Imaging mitochondria and plasma membrane in live cells using solvatochromic styrylpyridines

Article abstract of DOI:10.1016/j.jphotobiol.2019.111732

Investigating the dynamics of different biomolecules in the cellular milieu through microscopic imaging has gained paramount importance in the last decade. Continuous developments in the field of microscopy are paralleled by the design and synthesis of fluorophores that target specific compartments within a cell. In this study, we have synthesized four fluorescent styrene derivatives, a neutral styrylpridine, three cationic styrylpyridinium probes with and without cholesterol tether, and investigated their absorption, emission, and cellular imaging properties. The fluorophores show solvatochromic emission attributed to intramolecular charge transfer from donor to acceptor with an emission range of 500–600 nm. The fluorescent cholesterol conjugate labels plasma membrane effectively while the fluorophores devoid of the cholesterol tether label mitochondria. Cholesterol conjugate also shows strong interaction with liposome membrane. Furthermore, the fluorophores alsotrack the mitochondria in live cells with high specificity. Cell viability assay showed overall non-toxic nature of the probes even at higher fluorophore concentrations. Through sidearm modifications, keeping the fluorescent core intact, we successfully targeted specific subcellular compartments of neuronal (N2a) and non-neuronal (HeLa) mammalian cell lines. This strategy of using a single molecular scaffold with subtle substitutions could be ideal in generating a variety of fluorophores targeting other subcellular compartments.

Full text of DOI:10.1016/j.jphotobiol.2019.111732

Journal Pre-proof
Imaging mitochondria and plasma membrane in live cells using
solvatochromic styrylpyridines
Tarushyam Mukherjee, Aravintha Siva, Komal Bajaj, Virupakshi
Soppina, Sriram Kanvah
PII:
S1011-1344(19)31339-9
DOI:
https://doi.org/10.1016/j.jphotobiol.2019.111732
JPB 111732
Reference:
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Revised date:
Accepted date:
7 October 2019
20 November 2019
4 December 2019
Please cite this article as: T. Mukherjee, A. Siva, K. Bajaj, et al., Imaging mitochondria
and plasma membrane in live cells using solvatochromic styrylpyridines, Journal of
Photochemistry
&
Photobiology, B: Biology(2019), https://doi.org/10.1016/
j.jphotobiol.2019.111732
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
©
2019 Published by Elsevier.

Journal Pre-proof
Imaging Mitochondria and Plasma Membrane in Live Cells using
Solvatochromic Styrylpyridines
‡
†
‡
†
Tarushyam Mukherjee , Aravintha Siva , Komal Bajaj , Virupakshi Soppina* and Sriram
‡
#
Kanvah *
#‡ *Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar
3
82355, India. E-mail: sriram@iitgn.ac.in, kanvah@gatech.edu
†
*Department of Biological Engineering, Indian Institute of Technology Gandhinagar Palaj
Gandhinagar 382355 e-mail: vsoppina@iitgn.ac.in
Abstract
Investigating the dynamics of different biomolecules in the cellular milieu through microscopic
imaging has gained paramount importance in the last decade. Continuous developments in the
field of microscopy are paralleled by the design and synthesis of fluorophores that target specific
compartments within a cell. In this study, we have synthesized four fluorescent styrene
derivatives, a neutral styrylpridine, three cationic styrylpyridinium probes with and without
cholesterol tether, and investigated for their absorption, emission, and cellular imaging
properties. The fluorophores show solvatochromic emission attributed to intramolecular charge
transfer from donor to acceptor with an emission range of 500-600 nm. The fluorescent
cholesterol conjugate labels plasma membrane effectively while the fluorophores devoid of the
cholesterol tether label mitochondria. Cholesterol conjugate also shows strong interaction with
liposome membrane. Furthermore, the fluorophores were also used to track the mitochondria in
live cells with high specificity. Cell viability assay showed overall non-toxic nature of the probes
even at high concentrations. Through sidearm modifications, keeping the fluorescent core intact,
we successfully targeted specific subcellular compartments of neuronal (N2a) and non-neuronal
(HeLa) mammalian cell lines. This strategy of using a single molecular scaffold with subtle
substitutions could be ideal in generating a variety of fluorophores targeting other subcellular
compartments.
1

Journal Pre-proof
Keywords
Fluorescent Sterols, Intramolecular Charge Transfer, Mitochondrial Imaging, Plasma membrane
staining, Styrlpyridines.
Introduction
Targeted imaging of sub-cellular organelles is of paramount importance in cell biology and has
triggered numerous investigations towards the design of various fluorophores[1-5]. The basic
unit of life, a cell, is an intricately organized and highly dynamic entity, where the many
organelles carry out specialized functions inside and yet regularly interact with one another. To
get the accurate details of the cellular activities, visualization of these organelles becomes
necessary. This necessity steered the research towards fluorophore-based targeted imaging that
gives us visual evidence of the cellular world. For this purpose, Donor-π-Acceptor based
fluorescent organic molecules offer several tunable design strategies. A particularly exciting
scaffold based on styrylpyridine, offering strong environmental sensitivity was extensively
investigated for various applications including, DNA intercalation or groove binding[6-9],
analyte detection [10-12], imaging of plasma membrane[13] and mitochondria [14-16],
histological staining [17, 18], investigation of norepinephrine transportation[19, 20] formations
of supramolecular assemblies [21-26] and organic electronic applications[27-30]. In the current
study, we systematically investigate the utility of styrylpyridine and its derivatives to image
cellular compartments such as the plasma membrane and mitochondria. The plasma membrane
apart from maintaining cell integrity plays a vital role in various other processes such as signal
transduction, cell differentiation, and cell fusion. Thus, designing membrane probes that can
effectively label the plasma membrane might be beneficial for a better understanding of the
cellular structure. A typical probe must have red-emission to enable penetration depth, cellular
membrane compatibility and preferably possess a suitable recognition moiety. For this purpose,
we utilized styrylpyridine as an extrinsic fluorophore linked to the cholesterol moiety to
investigate cellular imaging. Cholesterol is an integral member of the mammalian cell
membrane, and it provides structural stability to the cell. Hence, targeting cell membrane or
cellular processes has been achieved through the tagging of the probe with cholesterol[31, 32].
Various reporter molecules such as NBD[33], Bodipy[32, 34], pyrene[35], Dansyl[36], and
2

Journal Pre-proof
others were conjugated to cholesterol and were used to study cellular processes such as
trafficking, model membrane studies, and biomolecular interactions[37].
Furthermore, among the various units of cell organelles, mitochondria, the powerhouse of the
cells, always hold particular importance due to its complex functionality that regulates cellular
physiological states[38, 39]. Consequently, monitoring of mitochondrial processes through the
use of fluorophores received significant attention [14, 40-42]. In this work, we utilize a single
fluorescent core to achieve targeted imaging of the plasma membrane and mitochondria. In short,
we synthesized four styrylpyridines (Scheme-1): the neutral styrylpyridine 1, cationic methyl
pyridinium derivative 2, hydroxyl propyl pyridinium derivative 3, and novel pyridinium-
cholesterol conjugate 4. The newer fluorescent cholesterol conjugate 4 exhibits large Stokes’
shift with excitation and emission in the visible region and shows high specificity to the plasma
membrane with no cytotoxicity. A comparison with existing cholesterol conjugates is given in
Table- S1. Interestingly the styrylpyridines 1-3 devoid of the cholesterol linker stain the
mitochondria in green to red regions, while the pyridinium cholesterol conjugate labels the cell
membrane. We have also demonstrated the ability to track live mitochondrial movement and
mitochondrial events such as fusion, fission with the help of our mitochondrial specific styrene
probes. The results of absorption, emission, and cellular imaging studies are presented in the
following sections.
Experimental Methods
All the necessary chemicals and reagents required for the synthesis of the probes were purchased
from Aldrich, Alfa Aeser, TCI Chemicals, Avra Synthesis, or SD Fine-Chem. The synthesized
1
13
samples were characterized using H and C NMR (Bruker Advance III-500 MHz) in
chloroform-D or dimethyl sulphoxide (D6) solvents. The absorption studies were performed on
Analytik Jena Specord 210 instrument, and the steady-state and the fluorescence studies were
measured using Horiba Jobin Yvon Fluorolog-3 spectrofluorimeter with a slit width of 2 nm. The
sample concentrations used for the fluorescence studies are in the order of 10 µM. The excitation
wavelength for recording the emission spectra is typically kept at the absorption maxima of the
samples under investigation. Mass spectral data is recorded using Water Synapt G2S ESI Q-ToF
mass spectrometer.
3

Journal Pre-proof
Model membrane preparation
DPPC model membrane was prepared by a solvent evaporation method. DPPC lipid was
dissolved in chloroform-methanol 2:1 (v/v) at the desired molar ratio, and after that, the solvent
layer was dissolved under reduced pressure. The dried lipid layer was hydrated soon after the
solvent layer was evaporated by the addition of triple distilled water and kept in 55-60 C to
form final vesicles. The required amount of probe (0.4 mM) was incorporated into the lipid layer
in the chloroform-methanol mixture. Imaging of model membrane was performed on a Leica
Laser Scanning Confocal microscope (TCS SP8) equipped with HyD and PMT detectors using a
4
58 nm laser.
Plasmids
Plasmids coding for fluorescently-tagged subcellular organelle specific markers such as Mito-
ECFP and Mito-DsRed were generated by sub-cloning into ECFP-N1 and DsRed-N1 plasmids
and used for labeling the mitochondria. ECFP-MEM was generated likewise by sub-cloning into
ECFP-C1 to label the plasma membrane. KDEL-BFP and LAMP1-YFP were also generated
similarly for the labeling of endoplasmic reticulum and lysosome respectively.
Cell culture and live-cell imaging:
HeLa and Cos-7 cells were grown in DMEM (Gibco) supplemented with 10% FBS (Gibco) and
2
mM GlutaMAX (Gibco) at 37 C in a humidified environment with 5% CO2. EMEM (Gibco)
5
was used in the place of DMEM for culturing N2a cells. For live-cell imaging studies, 1.5 x 10
cells were grown on glass-bottom dishes. These cells were subsequently transfected with 1 µg of
plasmid DNA using TurboFect transfection reagent (Thermo Scientific) and incubated overnight.
On the following day, cells were further treated by adding 2 µl of the probe from 1 mM stock
(prepared in DMSO) to 2 ml of DMEM and incubated for 10 min at 37C and washed three
times in HEPES buffer (pH 7.4) before imaging under the microscope. Live-cell imaging was
performed on a Leica Laser Scanning Confocal microscope (TCS SP8) at 37C temperature in
the presence of 5% CO2. Images were obtained using 405 nm, 458 nm and 561 nm lasers (for the
fluorescent proteins: 405 nm and 561 nm lasers used for Mito-CFP, ECFP-MEM, and Mito-
DsRed respectively; wherein for the styrene-1 we have used 405 nm and styrene-2 to styrene-4:
4
58 nm laser) and the emission signals were collected using HyD and PMT detector.
4

Journal Pre-proof
Mitochondrial tracking experiments live-cell imaging was performed in epifluorescence mode
O
using a Nikon inverted microscope (Nikon Eclipse Ti2, Japan) equipped with 37 C with 5% CO2
EMCCD camera (Andor iXon 897Ultra, USA). Images were acquired at ten frames/sec using
Nikon software (NIS element AR, Japan).
Mitochondrial membrane potential change Cells were seeded to the 35 mm glass-bottomed
live-cell dishes at a density of 1.5 × 105 cells per dish in culture medium. After 24 h, cells were
treated with 20 μM CCCP for 20 min and incubated with 1 μM probe before live-cell imaging
using Laser Scanning Confocal microscopy (Leica SPI8).
Cell Viability Assay
MTT assay was performed to calculate the toxicity of the probe in mammalian cells. Five
thousand HeLa cells were seeded per well and grown in 96-well plates. After 24 h of seeding, the
cells were treated with different concentrations (0.25, 0.5, 1, 2, 4, 8, and 10 µM) of the probes
respectively and incubated for 24 h. The cells that served as the negative control containing
respective concentration (v/v) of DMSO alone (without any probe) were replenished with fresh
media. After the treatment duration, the media was aspirated from all the wells and incubated
further with 0.5 mg/mL of MTT for 4 h. Media containing the MTT was then removed after
which, DMSO was added to solubilize the formazan. The absorbance values were recorded using
BioTek SYNERGYH1 plate reader at 570 nm. Absorbance values of cells treated with the
probes were normalized against the untreated cells (negative control). The percentage cell
viability was calculated with the help of the negative control as reference, where the cells were
treated with solvent (DMSO) alone. Finally, the percentage cell viability is plotted against probe
concentration as mean ± SD. The results presented are an average of three independent sets of
experiments.
Data Analysis
Analysis and quantification of co-localization were performed using ImageJ software (nih.gov).
The overlap coefficient and Pearson correlation coefficient (PCC) were evaluated by using the
co-localization finder plugin of the same software. Background fluorescence was subtracted from
this maximum value for the respective filter, and then the pixel intensity comparisons were
carried out.
5

Journal Pre-proof
Mitochondrial tracking Individual mitochondria were tracked frame-by-frame using the
custom-written plugin in ImageJ (nih.gov), and graphs were plotted in Origin 9.1 (Origin Lab).
Results and Discussion
Synthesis
The styryl probes (1-4) were prepared as per the scheme-1 given below. 4-picoline was
condensed with dimethylamino benzaldehyde to yield the neutral styrene 1. Methylation of 1
with methyl iodide produced styrene 2, and the reaction of 1 with 3-bromopropanol yielded
styrene 3 bearing a 3-carbon linker with a hydroxyl terminus. Reaction with 3-bromopropyl
cholesteryl formate, prepared by using cholesteryl chloroformate and 3-bromopropanol[43] with
styrene 1 yielded cholesterol conjugated styrene 4. Except styrene 1, the other styrenes 2-4
contain a single cationic charge that facilitated their solubility in aqueous media. The synthetic
procedures and the characterization spectral data are given in the supporting information.
Absorption and emission studies
The absorption and emission data of the styrenes 1-4 are summarized in Table-1. Styrene 1
bearing a dimethylamine donor and pyridine acceptor absorbs at ~370 nm in non-polar dioxane.
Styrene, 2, containing pyridinium group shows significant bathochromic shifts with absorption
maxima at 463 nm. The wavelength shifts are a result of extended conjugation owing to the
presence of a robust electron-withdrawing pyridinium group. Styrene 3 with a hydroxypropyl
unit and styrene 4 having a cholesterol moiety does not affect the absorption maxima [Fig 1a].
The molecules show moderate solvatochromic shifts with increasing solvent polarity. Styrene 1
absorbs at 370 nm in dioxane and shows absorption at 379 nm in polar DMSO. Styrenes 2-4 [Fig
1
4
b and Fig S1] also show moderate solvatochromic effects with an increase in solvent polarity:
63 to 471 nm for 2, 463 to 475 nm for 3 and 464 nm to 477 nm for 4. For cationic styrenes 2-4,
the absorption spectra are broad and show a hypsochromic shift in water. The broad absorption
spectra in water could be due to weaker water solubility, and the hypsochromic shift is attributed
to charge stabilization in water [12, 44].
6

Journal Pre-proof
Table 1: Absorption and emission of styrenes 1-4 in homogeneous solvents.
1
2
3
4
λa
λf
λa
λf
λa
λf
λa
λf
Solvent
(
nm)
370
372
380
375
370
377
-
(nm)
445
485
495
483
485
500
522
(nm)
465
470
470
470
470
476
450
(nm)
555
595
617
615
615
605
610
(nm)
463
474
475
475
473
480
455
(nm)
570
594
628
617
616
606
615
(nm)
465
470
478
478
478
483
455
(nm)
565
595
615
610
610
606
610
Dioxane
THF
DMSO
DMF
CH3CN
CH3OH
H2O
Scheme 1. Synthetic strategy for the Styrenes 1-4. Reaction Conditions: (i) KOH, 125C, 8 h; (ii)
Ethanol, 100C, 8-9 h; (iii) 3-bromopropanol, acetonitrile, 100C, 16 h; (iv) 3-bromopropyl
cholesterylformate, triethylamine, dry DCM, 0C, 12 h.
7

Journal Pre-proof
Fig. 1. Absorption spectra of styrenes 1-4 in (A) acetonitrile and absorption spectra of (B)
styrene 4 in different solvents.
Fig. 2. Emission spectra of (A) styrene 2 and (B) styrene 4 in different solvents
Analogous to the absorption data, the emission of neutral styrene 1 significantly differs from the
cationic fluorophores 2-4. Neutral styrene 1 emits at 445 nm in dioxane, but cationic compounds
show emission at 554-570 nm regions. Solvent polarity has a strong effect on the emission of the
styrenes. Upon increasing the solvent polarity, gradual emission changes were obtained with an
overall bathochromic shift of 50 nm (styrene 1) from dioxane to dimethylsulfoxide [Fig S2a].
The bathochromic shifts observed for styrenes 2-4 are similar to the neutral derivative with shifts
in the range of 50 -65 nm [Fig 2 and Fig S2b], and the shifts are attributed to the charge
stabilization in polar solvents[45]. On the other hand, substitution with hydroxyl alkyl or
cholesteryl tethers does not impact the emission behavior as compared to styrene 2.
8

Journal Pre-proof
Interaction with model membrane
Liposomes possess long-chain hydrophobic fatty acids and mimic the plasma membrane
in vitro[46, 47]. The cholesterol tether can contribute to the enhanced hydrophobicity and target
the membrane easily. Considering the inherent property, we have investigated the spectroscopic
behavior of the cholesterol-conjugate 4 in the liposome membrane to evaluate its membrane
binding characteristics. Styrene 4 exhibited ~40 fold increment (at 570 nm) in fluorescence
intensity [Fig 3A] in DPPC (dipalmitoylphosphatidylcholine) model membrane system along
with ~40 nm blue shift in the emission wavelength. This emission change is an indication of
probe-liposome interaction[48]. We presume that the cholesterol ‘head group’ in probe 4 is
anchoring the molecule into the membrane, and the remainder fluorescent core lies outside.
Styrene 4 is also able to sense the phase transition temperature of the membrane since the change
in the fluorescent intensity decrement is evident from the spectral behavior[48] [Fig S3]. To
further validate the specificity, we imaged the liposomes using laser scanning confocal
microscopy and probe (styrene 4) showed strong colocalization with liposome membrane [Fig
3
B]. The cumulative spectroscopic and microscopic response has thus inspired us to study
cellular imaging for the plasma membrane.
Fig. 3: Interaction of styrene-4 with in vitro model membrane: (A) Emission behavior of
styrene 4 in water and model membrane (DPPC) [Green and blue lines indicate the intensity
enhancement and blue shift of the styrene 4 after binding to model membrane], (B) Confocal
microscopy images of DPPC membrane tagged with styrene 4; scale bar: 5 µm.
9

Journal Pre-proof
Subcellular localization of the styrene probes:
Styrene probes label mitochondria and cell membrane in HeLa cells
Styrylpyridine and its derivatives have been explored for use in various cellular processes and
are also known for organelle-specific imaging[13, 14, 49]. Here, we aimed to label specific
subcellular compartments using fluorescent probes containing a single styrylpyridine core.
Generally, the cationic pyridinium core of styrenes enables better electrostatic interaction with
the mitochondrial membrane[39, 50]. To explore their specificity and competence of subcellular
localization, we performed fluorescence live-cell imaging using microscopy. In live HeLa cells,
all four styrenes efficiently crossed the plasma membrane within 10 min of incubation and
showed intense subcellular distribution. Styrenes 1-3 show dynamic long thread-like structures
that are uniformly distributed in the cytoplasm resembling mitochondrial localization, while
styrene 4 strongly labeled the cell periphery. The subcellular localization was further confirmed
by incubating the probes with cells expressing FP (fluorescent protein)-tagged organelle-specific
markers. The incubation of probes 1, 2, and 3 with HeLa cells expressing FP-tagged
mitochondria specific markers, Mito-DsRed and Mito-ECFP, showed robust co-localization with
the mitochondrial compartment (Fig 4 A-L). As compared to styrene 1 (PCC: 0.18), the styrenes
2
and 3 showed better mitochondrial specificity (PCC: 0.56 and 0.78, respectively) with a high
signal-to-noise ratio. Furthermore, we have also expressed other cell organelle specific
fluorescent markers. We transfected the Cos-7 cells with BFP-KDEL (Endoplasmic reticulum
marker) and LAMP1-YFP (Lysosome marker) plasmids before imaging. The observed
morphological pattern: mesh-like network of ER and circular structures of lysosome is distinct
from the thread-like mitochondrial structures obtained with styrenes 2 and 3 [Fig. S4]. Styrene 4
showed strong co-localization (PCC: 0.52) with plasma membrane labeled with ECFP-MEM
(Fig. 4 M-P), FP-tagged plasma membrane-specific marker. We also analyzed the intensity
profile of various cellular regions and compared the signal intensity of both compound and
marker channels (Fig. 4 D, H, L, and P). The results for styrenes 2 and 3 are consistent with the
previous findings of the cationic pyridinium derivative’s capability to exploit the electrochemical
potential of the mitochondrial membrane and label mitochondria specifically in vivo [51, 52].
10

Journal Pre-proof
Fig. 4. Labeling of subcellular compartments with styrenes in HeLa cells. Styrenes (1-4,
extreme left panel; 1 µM of each styrene) were incubated with HeLa cells expressing fluorescent
protein (FP)-tagged organelle-specific markers (second panel from left) and overlaid (third panel
from left). Representative images of cells incubated with (A-C) Styrene 1 (E-G) Styrene 2 (I-K)
Styrene 3, and (M-O) Styrene 4. (D, H, L and P) represent the intensity profile of line drawn in
the ROI shown in inset images of respective styrenes (extreme right panel). Dotted yellow lines
indicate the outline of transfected cells; white asterisks indicate nuclei. (scale bars, 10 µm).
11

Journal Pre-proof
Being a neutral derivative and owing to lower solubility, styrene 1 show high background noise
in the emission signal (Fig. 4 A). On the other hand, the presence of cholesterol moiety in styrene
4
provides extra leverage to target the plasma membrane by interacting with the lipid and
cholesterol-rich domains of the mammalian cell membrane making styrene 4 a lucrative probe.
Together the data suggest that the importance of sidearm modifications of the single fluorescent
core in targeting different cellular compartments.
Styrenes label mitochondria and cell membrane in N2a cells
For assessing the labeling ability of the styrenes in other cell types, we also performed live-cell
imaging with the N2a cell line (neuroblastoma cells). Although the foundational architecture is
the same as non-neuronal cells, neurons exhibit unique and specialized functionalities. Thus
imaging the various compartments hold promise in elucidating minuscule details of the neurons.
The results of the co-localization assay showed that styrenes 2 and 3 efficiently labeled the
mitochondria, while styrene 4 show robust localization in the plasma membrane, corroborating
our earlier observations in HeLa cells [Fig 5]. Apart from calculating the Pearson coefficient, we
have also analyzed the pixel intensities from each compound and marker channels, and data
indicated a healthy overlapping pattern [Fig 5].
Styrene probes are not cytotoxic
The suitability of a probe for its use in living cells can be ascertained by examining its cellular
toxicity. In this context, the cytotoxicity of the synthesized styrenes was assessed by a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. No relevant signs of
cytotoxicity were observed even while working at high concentrations (10 µM) over 24 h for all
styryl probes [Fig 6]. This further strengthens our claim to use them as cellular probes for
labeling subcellular compartments in live and fixed cells. Besides, the effect of the solvent used
for the imaging experiments was also found to be non-significant in toxicity experiment.
12

Journal Pre-proof
Fig. 5. Labeling of subcellular compartments with styrene probes in N2a cells. Styrenes (2-4
–
extreme left panel; 1 µM of each styrene) incubated with cells expressing fluorescent protein
(FP)-tagged organelle-specific markers (second panel from left) and overlaid (third panel from
left). Representative images of cells incubated with (A-C) Styrene 2 (E-G) Styrene 3 and (I-L)
Styrene 4. (D, H, and L) represent the intensity profile of line drawn in the images of respective
styrenes (extreme right panel). Dotted yellow lines indicate the outline of transfected cells; white
asterisks indicate nuclei. (scale bars, 10 µm).
13

Journal Pre-proof
Fig. 6. Cell viability assay of styrenes 1-4 investigated using the MTT assay; the dotted line
signifies the negative control where the cells were not treated with any probes.
Styrenes for live mitochondrial tracking in mammalian cells.
Mitochondria are highly motile and dynamic organelles. Their motility affects their functionality
in terms of cell division, development, and overall cellular growth. Interestingly they exhibit
different behavior at the onset of altered physiological and metabolic states that eventually lead
to several diseases such as Alzheimer’s, Parkinson’s, and cancer[53, 54]. Hence visualizing these
differences over a periodical change of time is pertinent through tracking them with specific
probes. The live-cell tracking of mitochondria using external fluorophores requires the probe’s
specificity towards mitochondrial membrane potential, low toxicity, high signal-to-noise ratio,
and high photostability[54]. By these parameters, we herein, track the mitochondrial movement,
mitochondrial fission and fusion events with the help of our mitochondria selective styrenes 2
and 3 [Fig 7 A and B, SI movie-1 and movie-2]. We were able to trace the mitochondrial
dynamics, such as microtubule-based bidirectional motion due to simultaneous presence and
activity of plus- and minus end-directed microtubule-based motor proteins of kinesins and
14

Journal Pre-proof
dyneins respectively on the mitochondrial membrane[55, 56], Representative tracks of
mitochondrial tracks for 2 and 3 are shown in Figure 8.
Fig. 7. Live mitochondria tracking in mammalian cells: (A1-A10) A montage of a representative
cell treated with 1 M styrene-2 showing live mitochondrial motion at different points. (B1-B10)
A montage of a representative cell treated with 1 M styrene-3 showing live mitochondrial
motion at different time points. Dotted yellow lines indicate the outline of cells treated with
styrene probe; white asterisks indicate nuclei; white arrow indicates the movement of single
mitochondria over time. (scale bars, 10 µm).
15

Journal Pre-proof
Fig 8: High-resolution live-cell tracking of mitochondria motion using Styrenes: Mitochondria
were fluorescently labeled with Styrene probes and used for tracking their dynamics in live cells.
Representative tracks of typical bidirectional motion of mitochondria in cells labeled with A)
Styrene 2 and B) Styrene 3.
Styrenes are sensitive to mitochondrial membrane potential
To test the sensitivity of the fluorophores to mitochondrial membrane potential, the cells were
treated with CCCP (Carbonyl cyanide m-chlorophenyl hydrazine), a mitochondrial membrane
potential uncoupler. At the onset of disturbed membrane potential, styrene 3 doesn’t show
specific mitochondrial binding as is evident from the fluorescence signal throughout the cells
(Fig 9). Thus the binding of the fluorophore is dependent on the membrane potential and its
electrostatic mode of interaction.
16

Journal Pre-proof
Fig 9: Styrene binding to mitochondria is membrane potential sensitive: (A) HeLa cells treated
with styrene 3 and (B) HeLa cells incubated with 3 in presence of CCCP. Scale: 5 µM.
Conclusion
In summary, we demonstrated the solvatochromic behavior and cellular localization of four
styrene probes in non-neuronal and neuronal cell lines. Mitochondria- specific localization was
observed for cationic styrenes 2 and 3, whereas the styrene 1 had a low signal to noise ratio in its
ability to stain mitochondria. Furthermore, we have also demonstrated tracking of the live
mitochondrial movements in cells and revealed that our dyes are sensitive to monitor live
mitochondrial dynamics in real-time. Styrene 4 with pendant cholesterol successfully targets the
plasma membrane and shows strong interaction with liposomes. We expect this work might give
a holistic approach to the scientific community towards designing various organelle-specific
probes keeping a single fluorescent core, which in turn enables us to work more efficiently in
multiple optical windows.
Supporting Information: Absorption, emission, and synthetic characterization data are
provided.
Conflicts of Interest: A provisional Indian patent aApplication (IP 201921044149 ) was filed.
17

Journal Pre-proof
Acknowledgments
Authors (SK, TM, KB) acknowledge funding support from SERB, Govt. of India
CRG/2018/004020). VS, AS acknowledges DBT (Grant No.: BT/PR15214/BRB/10/1449/2015
(
and BT/RLF/Re-entry/45/2015) and DST-SERB (Grant No.: ECR/2016/000913) for financial
support. IIT Gandhinagar for general infrastructural support is also acknowledged.
References
[
1] E. Kozma, P. Kele, Fluorogenic probes for super-resolution microscopy, Org. Biomol. Chem., 17 (2019)
15-233.
2] L. Truong, A.R. Ferré-D'Amaré, From fluorescent proteins to fluorogenic RNAs: Tools for imaging
cellularmacromolecules, Protein Science, 28(2019) 1374-1386..
3] H. Zhu, J. Fan, J. Du, X. Peng, Fluorescent Probes for Sensing and Imaging within Specif ic Cellular
Organelles, Acc. Chem. Res., 49(2016) 2115-2126.
4] W. Xu, Z. Zeng, J.-H. Jiang, Y.-T. Chang, L. Yuan, Discerning the Chemistry in Individual Organelles with
Small-Molecule FluorescentProbes, Angew. Chem. Int. Ed., 55 (2016) 13658-13699.
5] X.-P. He, Y. Zang, T.D. James, J. Li, G.-R. Chen, J. Xie, Fluorescent glycoprobes: a sweet addition for
improved sensing, Chemical Commun., 53(2017) 82-90.
6] D.V. Berdnikova, N.I. Sosnin, O.A. Fedorova, H. Ihmels, Governing the DNA-binding mode of styryl
2
[
[
[
[
[
dyes by the length of their alkyl substituents – from intercalation to major groove binding, Org. Biomol.
Chem., 16 (2018) 545-554.
[
7] B. Kumari, A. Yadav, S.P. Pany, P.I. Pradeepkumar, S. Kanvah, Cationic red emitting fluorophore: A
lightup NIR fluorescentprobe forG4-DNA, J. Photochem. Photobiol., B, 190 (2019) 128-136.
8] C. Allain, F. Schmidt, R. Lartia, G. Bordeau, C. Fiorini‐Debuisschert, F. Charra, P. Tauc, M.P. Teulade‐
[
Fichou, Vinyl‐Pyridinium Triphenylamines: Novel Far‐Red Emitters with High Photostability and Two‐
Photon Absorption Properties forStainingDNA, ChemBioChem, 8(2007) 424-433.
[
9] B. Dumat, G. Bordeau, E. Faurel-Paul, F. Mahuteau-Betzer, N. Saettel, G. Metge, C.l. Fiorini-
Debuisschert, F. Charra, M.-P. Teulade-Fichou, DNA switches on the two-photon efficiency of an
ultrabright triphenylamine fluorescent probe specific of AT regions, J. Am. Chem. Soc., 135 (2013)
1
2697-12706.
10] R. Badugu, J.R. Lakowicz, C.D. Geddes, Enhanced Fluorescence Cyanide Detection at Physiologically
Lethal Levels:ꢀReduced ICT-Based SignalTransduction, J. Am. Chem. Soc., 127 (2005) 3635-3641.
11] A.K. Vasu, S. Kanvah, Red-emitting cationic fluorophore as a probe for anionic surfactants, Dyes
Pigm., 142 (2017) 230-236.
12] P. Jana, M. Radhakrishna, S. Khatua, S. Kanvah, A "turn-off" red-emitting fluorophore for nanomolar
detection of heparin, Phys. Chem. Chem. Phys., 20(2018) 13263-13270.
13] L. Guo, R. Zhang, Y. Sun, M. Tian, G. Zhang, R. Feng, X. Li, X. Yu, X. He, Styrylpyridine salts-based red
[
[
[
[
emissive two-photon turn-on probe for imaging the plasma membrane in living cells and tissues,
Analyst, 141 (2016) 3228-3232.
[
14] C.S. Abeywickrama, K.J. Wijesinghe, R.V. Stahelin, Y. Pang, Bright red-emitting highly reliable styryl
probe with large stokes shift for visualizing mitochondria in live cells under wash-free conditions, Sens.
Act. B: Chem., 285 (2019) 76-83.
[
15] L. Yang, J.-Y. Niu, R. Sun, Y.-J. Xu, J.-F. Ge, Rosamine with pyronine-pyridinium skeleton: unique
mitochondrial targetable structure forfluorescentprobes, Analyst, 143(2018) 1813-1819.
18

Journal Pre-proof
[
16] Z. Ding, M. Tian, L. Guo, Z.-q. Liu, X. Yu, Modulate the structures and photophysical properties of
pyrene-based far-red fluorescentcationicdyes by regio-effect, Sens. Act. B:Chem., 276 (2018) 331-339.
17] L. Magrassi, D. Purves, J.W. Lichtman, Fluorescent probes that stain living nerve terminals, J.
Neuroscience, 7(1987) 1207-1214.
18] R. Krieg, A. Eitner, W. Günther, C. Schürer, J. Lindenau, K.J. Halbhuber, N, N-Dialkylaminostyryl dyes:
[
[
specific and highly fluorescent substrates of peroxidase and their application in histochemistry, J. Mol.
Histology, 39 (2008) 169-191.
[
19] J.W. Schwartz, G. Novarino, D.W. Piston, L.J. DeFelice, Substrate binding stoichiometry and kinetics
of the norepinephrine transporter, J. Biol. Chem., 280(2005) 19177-19184.
20] J.N. Wilson, A.S. Brown, W.M. Babinchak, C.D. Ridge, J.D. Walls, Fluorescent stilbazolium dyes as
probes of the norepinephrine transporter: structural insights into substrate binding, Org. biomol. chem.,
0 (2012) 8710-8719.
21] E.Y. Chernikova, D.V. Berdnikova, Y.V. Fedorov, O.A. Fedorova, F. Maurel, G. Jonusauskas, Light-
[
1
[
induced piston nanoengines: ultrafast shuttling of a styryl dye inside cucurbit[7]uril, Phys. Chem. Chem.
Phys., 19 (2017) 25834-25839.
[
22] S.-H. Li, X. Xu, Y. Zhou, Q. Zhao, Y. Liu, Reversibly Tunable White-Light Emissions of Styrylpyridiniums
with Cucurbiturils in Aqueous Solution, Org. Lett., 19 (2017) 6650-6653.
23] L. Sun, W. Zhu, W. Wang, F. Yang, C. Zhang, S. Wang, X. Zhang, R. Li, H. Dong, W. Hu, Intermolecular
[
Charge-Transfer Interactions Facilitate Two-Photon Absorption in Styrylpyridine-Tetracyanobenzene
Cocrystals, Angew. Chem. Int. Ed., 56 (2017) 7831-7835.
[
24] E.N. Ushakov, T.P. Martyanov, A.I. Vedernikov, S.K. Sazonov, I.G. Strelnikov, L.S. Klimenko, M.V.
Alfimov, S.P. Gromov, Stereospecific [2 + 2]-cross-photocycloaddition in a supramolecular donor-
acceptor complex, Tet. Lett., 60(2019) 150-153.
[
25] H. Wu, Y. Chen, X. Dai, P. Li, J.F. Stoddart, Y. Liu, In Situ Photoconversion of Multicolor
Luminescence and Pure White Light Emission Based on Carbon Dot-Supported Supramolecular
Assembly, J. Am. Chem. Soc., 141 (2019) 6583-6591.
[
26] A.K. Vasu, R. Khurana, J. Mohanty, S. Kanvah, pH-responsive molecular assemblies of
pyridylbutadiene derivativewith cucurbit[7]uril, RSCAdv., 8(2018) 16738-16745.
27] A.R. Marri, F.A. Black, J. Mallows, E.A. Gibson, J. Fielden, Pyridinium p-DSSC dyes: An old acceptor
learns new tricks, Dyes Pigm., 165 (2019) 508-517.
28] Y. Ooyama, S. Inoue, T. Nagano, K. Kushimoto, J. Ohshita, I. Imae, K. Komaguchi, Y. Harima, Dye‐
[
[
Sensitized Solar Cells Based On Donor–Acceptor π‐Conjugated Fluorescent Dyes with a Pyridine Ring as
an Electron‐WithdrawingAnchoringGroup, Angew. Chem. Int. Ed., 50 (2011) 7429-7433.
[
29] M. Cheng, X. Yang, J. Li, C. Chen, J. Zhao, Y. Wang, L. Sun, Dye-Sensitized Solar Cells Based on a
Donor–Acceptor System with a Pyridine Cation as an Electron-Withdrawing Anchoring Group, Chem. A
Eur.J., 18 (2012) 16196-16202.
[
30] J. Cui, J. Lu, X. Xu, K. Cao, Z. Wang, G. Alemu, H. Yuang, Y. Shen, J. Xu, Y. Cheng, Organic sensitizers
with pyridine ring anchoring group for p-type dye-sensitized solar cells, J. Phys. Chem. C, 118 (2014)
6433-16440.
31] Y.V. Faletrov, K.I. Bialevich, I.P. Edimecheva, D.G. Kostsin, E.V. Rudaya, E.I. Slobozhanina, V.M.
1
[
Shkumatov, 22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting
oxidoreductases, J. Steroid Biochem. Mol. Biol. 134(2013) 59-66.
[
32] M. Hölttä‐Vuori, R.L. Uronen, J. Repakova, E. Salonen, I. Vattulainen, P. Panula, Z. Li, R. Bittman, E.
Ikonen, BODIPY‐cholesterol: a new tool to visualize sterol trafficking in living cells and organisms, Traffic,
(2008) 1839-1849.
33] J.R. Robalo, J.P.P. Ramalho, L.M.S. Loura, NBD-Labeled Cholesterol Analogues in Phospholipid
Bilayers:Insights fromMolecularDynamics, J. Phys.Chem. B, 117 (2013) 13731-13742.
9
[
19

Journal Pre-proof
[
(
[
34] Z. Li, E. Mintzer, R. Bittman, First synthesis of free cholesterol− BODIPY conjugates, J. Org. Chem., 71
2006) 1718-1721.
35] C. Almeida, A. De Wreede, A. Lamazière, J. Ayala-Sanmartin, Cholesterol-pyrene as a probe for
cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths,
PloS one, 13 (2018) e0201373.
[
(
[
36] G. Gimpl, K. Gehrig-Burger, Probes for studying cholesterol binding and cell biology, Steroids, 76
2011) 216-231.
37] E. Sezgin, F.B. Can, F. Schneider, M.P. Clausen, S. Galiani, T.A. Stanly, D. Waithe, A. Colaco, A.
Honigmann, D. Wüstner, F. Platt, C. Eggeling, A comparative study on fluorescent cholesterol analogs as
versatile cellularreporters, J. Lipid Res., 57(2016) 299-309.
[
38] H.M. McBride, M. Neuspiel, S. Wasiak, Mitochondria: More Than Just a Powerhouse, Current
Biology, 16 (2006) R551-R560.
39] S. Wisnovsky, Eric K. Lei, Sae R. Jean, Shana O. Kelley, Mitochondrial Chemical Biology: New Probes
Elucidate the Secrets of the Powerhouseof the Cell, CellChem. Biol., 23(2016) 917-927.
40] X. Liu, L. Yang, Q. Long, D. Weaver, G. Hajnóczky, Choosing proper fluorescent dyes, proteins, and
imagingtechniques to study mitochondrialdynamics in mammalian cells, Biophys. Rep., 3(2017) 64-72.
41] P. Mishra, D.C. Chan, Mitochondrial dynamics and inheritance during cell division, development and
disease, Nature Rev. Mol. Cell Biol., 15(2014) 634.
42] S.O. Raja, G. Sivaraman, A. Mukherjee, C. Duraisamy, A. Gulyani, Facile Synthesis of Highly Sensitive,
[
[
[
[
Red-Emitting, Fluorogenic Dye for Microviscosity and Mitochondrial Imaging in Embryonic Stem Cells,
ChemistrySelect, 2(2017) 4609-4616.
[
43] K. Mebrouk, S. Debnath, M. Fourmigué, F. Camerel, Photothermal Control of the Gelation
Properties of Nickel Bis(dithiolene) Metallogelators under Near-Infrared Irradiation, Langmuir, 30 (2014)
592-8597.
44] H. Agnihotri, A.K. Vasu, V. Palakollu, S. Kanvah, Neutral and cationic pyridylbutadienes:
solvatochromism and fluorescence response with sodium cholate, Photochem. Photobiol. Sci., 14 (2015)
159-2167.
45] P. Jana, M. Radhakrishna, S. Khatua, S. Kanvah, A “turn-off” red-emitting fluorophore for nanomolar
detection of heparin, Phys. Chem. Chem. Phys., 20(2018) 13263-13270.
46] M. Collot, E. Boutant, M. Lehmann, A.S. Klymchenko, BODIPY with Tuned Amphiphilicity as a
FluorogenicPlasmaMembrane Probe, Bioconjugate chem., 30(2019) 192-199.
47] E. Rideau, R. Dimova, P. Schwille, F.R. Wurm, K. Landfester, Liposomes and polymersomes: a
comparative review towards cellmimicking, Chem. Soc. Rev., 47 (2018) 8572-8610.
48] H.S.P. Rao, A. Desai, I. Sarkar, M. Mohapatra, A.K. Mishra, Photophysical behavior of a new
8
[
2
[
[
[
[
cholesterol attached coumarin derivative and fluorescence spectroscopic studies on its interaction with
bile saltsystems and lipid bilayermembranes, Phys. Chem. Chem. Phys., 16(2014) 1247-1256.
[
49] L. Plougastel, M.R. Pattanayak, M. Riomet, S. Bregant, A. Sallustrau, M. Nothisen, A. Wagner, D.
Audisio, F. Taran, Sydnone-based turn-on fluorogenic probes for no-wash protein labeling and in-cell
imaging, Chem. Commun., 55(2019) 4582-4585.
[
50] A.C. Shaikh, M.E. Varma, R.D. Mule, S. Banerjee, P.P. Kulkarni, N.T. Patil, Ionic Pyridinium–Oxazole
Dyads:Design, Synthesis, and Application in Mitochondrial Imaging, J. Org. Chem., 84(2019) 1766-1777.
51] G. Zhang, Y. Sun, X. He, W. Zhang, M. Tian, R. Feng, R. Zhang, X. Li, L. Guo, X. Yu, S. Zhang, Red-
[
Emitting Mitochondrial Probe with Ultrahigh Signal-to-Noise Ratio Enables High-Fidelity Fluorescent
Images in Two-Photon Microscopy, Anal. Chem., 87(2015) 12088-12095.
[
52] Z. Wang, Y. Gu, J. Liu, X. Cheng, J.Z. Sun, A. Qin, B.Z. Tang, A novel pyridinium modified
tetraphenylethene: AIE-activity, mechanochromism, DNA detection and mitochondrial imaging, J.
Mater. Chem. B, 6 (2018) 1279-1285.
20

Journal Pre-proof
[
53] D.C. Chan, Mitochondria: Dynamic Organelles in Disease, Aging, and Development, Cell, 125 (2006)
1
241-1252.
54] Y. Chen, T.W. Rees, L. Ji, H. Chao, Mitochondrial dynamics tracking with iridium(III) complexes, Curr.
Opn. Chem. Biol., 43 (2018) 51-57.
55] V. Soppina, A.K. Rai, A.J. Ramaiya, P. Barak, R. Mallik, Tug-of-war between dissimilar teams of
microtubule motors regulates transport and fission of endosomes, Proc. Natl. Acad. Sci., 106 (2009)
9381-19386.
56] V. Soppina, S.R. Norris, A.S. Dizaji, M. Kortus, S. Veatch, M. Peckham, K.J. Verhey, Dimerization of
mammalian kinesin-3 motors results in superprocessive motion, Proc. Natl. Acad. Sci., 111 (2014) 5562-
567.
[
[
1
[
5
TOC Graphic
21

Journal Pre-proof
AUTHORSHIP STATEMENT
Manuscript title: Imaging Mitochondria and Plasma Membrane in Live Cells using
Solvatochromic Styrylpyridines
All authors have contributed to the working of the manuscript.
SK and VS: Supervision, Conceptualization, Methodology, Interpretation, Writing, Reviewing
and Editing. TM : Synthesis and biological experiments, acquisition of data, analysis and
interpretation, Original draft preparation, AS: Recombinant DNA tagging for plasmid
preparation and acquisition of data, KB: Synthesis of molecules and acquisition of data
22

Journal Pre-proof
Declaration of interests
☐
The authors declare that they have no known competing financialinterests orpersonal relationships
that could have appeared to influence the work reported in this paper.
☒
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
A provisional Indian Patent Application (IP 201921044149 ) was filed
23

Journal Pre-proof
Imaging Mitochondria and Plasma Membrane in Live Cells using
Solvatochromic Styrylpyridines
‡
†
‡
†
Tarushyam Mukherjee , Aravintha Siva , Komal Bajaj , Virupakshi Soppina* and Sriram
‡
#
Kanvah *
#‡ *Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar
3
82355, India. E-mail: sriram@iitgn.ac.in, kanvah@gatech.edu
†
*Department of Biological Engineering, Indian Institute of Technology Gandhinagar Palaj
Gandhinagar 382355 e-mail: vsoppina@iitgn.ac.in
Graphical Abstract

Cholesterol derivatives image plasma membrane and the fluorophores without
cholesterol tether image mitochondria.
24

Journal Pre-proof
25

Journal Pre-proof
Imaging Mitochondria and Plasma Membrane in Live Cells using
Solvatochromic Styrylpyridines
‡
†
‡
†
Tarushyam Mukherjee , Aravintha Siva , Komal Bajaj , Virupakshi Soppina* and Sriram
‡
#
Kanvah *
#‡ *Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar
3
82355, India. E-mail: sriram@iitgn.ac.in, kanvah@gatech.edu
†
*Department of Biological Engineering, Indian Institute of Technology Gandhinagar Palaj
Gandhinagar 382355 e-mail: vsoppina@iitgn.ac.in


Styrlpyridine with and without cholesterol tether were synthesized.
The fluorophores show solvatochromic emission attributed to intramolecular charge
transfer in green to red wavelength regions.


The neutral and the cationic styrylpyridines image and track mitochondria efficiently and
also
Cholesterol-tethered styrylpyridine stains plasma membrane and also shows robust
interaction with liposomes
26