NIR-emitting styryl dyes with large Stokes’ shifts for imaging application: From cellular plasma membrane, mitochondria to zebrafish neuromast

Article abstract of DOI:10.1016/j.dyepig.2021.109629

Near-infrared (NIR) emitting probes with very large Stokes' shifts play a crucial role in bioimaging applications, as the optical signals in this region exhibit high signal to background ratio and allow deeper tissue penetration. Herein we illustrate NIR-emitting probe 2 with very large Stokes' shifts (Δλ ≈ 260–272 nm) by integrating the excited-state intramolecular proton transfer (ESIPT) unit 2-(2′-hydroxyphenyl)benzoxazole (HBO) into a pyridinium derived cyanine. The ESIPT not only enhances the Stokes' shifts but also improves the quantum efficiency of the probe 2 (ф_fl = 0.27–0.40 in DCM). The application of 2 in live cells imaging reveals that compound 2 stains mitochondria in eukaryotic cells, normal human lungs fibroblast (NHLF), Zebrafish's neuromast hair cells, and support cells, and inner plasma membrane in prokaryotic cells, Escherichia coli (E. coli).

Full text of DOI:10.1016/j.dyepig.2021.109629

Dyes and Pigments 194 (2021) 109629
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
NIR-emitting styryl dyes with large Stokes’ shifts for imaging application:
From cellular plasma membrane, mitochondria to zebrafish neuromast
Dipendra Dahala, Krishna R. Ojhaa, Sabita Pokhrela, Sailaja Paruchuria, Michael Konopkaa,
,c,*
Qin Liub, Yi Panga
a Department of Chemistry, The University of Akron, Akron, OH, 44325, USA
b Department of Biology, The University of Akron, Akron, OH, 44325, USA
c Maurice Morton Institute of Polymer Science, The University of Akron, Akron, OH, 44325, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Near-infrared (NIR) emitting probes with very large Stokes’ shifts play a crucial role in bioimaging applications,
as the optical signals in this region exhibit high signal to background ratio and allow deeper tissue penetration.
Herein we illustrate NIR-emitting probe 2 with very large Stokes’ shifts (Δλ ≈ 260–272 nm) by integrating the
excited-state intramolecular proton transfer (ESIPT) unit 2-(2′-hydroxyphenyl)benzoxazole (HBO) into a pyr-
idinium derived cyanine. The ESIPT not only enhances the Stokes’ shifts but also improves the quantum effi-
ciency of the probe 2 (фfl = 0.27–0.40 in DCM). The application of 2 in live cells imaging reveals that compound
2 stains mitochondria in eukaryotic cells, normal human lungs fibroblast (NHLF), Zebrafish’s neuromast hair
cells, and support cells, and inner plasma membrane in prokaryotic cells, Escherichia coli (E. coli).
Near-infrared
ESIPT
Fluorescence imaging
Mitochondria
Neuromast
Plasma membrane
1. Introduction
forms a boundary to protect the cell from the extracellular environment
[20]. The dynamic changes in the plasma membrane, due to the
Fluorescent imaging plays an essential role in the visualization of the
specific organelles, organs, and organisms, as fluorescence imaging can
provide excellent spatial and temporal resolution. When coupling with
suitable imaging reagents, the technique can be used to selectively
display “targeting analytes” in cells, tissues, and even whole organisms
[1–7]. However, the fluorescence quality is strictly dependent on the
photophysical properties of the fluorescent probes used in staining the
sample [8]. An ideal fluorescence probe should have the ability to target
the specific organelles without perturbing their morphology and phys-
iology. For example, mitochondria are the membrane-bound organelles
that are considered as the powerhouse of eukaryotic cells and generate
entire energy in animal cells in the form of adenosine triphosphate
(ATP) by utilizing oxygen during oxidative phosphorylation [9–13].
Moreover, mitochondria are also responsible for various functions such
as cell signaling, cellular differentiation, cell cycle, and cell growth, and
cell death and apoptosis [14–16]. Therefore, the mitochondrial mal-
function is associated with various diseases such as Alzheimer’s dis-
eases, Parkinson’s diseases, ischemic and hemorrhagic stroke, cardiac
myocyte death, and cancers [17–19].
exchanging information and substances within a cell and surrounding, is
the basic condition for endocytosis, exocytosis, nutrient transport, cell
proliferation, and signal transmission, etc. [21,22] The morphology of
the plasma membrane is the direct indication of the cell status, such as
partial swallowing, and rupture of the plasma membrane is the sign of
cell apoptosis induced by drugs [22,23]. In Prokaryotic cells, such as
Escherichia coli (E.coli) (a Gram-negative bacteria), the morphology of
the membrane consists of three layers: the outer membrane, the pepti-
doglycan layer, and the inner membrane. The peptidoglycan layer is
occupied by viscous periplasmic space [24]. A membrane is a
lipid-bilayer that contain integral proteins. The outer membrane is made
up of complex lipopolysaccharides, which are different from the inner
membrane [25]. Therefore, there is great interest in understanding the
structure and functions of various cell organelles, such as the inner
cytoplasmic membrane in prokaryotes or mitochondria in eukaryotes.
Hearing loss is one of the significant public health problems, and the
degeneration of mechanosensory hair cells of the inner ear sensory
epithelia is one of the leading causes of hearing loss in humans [26,27].
The Zebrafish is emerging as a powerful tool for identifying the verte-
brate gene and their functions [27,28]. The Zebrafish lateral line has
Another crucial cellular component is the plasma membrane that
* Corresponding author. Department of Chemistry, The University of Akron, Akron, OH, 44325, USA.
E-mail address: yp5@uakron.edu (Y. Pang).
https://doi.org/10.1016/j.dyepig.2021.109629
Received 26 March 2021; Received in revised form 4 July 2021; Accepted 5 July 2021
Available online 6 July 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

D. Dahal et al.
Dyes and Pigments 194 (2021) 109629
emerged as a powerful tool for drug-screening assays that cause or
prevent the hair cells’ death [26]. The lateral line consists of numerous
mechanosensory organs called neuromast that comprise sensory hair
cells and surrounding support cells. Although transgenic methods can be
utilized for recognition of neuromasts [26], [29] the fluorescent dyes
remains the easiest and the most accessible reagents used to visualize the
lateral lines.
hence help in cell penetrations. Thus, compound 2 stained mitochondria
in eukaryotic cells (on normal human lungs fibroblast (NHLF)), neuro-
mast hair cells and support cells of Zebrafish in vivo and inner plasma
membrane of prokaryotic cells (on Escherichia coli (E.coli)). Our study
also revered that lipophilic dye 2 could be an excellent alternative for
the investigation of protein diffusion in prokaryotic cells.
Fluorescence probes play a pivotal role in the study of biological cell
organelles, as they exhibit high sensitivity and allow real-time obser-
vation [30–33]. However, most of the fluorescence probes emitting in
the visible region have limited application because of high phototoxicity
and interference from the background autofluorescence of biological
samples [34]. Near-infrared (NIR) emitting fluorescence probes are
useful for bioimaging applications, as they have a high signal-to-noise
ratio, reduced photon scattering, and deeper tissue penetration depth
[35,36]. Current small-molecule fluorophores for NIR emission include
BODIPY, rhodamine, benzofuran, and cyanines [37,38]. However, these
fluorophores exhibit a small Stokes’ shift (Δλ = 10–60 nm), which
prevents the optimum collection of the fluorescence signal and hamper
the bioimaging application [39–41]. Therefore, developing NIR emitting
fluorescent probes with large Stokes’ shifts is desirable for improved
sensitivity in fluorescence imaging. Also, NIR emitting probes with large
Stokes’ shifts are useful for single excitation multicolor live-cell imag-
ing, which plays an essential role in biological studies [42].
2. Result and discussion
2.1. Synthesis and characterization of compound 2
In a 50 mL round-bottomed flask, 120 mg (0.51 mmol) of compound
6 was dissolved in methanol (20 mL), and 0.5 ml of piperidine was
◦
added. The mixture was heated to 60 C, and 152 mg (0.60 mmol) of
compound 5 were added, and the mixture was stirred at 60 ◦C overnight.
The solvent was then dried on a rotary evaporator, and the residual solid
was washed with 50 ml ethyl acetate. The residue was filtered and dried
to get 216 mg (~85% yield) of compound 2 (Scheme 1). The details for
the synthesis of compound 5 can be found in our recent publication [45].
The structure of 2 was confirmed by NMR (1H and 13C) (ESI Figure S1
and S2) spectroscopies and mass spectrometry (ESI Figure S3; calc mass:
343.1415). The resonance signal at 11.906 ppm was observed in 1H
NMR, indicating that the hydroxyl proton is involved in the intra-
molecular hydrogen bond (Figures S1).
Recently, our group and others have synthesized NIR emitting probes
by the integration of excited-state intramolecular proton transfer
(ESIPT) group; 2-(2′-hydroxyphenyl)benzoxazole (HBO) and 2-(2′-
hydroxyphenyl)Benzothiazole (HBT), with hemicyanines via sharing
meta-phenylene bridge, which gives NIR emission with very large
Stokes’ shifts (Δλ = 234–272 nm) [43–48]. In this report, we present the
synthesis of NIR-emitting molecular probe 2 by coupling ESIPT unit
HBO with pyridinium styryl group via sharing a meta-phenylene bridge
for biological applications. Compound 2 exhibited NIR emission with
very large Stokes’ shifts (Δλ = 260–272 nm), increased photostability,
and improved quantum efficiency than its model compound 3 (pyr-
idinium styryl hemicyanine chromophore without ESIPT). The substi-
tution of ESIPT unit also increases the lipophilicity of the compound and
2.2. Optical properties
Compound 2 exhibited UV-absorption (λmax) at 415 nm (ε = 25,809
Mꢀ 1cmꢀ 1). The emission was observed at λem ≈ 675 nm for 2 in DCM,
respectively, with a large Stokes’ Shifts (Δλ ≈ 260) (Fig. 2). As a
consequence of excited-state intramolecular proton transfer (ESIPT), the
emission spectra of 2 were well separated from its absorption, leaving an
optical window between 490 nm–580 nm. This was in sharp contrast to
compound 3, whose emission had a small Stokes’ shift. The emission
wavelength of 2 was slightly affected by the polarity of solvents, with
λem varying from 672 to 695 nm (Table 1 and ESI Figures S6). It should
be noted that the
π-conjugation length of 2 could be estimated by
Scheme 1. Synthesis of Compound 2 (pyridinium cyanine coupled with HBO).
2

D. Dahal et al.
Dyes and Pigments 194 (2021) 109629
Fig. 1. Structure of NIR emitting ESIPT probe.
Fig. 2. Photophysical properties of compounds 2 and 3 in DCM. Compound 3 shows normal emission, and 2 shows ESIPT emission.
different solvents with varying polarity (Table 1), which are signifi-
Table 1
cantly higher than that of 3 (ф ≈0.003 to 0.10) [44]. The results further
fl
Optical properties of compounds 2 and 3 in different solvents.
illustrated the impact of the ESIPT unit.
Solvents
Compound 2
Compound 3
Abs.
Fl. Em.
Q.Y.
Abs.
Fl. Em.
Q.Y.
2.3. Low-temperature fluorescence
(λAbs
)
(λem
)
(ф )
(λAbs
)
(λem
)
(ф )
fl
fl
DCM
415
392
392
391
386
394
394
384
674
695
695
694
683
693
690
672
0.27
0.21
0.27
0.25
0.28
0.14
0.15
0.05
428
389
398
576
577
588
0.08
0.01
0.02
The observed large Stokes’ shifts of compound 2 could be associated
with the ESIPT in structure 2, which led to its keto structure 2a. After
undergoing ICT, 2a was transformed into 2b (Scheme 2). Low-
temperature fluorescence spectra of 2 were acquired in order to shed
some light on the photophysical process associated with the ESIPT and
ICT. A dilute solution of 2 in ethanol was quickly cooled in liquid ni-
trogen in a quartz Dewar, and fluorescence spectra were acquired at
different temperatures by gradually increasing the temperature over
2–3 h. At room temperature, the emission was observed at 692 nm,
DMF
DMSO
EtOH
MeCN
MeOH
THF
389
398
557
570
0.100
0.02
Water
398
575
0.003
pyridinium styryl hemicyanine 3 (Fig. 1), as they exhibited nearly
identical λmax. However, the presence of an HBO unit enabled the ESIPT
emission, which was responsible for the observed large Stokes shift.
◦
which was blue-shifted to 592 nm at ꢀ 189 C. When the temperature
was gradually increased after cooling to ꢀ 189 ◦C, the spectral redshift
Fluorescence quantum yield of 2 (ф ≈0.05 to 0.27) was measured in
◦
fl
was observed within a small range of temperatures from ꢀ 110 C to
3

D. Dahal et al.
Dyes and Pigments 194 (2021) 109629
Scheme 2. Structures of the enol and keto tautomers of 2 that undergo ESIPT and ICT. The dotted squire is the effective chromophore of compound 2.
ꢀ 75 ◦C (Fig. 3), it was assumed ICT was not occurring when compound 2
was in the frozen solvent matrix that prevents the bond rotation and
reorganization. However, ESIPT was not expected to shift the spectra at
low temperature [49]. Therefore, the large spectral shift (Δλem ≈ 100
nm) was attributed to the ICT within keto-tautomer 2b formed from 2
(Scheme 2).
Compound 2 was further used to stain the prokaryotic cells, E. coli, in
order to discover its broader applications. Thus E. coli cells were incu-
◦
bated with 2 at 37 C for 15 min and the images were recorded on a
confocal fluorescence microscope. Interestingly, the bright fluorescence
signals were observed from the peripheral region of the E. coli cells
indicating that it stained the membrane of the bacterial cells (Fig. 5A,
and ESI Figures S10 and S11A).). It should be noted that the cytoplasmic
membrane of gram-negative bacteria, E. coli bacteria, possesses outer
and inner membranes. In order to further identify the location of the
fluorescent dye, the E. coli (DGC-102) cells were plasmolyzed by using a
hypertonic NaCl solution (Fig. 5A–C) and 0.6 M sucrose solution
(Fig. 5D–G). Thus, the cells were treated with 2, and 0.4 M NaCl solution
and/or 0.6 M sucrose solution were added to induce osmotic shock [50].
Addition of the hypertonic NaCl/sucrose solutions would induce water
efflux through a semipermeable membrane, causing the shrinkage of the
inner membrane of cells [51]. The plasmolyzed bays were observed
upon the addition of the NaCl solution and/or sucrose solution (Fig. 5 B,
C, and D ESI Figure S11B – S11D), indicating that the dye selectively
stained the inner cytoplasmic membrane. This study implies that
different membrane properties, including lipid and protein diffusion,
2.4. Cell imaging study by using compound 2
The attractive photophysical properties such as high fluorescence
quantum yield, NIR emission with large Stokes’ Shifts, and low toxicity
of compound 2 (IC50 = 66.45 μM) (ESI Figure S7) encouraged us to
investigate its possible applications. Therefore, compound 2 was used to
stain normal human lungs fibroblast (NHLF) cells and E. coli bacterial
cells. The confocal fluorescence microscopic imaging of NHLF cells
revealed strong non-uniform fluorescence signals from the perinuclear
region (Fig. 4 second column, ESI Figure S11). Co-staining of 2 with
MitoTracker red confirmed that 2 exhibited good selectivity to stain
mitochondria (Fig. 4 third column, ESI Figure S12) with the Pearson’s
colocalization coefficients of 0.92.
Fig. 3. Low-temperature fluorescence of compound 2 in EtOH.
4

D. Dahal et al.
Dyes and Pigments 194 (2021) 109629
Fig. 4. Confocal fluorescence images of Normal Human Lungs Fibroblast (NHLF) cells co-stained with compound 2 (1
μM) and MitoTracker Red (200 nM) (Top row)
acquired at 60× magnification of the oil-drop objective lens. The bottom row is four times digitally enhanced images of a portion enclosed in a white box of the top
row The compound 2 was excited at 405 nm laser with emission filters of 700–720 nm, and the Mitotracker Red was excited at 561 nm laser with the emission
bandpass filters of 595/50 nm.
an extended time in the microscope. Therefore, photostability is a sig-
nificant parameter in real-time live-cell imaging. In order to evaluate
how fast or slow the fluorophore is bleached, photobleaching behavior
(photostability) of compound 2 was examined by illuminating the bac-
terial (E. coli) cells stained with dye 2 with a high-intensity laser beam
over 60 min with continuous scanning. Since the scanned sample area
contained about 40 bacteria, each of the individual cells was irradiated
for about 1.5 min in average. The compound 2 exhibited relatively
constant stability for about 60 min for individual cells when analyzed in
real-time cell imaging (Fig. 6A and B). The relative photostability of
compound 1 was also compared with compound 2 (Fig. 6C and ESI
Figure S16). It was observed that compound 1 initially had brighter
emission than compound 2; however, the intensity of the cell imaging
stained with compound 1 dropped significantly than compound 2 over
time with continuous laser scanning. This implies that the presence of
HBO in compound 2 makes it more photostable than the HBT in com-
pound 1.
2.6. Fluorescence imaging of zebrafish using compound 2 in vivo
Our previous work showed that compound 1 was useful for the
developmental study of the neuromast hair cells and supporting cells of
Zebrafish [26]. Encouraged by its low toxicity and improved photo-
stability, we decided to explore the further use of 2 for zebrafish im-
aging. Thus, the zebrafish embryos of 36–96 h of post-fertilization (hpf)
Fig. 5. E. coli cells labeled and plasmolyzed. (A) Healthy cell stained with
were incubated with 2 for 30 min at room temperature, and confocal
compound 2 without using NaCl solution, (B & C) plasmolyzed Cells with 0.4 M
fluorescence microscopic imaging was acquired. The microscopic im-
aging revealed that strong fluorescence signals were observed from the
head region around the eyes and along the lateral lines of the body from
head to tail of the embryos.(48–96 hpf) (Fig. 7 for 72 hpf). The structure
in the digitally enhanced image (Fig. 7 bottom row and Figure S17) also
revealed that the compound 2 selectively labeled the neuromast hair
cells and support cells. Staining neuromast hair cells and support cells by
compound 2 were also confirmed by colocalizing with commercially
available neuromast selective dye 4-(4- Diethylaminostyryl)-1- methyl
pyridinium iodide (4-Di-2-ASPI) (Fig. 7, D – F).
NaCl solution, which showed dark plasmolyzed bays in the region of plasmol-
ysis shown with the arrows, and (D–G) fluorescence images of plasmolyzed cells
with 0.6 M sucrose solution along with bright fields. Some cells have multiple
bays indicating more than one plasmolyzed area (B & F). The original images
were acquired under 100× magnification of the oil-drop objective lens. The
compound 2 was excited at 405 nm laser with emission filters of 700–720 nm.
could be investigated using membrane targeting compound 2 [52].
2.5. Stability of dye
Rapid photobleaching of a dye is a common problem for fluorescence
dyes since long-term live-cell imaging requires excitation with laser for
5

D. Dahal et al.
Dyes and Pigments 194 (2021) 109629
Fig. 6. A) Confocal fluorescence images of E. coli cells taken within 1 h period to observe the relative intensity of compound 2, B) The plot of the relative intensity
measured over 90 min for compound 2 vs time, and C) Comparison of relative intensities between compound 1 and 2. The images of cells in “A” were acquired under
100× magnification of the oil-drop objective lens. The compound was excited at 405 nm laser with emission filters of 700–720 nm.
Fig. 7. Confocal fluorescence microscopic imaging of 72 hpf zebrafish stained with compound 2 and commercial dye 4-Di-2-ASPI, A) 4-Di-2-ASPI, B) Compound 2, C)
merged A and B under 10× magnification and D – F) High magnification (digitally enhanced) images of the spots in head and tail regions shown with yellow arrows
in A – C respectively. The excitation wavelength for compound 2 was 405 nm with the emission filters of 700–720 nm, and the excitation/emission wavelength for 4-
Di-2-ASPI was 488/561 nm, respectively.
3. Conclusion
with a pyridinium styryl-derived cyanine unit, which exhibited very
large Stokes’ shifts (Δλ ≈ 260 nm). The dye showed high quantum ef-
NIR emitting probe 2 was synthesized by integrating the ESIPT unit
ficiency (ф = 0.27 in DCM), increased photostability, and high
fl
6

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Dyes and Pigments 194 (2021) 109629
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The authors declare that they have no known competing financial
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We thank the support from NIH (Grant no. 1R15GM126438-01A1).
Y. P. also acknowledges partial support from Coleman Endowment from
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
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