carbon nanotubes (SWNTs) as an
imaging agent.[10] Following this work, many photostable inorganic
NIR-II imaging agents such as quantum dots (QDs) and
lanthanide nanoparticles with tunable emission wavelengths and
brightness were also reported.[11] However, considering the
potential unknown long term toxicity of these inorganic agents for
clinical translation, organic fluorophores are progressively
attractive from a practical point of view because of their compact
molecular structures, minimal concerns over toxicity, synthetic
reproducibility, and facile derivatization. [12, 13] According to their
chemical structures, the reported NIR-II fluorophores up to now
can be roughly divided into two groups, i.e., donor-acceptor-donor
(D-A-D) type fluorophores and polymethine fluorophores (D--A).
In general, to improve the bioimaging and biosensing
performance, three crucial guidelines should be considered in
NIR-II fluorophores design: (1) absorption and emission
wavelength, (2) NIR-II fluorescence brightness and (3)
fluorophore stability (chemostability and photo-stability).
In early research, many strategies have been tried to
bathochromically shift the emission wavelength so that the
maximum emission wavelength can reach the NIR-II window.
Undeniably, some fluorophores emitting beyond 1000 nm work
well in NIR-II bioimaging. However, some NIR-I fluorophores such
as methylene blue (MB, 1)[14] and indocyanine green (ICG, 5)
[15]
had also been verified for NIR-II bioimaging with their off-peak
tail emissions beyond 1000 nm (figure 1). Some of them
performed even better than several NIR-II fluorophores with
maximum emission beyond 1000 nm, which suggests that
wavelength is not the only factor affecting NIR-II imaging
performance. The NIR-II fluorescence brightness, which is a
useful parameter for making comparisons between different
fluorophores and defined as the product of the quantum yield and
extinction coefficient (  ), is a very important factor while the
a
Department of Chemistry, State Key Laboratory of Molecular
Engineering of Polymers, Shanghai Key Laboratory of Molecular
Catalysis and iChEM, Fudan University, Shanghai 200433, P. R. China.
E-mail: zhang_fan@fudan.edu.cn;
b
School of Pharmacy, Fudan University, Shanghai 200433, P. R. China.
Angewandte Chemie International Edition 10.1002/anie.202007040

REVIEW
researchers try to bathochromically shift the maximum
absorption/emission wavelength (max,abs/em) to the NIR-II region.
A plot of    versus max,abs for the major classes of significant
fluorophores (1-19) used in NIR-II imaging is displayed in Figure
1 and table 1. Furthermore, the complex biological environment
requires that the fluorophores should be stable and not susceptive
to reactive biological species. Meanwhile, they also should have
suitable photo-stability under the excitation of external light
source. In short, an optimal biocompatible NIR-II fluorophore
should be (i) bright, (ii) stable in aqueous solution and (iii)
available for a long excitation/emission wavelength. In this section,
we will discuss the detailed molecular engineering strategies for
NIR-II fluorophores design based on the above three crucial
factors.

15
8
3 LZ 1105
ECX
NIR-II FL. Brightness ( x , M-1cm-1)

Accepted Manuscript
CX-2 14
16
3 12
10 IR 820 10
FD 1080
9
Flav7
NJ1060 IR 1061
ICG 5H5
71
6 11
3 CX-3
5
BTC 1070 19
102 17
13
FTX
18
CH-4T 2
3 IR 26
4
MB
1 IR-BGP IRDyes 800CW Rh 1029

101
600 800 1000 1200
Wavelength (nm)

Figure 1. Plot of NIR-II fluorescence brightness ( x ) vs. the maximum absorption wavelength (max) for the representative fluorophores used for NIR-II bioimaging.

Table 1 The photophysical data of fluorophores in figure 1.

Zuhai Lei received his PhD degree (2017) from

λabs/λem ε (× 10 5
ΦNIR-II εΦNIR-II (M - East China University of Science & Technology
NO. Solvents Ref. (ECUST) followed by 2 years postdoctoral
(nm) M-1 cm-1) (%) 1
cm-1)
a experience in the Fudan University. He is
1 H2O 665/683 0.7 0.02 14 14b
2a HSA 748/1005 0.1 0.48 48 15a currently a tenure-track professor at Fudan
3a PBS 763/1047 0.108 0.3 32 32c University, where his research interests focus on
4# H2O 774/789 2.4 N. A. 21 15b the design and synthesis of NIR-I/II fluorescent
5b H2O 785/822 1.2 0.1 120 15c probes for bioimaging and biosensing.
6 H2O 815/829 1.5 0.09 135 52
7a toluene 889/1080 0.24 1.2 288 30
8b DCM 879/920 1.32 3.1 4000 31 Prof. Fan Zhang received his PhD in 2008 from
9a H2O 910/1060 1.7 0.5 850 24a Fudan University followed by more than 2 years
10a CHCl3 981/1032 1.9 0.45 855 8f postdoctoral experience in the University of
11a DCM 1014/1070 1.15 0.09 104 20 California at Santa Barbara before joining as a
12a DCM 1026/1045 2.36 0.53 1200 23 professor in the Chemistry Department of Fudan
13a DCM 1029/1093 0.19 0.33 63 50 University in 2010. His current research
14a FBS 1046/1080 0.29 5.9 1700 34
interests include bioanalysis, bioimaging and
15a DMSO 1058/1100 1.48 3.89 5700 54
drug delivery. Prof. Zhang has authored a
16b DCM 1061/1100 2.38 0.59 1400 47c
number of book chapters, patents, and more
17a MeCN 1069/1125 0.34 0.26 88 49
18b DCE 1080/1130 1.03 0.05 51 27b than 100 peer-reviewed research papers.
19a CHCl3 1089/1140 1.05 0.09 95 8f

Note: NIR-II means NIR-II fluorescence intensities were integrated beyond 1000
nm. a: Relative quantum yield with IR 26 as a reference (IR 26 = 0.05% in DCE); 2.1. Molecular engineering for longer absorption/emission
b: Absolute quantum yield. #The NIR-II brightness of IRDyes800CW is a relative wavelength
brightness to IR-E1050 since no direct data about its NIR-II Φ is available. The
IR-E1050 brightness = 0.8 x 104 M-1 cm-1*0.2%=16 M-1 cm-1, IRDyes
800CW=1.3*E1050=21 M-1 cm-1.

3
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Angewandte Chemie International Edition 10.1002/anie.202007040

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Generally, a fluorophore is a push-pull conjugate system.
NIR-II fluorophores exhibit a smaller HOMO-LUMO gap than the
NIR-I fluorophores. In principle, this can be accomplished by
raising the HOMO or lowering the LUMO of a NIR-I fluorophore
(figure 2A). Although the structures of D-A-D fluorophores and
polymethine dyes (D--A) are quite different, they share the same
strategies to bathochromically shift wavelength: (1) elongating the
conjugated chain, (2) increasing the electron density of donor and
(3) decreasing the electron density of acceptor.
Lengthening the conjugated chain can be used to
bathochromically shift the wavelength apparently.[16] For example,
as shown in figure 2B, when a vinylene unit is incorporated into
the polymethine fluorophore 20 to get 21, a large bathochromic
shift is obtained.[17] This strategy is also suitable for the D-A-D
fluorophores. The D-A-D fluorophore CH-1055-PEG is known as
the pioneering small organic fluorophore used for NIR-II
bioimaging.[18] The high-resolution images with superior signal-to-
background ratio (SBR) at depths of millimeters attract much
attention from then on. Following this fascinating work, many
improved chemical structure modifications were reported, such as
bathochromic shift the wavelength by incorporating conjugated
thiophene.[19]

Figure 2. Schematic illustration of structural engineering for longer wavelength (A, the blue emission tail represents those NIR-I fluorophores that can be used for
NIR-II bioimaging with their tail emissions) and representative strategies: (B) elongating the conjugated chain, (C) donor modification by increasing the electron
Accepted Manuscript
density, (D) acceptor modification by decreasing the electron density, (E) Exchange of heteroatom, (F) formation of J-aggregate. F: Reproduced with permission
from Ref. 29. Copyright 2019, American Chemical Society. The key change points are marked with red.

Besides, donor modification can also bathochromically shift
the wavelength apparently (figure 2C). For a D-A-D or D- −A
fluorophore, increasing the electron density of the donor usually
results in lower HOMO-LUMO gap.[20] As shown in the seminal
work of NIR-II D-A-D fluorophore, when the donor was changed
from fluorene to triphenylamine (TPA), 93 nm bathochromic shift
was realized (22 to 23).[21] When an amino was introduced to the
donor fluorene, 140 nm emission bathochromic shift was
observed (24 to 25).[22] Addition of an electron-donating
dimethylamino group to the benzopyrylium of IR 27 (26) can get
Flav7 (12).[23] The maximum absorption and emission of Flav7 are
much longer than these of IR 27. Besides, shortening the
polymethine chain from heptamethine to pentamethine
meanwhile introducing an electron-donating diethylamino group
to the benzothiopyrylium unit can get an elegant long-wavelength
NIR-II fluorophore BTC1070 (11) with maximum absorption
beyond 1000 nm. More importantly, BTC1070 does not cross the
cyanine limit in aqueous solution.[20] The cyanine limit is
characterized by intense and narrow absorption with minimal
bond length alternation and delocalized charge on the conjugation
chain. Beyond the cyanine limit, i.e., the linear system of
polymethine fluorophore undergoes symmetry collapse and

4
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Angewandte Chemie International Edition
REVIEW
charge localization, is characterized by weak and broad
absorption. Fluorophores with long-wavelength but cross the
cyanine limit cannot be used for bioimaging. In addition to these
D-A-D fluorophores and polymethine structures, some aza-
BODIPY with strong electron donors also have long-wavelength
extending to the NIR-II window (9). At the same time, they also
have a large Stokes shift, excellent photo-stability and good
fluorescence brightness(figure 1).[24]
Furthermore, acceptor modification can also
bathochromically shift the wavelength apparently (figure 2D).
Decreasing the electron density of the acceptor usually
results in lower HOMO-LUMO gap.[21] For the D-A-D
fluorophores, when the sulfur atom in the acceptor is
replaced by selenium atom, large bathochromic shift is
realized (28 to 29).[21] Interestingly, replacing thiophene with
pyrrole results in a large bathochromic shift. This should be
due to pyrrole is more electron-donating than thiophene.
More importantly, the hydrogen of pyrrole can form an
intramolecular hydrogen bond with the acceptor, which
results in decreased acceptor electron density. [25]
Exchange of heteroatom is also a practical molecular
engineering strategy to bathochromically shift the
wavelength. Usually, varying the oxygen atom of heterocycle
to carbon group atom or other chalcogen atom results in red-
shifted wavelength.[26] For example, when the oxygen of
fluorophore 26 was replaced by sulfur atom to get
fluorophore 18, about 100 nm bathochromic shift was
realized (figure 2E).[27]
To fabricate J-aggregate is also an effective method to obtain
long-wavelength imaging agents. Fluorophore with a conjugated
plane structure may form aggregate in solution sometimes. One
of which is J-aggregate, in which fluorophores orient in head to
tail fashion. Presenting with bathochromically-shifted absorption
and emission spectra, enhanced extinction coefficient (),
shortened fluorescence lifetime () and small Stokes shift, J-
aggregate is a desirable in vivo imaging agent although it is
challenging to obtain and stabilize the fluorophore alignment in
complex settings.[28-29] Fortunately, the aggregates can be
stabilized in a complex biological environment if they are loaded
in appropriate vehicles. For example, based on the self-assembly
of FD-1080 (14) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC), stable FD 1080 J-aggregate with absorption and
emission beyond 1300 nm was obtained (figure 2F).[29a] The IR
140 J-aggregate can be formed and stabilized when loading
fluorophore IR 140 (30) into the hollow mesoporous silica
nanoparticles (figure 2F).[29b] The maximum absorbance of the
aggregate is 1040 nm. Compared with its monomer fluorophore
IR 140 which emitting at 826 nm, 114 nm bathochromic-shift was
realized. Both these two J-aggregates mentioned above are
elegant NIR-II imaging agents and stable during the process of in
vivo fluorescence bioimaging
2.2. Molecular engineering to enhance NIR-II fluorophores
brightness
The fluorescence quantum yield of a NIR-II fluorophore is
typically low owning to small HOMO-LUMO bandgap and poor
structural rigidity. The brightness of most existing fluorophores
used for NIR-II imaging is less than 103 M-1 cm-1(figure 1), which
is still much lower than that of visible to NIR-I fluorophores (up to
105 M-1 cm-1).[12g] The low brightness of NIR-II fluorophores in
5
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10.1002/anie.202007040
aqueous solution are significantly related to the interactions with
water molecules, which result in energy transfers from the excited
states of molecules to the surrounding water. [30] Generally, the
intermolecular interactions can be efficiently reduced by steric
hindrance (figure 3A). Fluorophores with reduced intermolecular
interactions are usually much brighter. [7, 31] There are two main
methods to introduce steric hindrance to the fluorophores. The
first strategy is to directly connect bulky side-groups to the
conjugated plane of the fluorophore by covalent bonding. For
example, to improve the NIR-II fluorescence performance of the
D-A-D fluorophores, the side chains were introduced to the
donor(32 to 33), i.e., thiophene and fluorene (figure 3B).[19a, 30,
32]
Accepted Manuscript
The side chains on the shielding units extend out of the D-A-D
core structure backbone and form a nonpolar hydrophobic cavity
that enfolds the core skeleton. The cavity can efficiently prevent
the intermolecular interactions between the core backbone and
water. Besides, 3,4-ethoxylene dioxythiophene (EDOT) or 3-(2-
(2-(2-methoxyethoxy)ethoxy) ethoxy) (TEG) or 3-alkyl (octyl)
substitutions were also installed into the donor thiophene. These
substitutions can efficiently distort the conjugated backbones,
which could efficiently reduce intermolecular interactions and
energy transfer from the excited states of molecules to the
surrounding water.[33] In short, both side chains on the fluorene
and substitutions on the thiophene can form a hydrophobic
environment that could reduce the energy of the excited state lost
to the surrounding water through the non-radiative way.
Packing the fluorophores with protein to form a stable
protein-fluorophore complex via supramolecular interactions is
also an effective method to introduce steric hindrance (figure
3C, D). Usually, the protein is albumin while the fluorophore is a
fluorescent dye that bearing sulfonate. The complex is usually
much brighter than fluorophore alone since the intramolecular
rotation of fluorophore was restricted and the intermolecular
interaction between the core structure of fluorophore and water
was reduced. Following this strategy, some bright fluorophore-
protein complexes were fabricated with sulfonate bearing
fluorophores such as FD1080 (14)[34], CH-4T (2)[15a], CQ-4T
(31)[35], ICG (5), IR12N3 and IR783[36] (figure 3c). Among these
complexes, some of them can produce a more than 100-fold
increase in NIR-II fluorescence.
Another method to improve the brightness is to shorten the
emission wavelength. It sounds like a dilemma at first. On the one
hand, we want a longer emission wavelength since longer
wavelength means lower background fluorescence and less bio-
tissue scattering. On the other hand, fluorophores with longer
emission wavelengths are much more likely to deactivate the
excited state through rotation or vibration, which results in low
quantum yield. Molecular engineering should balance the trade-
off between fluorescence emission and non-radiative internal
conversion. For example, when the donor TPA is modified with
the strong electron-withdraw phenylazo group to get a new
fluorophore TPB-AZO (35), the bandgap increases since the
intramolecular charge transfer is weakened, which results in
hypochromatic shift. At the same time, the quantum yield and
brightness increase significantly (figure 3E).[37] Besides,
suppressing the excited state twisted intramolecular charge
transfer (TICT) can also improve the brightness. The excited state
TICT often happens when the donor is a tertiary amine especially
an aliphatic tertiary amine, which results in decreased quantum
yield. For example, the quantum yield Q8P (36) is quite low
because the dimethylamine will form a TICT at the excited state.
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REVIEW
When the dimethylamine is protonated or alkylated (37), the TICT
can be well suppressed, which results in increased quantum yield
(figure 3E).[38]
Besides, most NIR-II fluorophores suffer from aggregation-
caused quenching (ACQ) in the aqueous solution (38-40). In
contrast, fluorophores with aggregation-induced emission (AIE)
phenomenon emit intensely when aggregated in aqueous solution
because of the restriction of intramolecular motion (RIM)
mechanism. Therefore, the molecular engineering of fluorophores
for AIE is an effective strategy to obtain high bright NIR-II
fluorescence. To improve the AIE performance of these NIR-II
fluorophores, many strategies were applied. [39] For example, the
AIE effects of those D-A-D fluorophores can be finely tuned by
changing the substitutions of donor thiophene, such as HL3
(41)[40], 2TToC6B (42)[41]. Besides, excellent AIE performance can
also be obtained by introducing the classical tetraphenyl ethylene
group to the donor thiophene, such as TB1 (43)[42] and HLZ-BTED
(44)[43] (figure 3F).

Figure 3. Schematic illustration of molecular engineering to enhance fluorophore brightness and representative strategies. (A) Schematic illustration of fluorophore
protection and modification. Briefly, introducing steric hindrance covalently (i), assembly with protein (ii), and structural modification (iii) are three effective strategies
to enhance brightness. (B) Introducing steric hindrance by donor modification. (C) Assembly of NIR-II fluorophores and protein to get better performance. (D)
Emission spectra and NIR-II fluorescence images of FD1080 in PBS and FBS. (E) Donor modification to increase the quantum yield. (F) Structural modification for
Accepted Manuscript
AIE. The key points are marked with red. The quantum yields are relative quantum yield with IR 26 as a reference (IR 26 = 0.05% in DCE).

wavelength fluorophores is notoriously poor, as well. They may

2.3 Molecular engineering to improve NIR-II fluorophores
stability
An outstanding fluorophore used for bioimaging should have
suitable chemostability and photo-stability in aqueous solution.
However, the chemostability of long-wavelength fluorophores are
intrinsically poor because of a small HOMO-LUMO gap. A low
LUMO orbital leads to susceptibility to nucleophilic attacks, such
as intracellular thiols (cysteine, glutathione, etc.).[3a] A high HOMO
orbital leads to susceptibility to electrophilic attack and
oxidation.[44] At the same time, the photo-stability of most long-
readily decompose or isomerize upon photoexcitation. [45]
Therefore, reducing the possibility of reactive species attack may
increase chemostability. Steric hindrance is a generic method to
protect organic molecules from attacking by reactive species
(figure 3A). Based on this idea, the bulky dioctyl chain-substituted
3,4-propylenedioxy (PDOT) thiophene was introduced as the
donor of the D-A-D fluorophores. Compared with the EDOT or
unsubstituted thiophene, the bulky steric hindrance of PDOT can
afford better protection of the conjugated backbone from
interaction with reactive species, and thereof results in improved
stability.[32a, 46] Besides, a micelle is also a competent shelter for

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Angewandte Chemie International Edition 10.1002/anie.202007040

REVIEW
NIR-II fluorophores.[47] Furthermore, packing the fluorophores
with protein to form a protein-fluorophore complex via
supramolecular interactions is also an effective method to
improve NIR-II fluorophores stability, such as CH-4T[15a].
Since isomerization can result in low photo-stability,
fluorophores with the more rigid conjugated planes should be
more stable. Therefore, it is not surprising that the
performance of polymethine fluorophores can be significantly
improved by rigidifying the conjugated backbone
structures.[48] Inspired by the rigid xanthene structure, CXs
(10, 19) (figure 1), characterized by two xanthene units linked
by an odd number of methine units, are partially rigidified
imaging, small vessel imaging, lymph node imaging, etc.
Water solubility is an essential issue to address in using NIR-
II fluorophore for bioimaging. To modify the hydrophobic core
of the NIR-II fluorophores, many chemical routes have been
developed. The hydrophilic groups such as sulfonate,
carboxyl and polyethylene glycol (PEG) with different
molecular weights are widely used to modify the core
fluorophore structures to obtain good solubility. Besides, to
encapsulate the hydrophobic fluorophore into the micelle or
other carriers is also widely used, in which the NIR-II
fluorophores perform like hydrophilic nanoparticles (figure
4A).[49] Generally, sulfonated fluorophores or fluorophores

polymethine fluorophores and stable in aqueous solution
with maximum absorption wavelength at 1090 nm. [8f] 5H5 (17)
is also a partially rigidified polymethine fluorophore with
outstanding performance in NIR-II fluorescence bioimaging.
Intriguingly,[49] although the Rhs (13) fluorophores which
constructed by the integration of xanthene and
benzo[cd]indolium are asymmetric, they also performed well
in NIR-II bioimaging.[50]
2.4 Fluorophores with off-peak fluorescence extends to NIR-
II window
Accepted Manuscript
with large molecular weight PEGlation are excreted rapidly
and non-targeted.[18, 34] Therefore, they are suitable for
dynamic in vivo bioimaging (figure 4B). For targeted
bioimaging, the target molecular ligand, such as affibody,
hormone or peptide can be conjugated to the fluorophores
via carboxyl (45), maleimide or click reaction (figure 4C).[18,
32e, 33a, 53]
At the same time, multiplexed imaging can be
realized by using different fluorophores with non-overlapping
emission spectra.

During the early stage of NIR-II bioimaging, most

fluorophores used as imaging agents are those emitting
beyond 1000 nm. However, the brightness of most of these
fluorophores is quite low (figure 1). The following researches
show that the emission spectrum of ICG on InGaAs detector
can extend even beyond 1500 nm with a peak at 820 nm. [15,
36, 51]
Since the NIR-II fluorescence brightness of ICG is much
higher than most NIR-II fluorophores (figure1), impressing in
vivo NIR-II images are obtained with its off-peak tail emission
beyond 1000 nm. The success of ICG indicates that many
other traditional NIR-I fluorophores may be used as NIR-II
imaging agents directly with a high quantum efficiency NIR-
II detector such as InGaAs camera. Indeed, following ICG, Fl

IR 820[52] and IRDye 800CW[15b] are also successfully applied Fl Fl

for NIR-II imaging. More recently, MB (FDA approved) which

peaks at 665 nm with a low  of 3.8 % also been successfully
applied for NIR-II in vivo mice imaging.[14] The success of
NIR-II bioimaging of MB may be regarded as a landmark
event. Many traditional NIR fluorophores with better
fluorescence performance than MB, such as silica
rhodamine, ECXs, Cy5, Cy5.5, BODIPY, et al., will be
potential options for NIR-II bioimaging.[12g, 31] However, the
tissue penetration depth and imaging resolution, which
predominantly depend on the absorption and scattering of
both excitation and emission light, will be remarkably Figure 4 Representative NIR-II fluorophores for bioimaging. (A) The
hydrophobic structure 5H5 was used for vessels and tumor imaging via
improved by bathochromic shift of excitation wavelength
hydrophilic encapsulation, in which the fluorophore performs like a
from NIR-I to NIR-II.[34] The short excitation wavelength may nanoparticle. Reproduced with permission from Ref. 49. Copyright 2018
become the bottleneck of those NIR-I fluorophores for deep American Chemical Society. (B) The water-soluble LZ1105 was used for
tissue imaging. Therefore, NIR-II fluorophore with a longer dynamic molecular imaging of the carotid artery, the dynamic process of
thrombolysis after injection of thrombolytics was clearly visualized (yellow
wavelength and high brightness is still urgently needed for
arrows). (C) CH1055 was modified for targeted tumor imaging.
imaging in deeper depth with higher resolution. Reproduced with permission from Ref. 18. Copyright 2016, Springer
Nature. The key points are marked with blue. FL.: Fluorophore.

3 NIR-II bioimaging
3.1 Dynamic bioimaging
The last several years have witnessed a myriad of
impressive NIR-II bioimaging such as tumor imaging, liver

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Angewandte Chemie International Edition
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As a proof-of-concept bioimaging application, the vessel
in deep tissue is a well-established dynamic imaging object.
Many NIR-II fluorophores have been successfully used for
non-invasive in vivo cerebrovascular imaging. The brain
vasculature and abdominal vessels of mice can be clearly
visualized. Besides, based on the respiratory craniocaudal
motion of the liver, the mouse’s respiratory rate can also be
quantified via NIR-II fluorescence imaging.[34]
NIR-II fluorophore with a long-term circulation time can
be used for real-time monitoring of the thrombolysis in the
carotid artery. For example, LZ1105 (15), which bearing four
sulfonate groups, has long-term blood circulation time and
can be used for continuous monitoring of the dynamic
vascular process.[54] In a FeCl3 induced carotid artery
thrombosis model mouse, the dynamic processes of
thrombolysis and blood flow recovery after injection of
recombinant tissue plasminogen activator (rt-PA, a drug for
treating thromboembolism) were undoubtedly visualized by
NIR-II fluorescence of LZ1105 (figure 4b, yellow arrows).
Through dynamic real-time NIR-II fluorescence imaging, the
potential incurable massive hemorrhage could be avoided by
controlling the administration of rt-PA.
Fluorescence-guided surgery is also, to some extent, a
dynamic imaging process. Since NIR-II fluorescence imaging
is barely affected by the illumination of the operating room
and has improved image resolution, it should be a reliable
modality for dynamic fluorescence-guided surgery. For the
last several years, some NIR-II fluorescence-guided
surgeries were successfully performed with NIR-II
fluorophores. Different kinds of tumors including U87MG
tumor, glioma, human liver-tumor, etc., were resected with
the help of NIR-II fluorescence.[6, 18-19] Generally, the tumor-
to-normal-tissue ratios (TNRs) of NIR-II are much higher
than those of NIR-I. Moreover, NIR-II imaging can
discriminate tumor lesions more effectively compared with
NIR-I imaging and can visualize some tumor lesions that are
missed by NIR-I imaging.[6]
3.2 Multiplexed bioimaging
Multiplexed bioimaging allows us to simultaneously
analyze multiple targets, which is very important for
biomedical research. The signals of multiplexed bioimaging
for readout are usually based on fluorescence parameters
such as wavelength, intensity, and lifetime. [55] The
fluorescence wavelength is the most popular readout signal
for multiplexed bioimaging with NIR-II fluorophores. For
multiplexed imaging in the visible window, multicolor can be
conventionally achieved since numerous fluorophores with
high quantum yield and different wavelengths are available.
However, up to now, only dual-color[32e] and three-color[33a]
bioimaging in the NIR-II window have been reported.
Meanwhile, most NIR-II multicolor bioimaging was realized
by combining organic fluorophores with inorganic imaging
agents such as QDs and SWCNTs.[33a] That is because all
NIR-II fluorophores available now suffer from limitations,
such as low quantum yields, small Stokes shift, broad
absorption and emission spectra, and no organic fluorophore
of which maximum emitting beyond 1500 nm in aqueous
solution is available.
8
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10.1002/anie.202007040
Generally, different imaging agents with non-overlapping
emission are needed for multiplexed bioimaging in vivo. For
example, the fluorophore IR-FEPC and PbS QD were used
for dual-color imaging of hormone receptors. IR-FEPC and
PbS were conjugated with hCG and FSH to form hCG@IR-
FEPC and FSH@PbS respectively. The 1100 nm long pass
(LP) signal of hCG@IR-FEPC and 1500 nm LP signal of
FSH@PbS reveal hCG receptor and FSH receptor
respectively.[32e] The Depp Red (850 – 900 nm), IR-FGP
(1050 -1300 nm) and SWCNT (1500 -1700 nm) were used
for three-color imaging of brain tissue, in which cell nuclei,
neuron and blood vessel were labeled with Depp Red,
lgG@IR-FRP and SA@SWCNT, respectively.[33a] The IR-
Accepted Manuscript
783@BSA complex (900 – 1000 nm), IR-FD (1100 – 1300
nm), and PbS/CdS QD (>1500 nm) were simultaneously
used for depicting tumor, lymph nodes, and blood vessels,
respectively.[32a] For three-color NIR-II imaging with organic
fluorophores, the emission spectra of fluorophores should be
overlapped as less as possible. Besides, their brightness
should also be at the same level. Unfortunately, up to now,
fluorophores that meet the requirements are still limited.
4 NIR-II fluorescent biosensing probes
Fluorescence bioimaging in the NIR-II window allows us
to picture deep tissue with ultrahigh resolution. At the same
time, it is also urgent to realize biosensing in deep tissue,
which requires that the fluorescent probe should be able to
respond to the specific stimuli selectively. With the help of
NIR-II fluorescent probes, biological or pathological
processes in deep tissue would be visualized. Up to now, two
kinds of NIR-II fluorescent probes have been reported, i.e.,
off-on probes and ratiometric probes.
4.1 Molecular engineering for Off-On probes biosensing
The fluorescence of the off-on fluorescent probe would be lit
up after the probe interacts with the targets of interest (figure 5A),
which is the most widely used molecular engineering strategy.[56]
The photophysical property of some fluorophores can be
dramatically changed with different substitutions on the core
structure. For example, the spectra of BODIPY in figure 5B
change obviously with the alteration of the substitution R group
(49-52). Therefore, this BODPY based platform can be
conveniently turned into off-on NIR-II fluorescent probe, such as
H2S[57], -galactosidase (-gal), nitroreductase (NRT), alkaline
phosphatase (ALP), NAD(P)H:quinone oxidoreductase isozyme
(NQO), or viscosity probe (Figure 5B).[58] Beside substitute
modification, the general fluorescence quenching group nitro can
also be applied in the NIR-II probe. For example, the fluorescence
of IR 1048 is quenched by the nitro-imidazole and can recover
after nitro reduction (53 to 54, Figure 5C).[59] Most fluorophores
suffer from aggregation caused quenching (ACQ). While ACQ
nanoparticles also can be turned into off-on type probe if the
aggregate can decompose into monomer at some specific
situation. For example, HISSNPs which self-assembled by an
amphipathic IR 1061-pendent hyaluronic acid polymer in aqueous
solution and further crosslinked by disulphide can be
decomposed by hyaluronidase and thiols. At the same time, the
Angewandte Chemie International Edition 10.1002/anie.202007040

REVIEW
fluorescence of IR 1061 recovers when the aggregate is
disassembled (Figure 5D).[60]
Another strategy to light up fluorescence is covalent-
assembly. The fundamental feature of the covalent-
assembly principle is that the conjugative backbone of a
fluorophore is split into two fragments in the probe. These
two fragments are covalently assembled to reconstruct the
fluorophore through a chemical cascade, which can be
selectively triggered by the analyte of interest. This feature
warrants a turn-on fluorimetric signal from a zero background,
that is, the probe is highly sensitive. [61] For example, the
acceptor of the probe AOSNP (55) is split into two aminos,
5E). [62] Just like covalent-assembly, “conjugate-disruption” is
also a general strategy to fabricate off-on type fluorescent
probe. The fundamental feature of the “conjugate-disruption”
principle is that the conjugative double bond of a fluorophore
is disrupted into a single bond in the probe. The single bond
is turned into a conjugative double bond through a chemical
cascade, which is selectively triggered by the analyte of
interest. This feature warrants a turn-on fluorimetric signal
from a zero background or bathochromicly-shifted emission.
For example, the probe IRBTP-P (57) which has not
fluorescence in the NIR-II region since its conjugation chain
is disrupted can be turned into fluorophore IRBTP-O (58)

Accepted Manuscript
which results in little fluorescence since the D-A-D structure which recovers fluorescence in the NIR-II region when the
is broken. The whole D-A-D structure (AOSNP-NO, 56) is masking group phenyl borate is removed by OONO-, so does
restored when the probe reacts with the analyte NO (Figure Hydro-1080 (59 to 60, Figure 5F).[63]

F
Fluorescence Intensity

F0
 (nm)

Figure 5． Representative OFF-ON NIR-II fluorescent probes and schematic illustration. A) Illustration of the off-on mechanism and the fluorescence
change pattern. B) Fluorophore substitution probe. The mechanism and scope of NIR-II fluorescent probe based on the BODIPY platform C) Nitro
quenching probe. D) Aggregate disassembly probe. E) Covalent-assembly probe. F) Conjugate-disruption probe. G) Energy transfer (FRET and CRET)
probe.

The principle of Förster resonance energy transfer (FRET) window is also an efficient strategy to develop an off-on
is also an efficient strategy to construct an off-on probe. In probe, which can be realized by energy transfer (BRET and
the FRET system, if the acceptor is a fluorescence quencher, FRET) with little background fluorescence interference.[65]
the fluorescence of the donor will recover when the acceptor For example, the NIR-II chemiluminescence of NIR-II
is destructed or the donor and acceptor are separated from chemiluminescence sensor (CLS, energy transfer from 63 to
each other. For example, the NIR-II fluorescence of probe 64 then to 65) can be selectively triggered by H2O2, which
SNP25 (61, 62) recovers when the FRET acceptor (62) is can be used for inflammation detection.[65]
destructed by ClO-(Figure 5G).[64] Unfortunately, the
available biocompatible NIR-II fluorescence quenchers are 4.2 Molecular engineering for ratiometric probes
still limited. Besides, chemiluminescence in the NIR-II biosensing

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REVIEW
The off-on probes are widely used in qualitative
biosensing. Compared with off-on probes, ratiometric probes
are more accurate and reliable for quantitative or semi-
quantitative biosensing with built-in self-calibration (Figure
6A).[66] For example, different pHs at different tissue depths
were measured by a NIR-II fluorescent probe BTC1070
(Figure 6B).[20] BTC1070 bears two amino groups with the
pKa values are calculated to be 0.29 and 3.81, which shows
ratiometric fluorescence change from 1065 nm to 980 nm
between pH 1-4. The maximum emissions of 1065 nm and
980 nm correspond to BTC1070 and BTC1070H 2+2 ,
respectively. Therefore, by dividing NIR-II ratiometric
reliable accuracy comparable to standard pH meter was
realized. Both BTC1070 and BTC1070H 2+2 are symmetric
structures with strong emission, while the asymmetric
BTC1070H+ emits weakly. That is to say, the fluorescence
of NIR-II polymethine fluorophores can be manipulated by
structural symmetry modification. From the fluorescence
mechanism point of view, the wavelength of BTC1070 is
strongly affected by the amino groups due to the
intramolecular charge transfer (ICT) effect of nitrogen atoms.
Nevertheless, the ICT process is suppressed when the
amino groups are protonated. Therefore, the wavelength of
BTC1070H 2+2 is blue-shifted. Following this strategy, many

Accepted Manuscript
imaging sub-regions and drawing calibration curves, non- new NIR-II ICT probes could be constructed by ICT
invasive gastric pH evaluation at a depth of 4 mm with manipulation.

Figure 6． Representative ratiometric NIR-II fluorescent probes and schematic illustration. A) Illustration of the ratiometric probe mechanism and the
fluorescence change pattern. B) Structure of BTC1070 and its fluorescence spectra at various pH values and digital photographs of mice stomach . C)
The structure and spectra of CXs, the schematic illustration of the detection mechanism of PN1100 and images of mice liver. Reproduced with
permission. Copyright 2019, Wiley-VCH[8f]. The schematic illustration of the detection mechanism of NdMY and images of mice liver. Reproduced with
permission. Copyright 2020, Wiley-VCH[67]. D) Schematic illustration of the ratiometric probe fabricated by hybridizing inorganic particl es with organic
fluorophores, the fluorophore in a and b is SeTT and Cy 925, respectively. Reproduced with permission. Copyright 2020, American Chemical Society [68-
69]
.

FRET is also an effective strategy to develop a ratiometric
NIR-II fluorescent probe. The FRET efficiency which defined
as the proportion of the donor molecules that have
transferred excitation state energy to the acceptor molecules
decreases with increasing intermolecular distance (typically
in the range of 1–10 nm). The emission spectra of the FRET
pair and the donor’s lifetime change obviously with the
distance or the ratio of donor to acceptor. Therefore, a ratio
signal can be tuned by donor or acceptor manipulation. For
example, the donor’s emission strength and lifetime increase
when its counterpart is destructed or departs. Following this
strategy, a ratiometric NIR-II fluorescent probe PN1100 was
developed by simultaneously loading CX-1 and CX-3 into a
micelle (Figure 6C)[8f] and a NIR-II fluorescence lifetime
based probe NdMY was constructed by incorporating the
fluorophore MY-1057 (63) into the Nd3+ nanoparticle (Figure
6C)[67]. Both PN1100 and NdMY showed high selectivity
towards OONO- and they have been successfully verified in
drug-induced hepatotoxicity and hepatic carcinoma mice,
respectively. Significantly, lifetime imaging is independent of
tissue penetration depth. Therefore, the development of the
NIR-II FRET-based lifetime probe is an attractive strategy for
the detection of targets in deep tissue.
Inorganic-organic hybrid is another strategy to develop a
NIR-II ratiometric probe. Inorganic nanoparticles (NP) such
as those doped with rare-earth ion have NIR-II fluorescence

10
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Angewandte Chemie International Edition
REVIEW
emission under laser excitation. They are usually stable and
not susceptive to reactive species, such as HClO, OONO -,
H2O2. On the other hand, organic fluorophores are relatively
more susceptive to the reactive species. Therefore, a
ratiometric NIR-II probe can be conveniently constructed by
incorporating a fluorophore into a nanoparticle. Usually, they
should have overlapped absorption but non-overlapped
emission spectra in order to get a ratio output with the same
excitation. For example, when the surface of Er3+ doped
nanoparticle (DCNP) is encapsulated with fluorophore SeTT
(64)[68] or Cy925 (66)[69], a NIR-II ratiometric fluorescent
probe for the detection of HClO is fabricated (figure 6D). Both
SeTT and Cy920 react with HClO selectively, which results
in fluorescence loss, while the emission of DCNP is not
influenced. Therefore, the ratio signal of fluorophore and
DCNP is a good indication for the presence of a different
concentration of hypochlorous acid, and the rationality of the
probes has been verified in the arthritis mice model.
5 Conclusions & outlook
NIR-II organic fluorophores have several advantages,
such as compact molecular structures, minimal concerns
over toxicity, and facile derivatization. Up to now, many
molecular engineering strategies of NIR-II fluorophores and
fluorescent probes have been established for bioimaging and
biosensing in deep tissue with high resolution and sensitivity.
Nevertheless, the majority of existing NIR-II fluorophores and
probes are still in their infancy, with limitations for clinical
application. To expand the applications of NIR-II
fluorophores in bioimaging and biosensing, some challenges
have to be overcome.
First, high-performance fluorophores are still limited. As
discussed above, a high-performance fluorophore should be
bright enough in the NIR-II window with appropriate chemical
and photo-stability. The D-A-D fluorophores, represented by
CH1055, are characterized by large Stokes shift and long
emission wavelength. Up to now, they have been thoroughly
studied. Many impressing images were recorded with these
D-A-D fluorophores. The emission wavelengths of D-A-D
fluorophores range from 1000 nm to 1450 nm with different
combinations between donors and acceptors. At the same
time, the fluorescence brightness is proportional to  and .
However, both  and  of these D-A-D fluorophores are far
from perfect.[46] Of course, one practical strategy to produce
a brilliant multi-fold increase in fluorescence is that packing
these fluorophores with protein to form a protein-fluorophore
complex.[15a] Future research should focus on balancing the
fundamental trade-off between large Stokes shift and
brightness. For polymethine fluorophores, the cyanine limit
should be considered since flexible long-wavelength
heptamethine usually beyond the cyanine limit. [70] One
practical strategy to fix this challenge is to shorten the
methine chain meanwhile enhancing the heterocyclic end
unit. Probably, conformational restraint may also be an
option to acquire NIR-II emission while not beyond the
cyanine limit. At the same time, many NIR-II fluorophores are
not stable in aqueous solution because the conjugative
backbone can be attacked by reactive species such as water.
This may be overcome by introducing a steric hindrance to
11
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10.1002/anie.202007040
the fluorophore skeleton. Meanwhile, steric hindrance may
also reduce the possibility of fluorophore aggregation in
aqueous solutions.
Second, biocompatible fluorophores with maximum
emission wavelengths beyond 1200 nm are still lacking. Up
to now, the reported NIR-II fluorophores including the D-A-D
fluorophore, polymethine and some aza-BODIPY are
spectrally active in 1000-1200 nm window. Few
biocompatible fluorophores emitting beyond 1200 nm are
available nowadays, which limits many applications in the
NIR-II window, such as multiplexed bioimaging. Besides,
most of the NIR-II fluorophores have a large molecular
Accepted Manuscript
weight and poor solubility in aqueous solutions. Efforts
should be taken to lower the bandgap of the fluorophore to
bathochromic shift wavelength meanwhile keep it stable in
aqueous solution.
Third, more strategies are desired for the development of
NIR-II fluorescent biosensing probes. With the help of NIR-II
fluorescent probes, biological or pathological processes in
deep tissue can be visualized. However, most of the reported
probes can only respond to one detection analyte. While the
biological environment is a complex network, many species
are highly related in vivo. It will be fascinating that we can
visualize or even quantify the related species in vivo
simultaneously with one probe. Therefore, future efforts
should be taken to develop multi-response NIR-II probes,
especially those probes that can respond to different
analytes and have differentiable signals for various analytes.
Fourth, bioluminescence and chemiluminescence probes
spectrally in the NIR-II window are still challenging.[65]
Because they do not require external excitation, they can
better improve the signal-to-noise ratio. Bio-tracking
experiments can be performed with bioluminescence since
luciferase can be transfected.[71] On the other hand,
chemiluminescence probes are also sought-after for their
capacities in responding to biological microenvironment. [72]
To bathochromic shift the emission wavelength of the
bioluminescence or chemiluminescence probes, new
substrates, efficient energy transfer system or mutant
luciferase are highly desired.
Last but not least, fluorescence lifetime imaging allows
quantitative detection, however, NIR-II fluorescence lifetime
imaging is currently only available for inorganic rare-earth
nanoparticles, and the detection speed is limited by the long
fluorescence lifetime of rare-earth ions.[73] The fluorescence
lifetime imaging with NIR-II molecular probes has not yet
been achieved. Future efforts should be taken to develop
appropriate lifetime probes with fluorescence lifetime that
can be distinguished from the biological background.
Moreover, organic probes with different fluorescence lifetime
are also desired for multiplexed detection. Of course, these
assays also need the support of the corresponding imaging
instruments development.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Angewandte Chemie International Edition
REVIEW
This work was supported by the National Key R&D Program of
China (2017YFA0207303), the National Natural Science
Foundation of China (21725502, 51961145403, 21908030), the
Key Basic Research Program of Science and Technology
Commission of Shanghai Municipality (17JC1400100,
19490713100).
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Entry for the Table of Contents

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Accepted Manuscript
By virtue of the combined merits of diminished background fluorescence and deep penetration, fluorescence imaging in the NIR-II
window has become a powerful method for biomedical research. NIR-II fluorophores occupy a central position in NIR-II bioimaging.
This review aims at discussing the strategies applied for designing NIR-II fluorophores and NIR-II fluorescent probes.

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