Live-cell imaging of lipid droplets using solvatochromic coumarin derivatives

Article abstract of DOI:10.1039/d0ob01277g

Lipid droplets (LDs), the lipid-rich intracellular organelles, serve to regulate many physiological processes and therefore attention has been attracted towards their selective detection. We report positively solvatochromic lipophilic dyes, based on the push-pull framework containing coumarin-pyridine heterocycles for selective live-cell imaging of lipid droplets (LDs) in Cos-7 and McA-RH7777 cells at ultralow concentrations (200 nM). The fluorescent probes show a remarkable increase in fluorescence intensity with time with the hydrophobic core of the lipid droplets contributing to the observed intensity enhancement. The simple structural framework, red emission, strong Stokes shift (>80 nm), and excellent biocompatibility highlight their significance as a versatile imaging tool for studying lipid droplets (LDs).

Full text of DOI:10.1039/d0ob01277g

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Live-cell Imaging of Lipid Droplets by Slvatochromic CoumDaOrI:i1n0.1039/D0OB01277G
Derivatives
Palash Jana#, Aravintha Siva†, Virupakshi Soppina†* and Sriram Kanvah#*
#Department of Chemistry, Indian Institute of Technology Gandhinagar,
Palaj, Gandhinagar 382355, 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, vsoppina@gmail.com
Abstract
Lipid droplets (LDs), the lipid-rich intracellular organelles, serve to regulate many
physiological processes and therefore attracted attention towards selective detection of lipid
droplets. We report positively solvatochromic lipophilic dyes, based on the push-pull
framework containing coumarin-pyridine heterocycles for selective live-cell imaging of lipid
droplets (LDs) in Cos-7 and McA-RH7777 cells at ultralow concentrations (200 nM). The
fluorescence probes show a remarkable increase in fluorescence intensity with time with the
hydrophobic core of the lipid droplets contributing to the observed intensity enhancement. The
simple structural framework, red-emission, strong Stokes shift (>80 nm), excellent
biocompatibility highlights their significance as a versatile imaging tool for studying of Lipid
droplets (LDs).
Introduction
Lipid droplets (LDs) are specialized intracellular organelles in cells and organisms, which
evolved as a universal energy reservoir mechanism in both eukaryotes and prokaryotes1-5.
These ubiquitous organelles are composed of a phospholipid monolayer that encapsulates
triglycerols and sterol esters. A plethora of proteins decorates the surface of these organelles,
including the membrane protein perilipin, membrane trafficking proteins like Rab5 and Rab18,
and enzymes such as triglycerol lipases that are involved in lipid metabolism6-9. Although
different models postulate different mechanisms of lipid droplet formation, the common belief
is that these organelles originate from the endoplasmic reticulum10. LDs play essential roles in
several cellular processes that include the production of energy, formation of membranes,
carriers of lipids, protein-protein interactions, and metabolism of fats 5, 11. Besides, they also

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play crucial roles in other cellular processes including lipid ligand generation and transcription
DOI: 10.1039/D0OB01277G
factor sequestration12. The dynamics and functions of these droplets are further complicated by
their interaction with multiple other organelles like the mitochondria, endosomes, and
endoplasmic reticulum13. Due to their critical role in numerous such cellular events, any
abnormalities in these organelles are implicated in the development of metabolic diseases.
Multiple reports state the involvement of these organelles in health conditions such as obesity,
diabetes, atherosclerosis, neurodegeneration, and fatty liver diseases12, 14, 15. Despite the vast
influx of information on the importance of these dynamic structures in the recent past,
fundamental questions on the composition, function, and biogenesis of these organelles remain
mostly unanswered.
This shortage of information is partially attributed to the lack of techniques that help us
visualize these structures inside the cell. While conventional microscopic methods like light
microscopy do contribute to the study of the biophysical properties of these organelles, they
usually require the cells to be fixed and processed. On the other hand, imaging of these
organelles with fluorescent dyes aids significantly in understanding their dynamics and
localization in live-cell conditions16, 17. Therefore, to visualize real-time and get insights on
their interactions, highly sensitive fluorescence microscopy-based imaging tools were much
sought18. The use of organelle-specific fluorophores will allow the study of precise localization
and dynamics of different sub-cellular components. In particular, the use of donor or acceptor
substituted fluorophores have attracted significant attention owing to their potential
applicability for sensing and bioimaging19-22. The research on lipid droplets gained momentum
with the simultaneous synthesis of a variety of fluorescent dyes, including Nile Red 23, Bodipy
17
26
27-30
24, 25, cyanine dyes , Lipid Green , push-pull
and amphipathic derivatives 31 32, and
others33, 34. Some limitations of the dyes include narrower Stokes shift, photostability,
complicated or expensive synthetic strategies, interfering background signals and low
specificity in live cells. Dyes such as Oil Red O and Sudan III also suffer from such drawbacks
in addition to being disruptive to the cells in a few cases, resulting in false-positive
observations. Despite the rapid development of organic fluorophores, newer molecules are
continually being developed for stronger and selective imaging of the lipid droplets. A
summary of some recent imaging lipid droplets using organic fluorophores is shown in table
S1.

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In this report, we propose a simple coumarin-substituted styrylpyridines as potential molecules
DOI: 10.1039/D0OB01277G
for imaging lipid droplets [Fig-1]. Both coumarin and pyridine moieties are extensively used
for biological applications and form a crucial structural component of many natural product
scaffolds. The presence of diethylamino or julolidine electron donating groups helps in shifting
the intramolecular charge transfer bands to the red-wavelength regions. Further, the donor-
acceptor structural framework separated by the styryl bridge also yields a relatively significant
Stokes shift. Moreover, suitably substituted coumarin derivatives are also used as excellent
fluorescent reporters 35-39. In the present report, we synthesized and characterized two simple
styrylcoumarin probes and demonstrated the tracking of lipid droplets inside the cell and the
study enables future investigations in understanding the dynamics of these organelles and
studying disease models.
Experimental
The chemicals required for the synthesis of pyridine acrylonitrile derivatives were obtained
from Aldrich, Alfa Aesar, Sigma-Aldrich, and S. D. Fine. The UV-Vis absorption spectral data
were measured using Analytic Jena, Specord 210 spectrophotometer. The emission spectra
were recorded using the Horiba Fluorolog-3 spectrofluorimeter. The synthesized samples were
characterized using NMR and HRMS obtained using Bruker Avance 500 MHz NMR
spectrometer and Waters-Synapt G2S (ESI-QToF) mass spectrometer, respectively. For the
absorption or fluorescence studies, 10 μL of the desired fluorophore-stock solution in dioxane
(∼10−3 M) was added to 1 mL (effective concentration ~10 µM) of the desired solvent media,
and the samples were excited at their respective absorption maxima (λabs).
Reagents and plasmids
All reagents for molecular biology, cell culture, and imaging experiments were purchased from
Sigma-Aldrich, Thermofisher Scientific, and New England Bio Labs (NEB) unless otherwise
mentioned. HPos is a peptide that is putatively used as a lipid droplet-targeting sequence. The
plasmid DNA for HPos and the commercial lipid droplet stain, Oil Red O were a kind gift from
Prof. Roop Mallik, TIFR, Mumbai. We generated the HPos-mCherry plasmid by sub-cloning
the HPos using appropriate restriction sites into mCherry-N1 backbone plasmid. All the
plasmids used in the study were verified by DNA sequencing.

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DOI: 10.1039/D0OB01277G
Cell culture and transfection
Cervical adenocarcinoma (HeLa), COS-7 (monkey kidney fibroblast, ATCC), and rat liver
cells (McA-RH7777) cells were grown in DMEM supplemented with 10% fetal bovine serum
(FBS) and 2 mM L-glutamine at 37 C with 5% CO2. Cells were transfected with 1.0 g of
plasmid DNA using Lipofectamine-3000 transfection reagent.
MTT assays
For MTT, five thousand HeLa cells were seeded (per well) in a 96-well plate. These cells were
subsequently treated with varying concentrations of the probes for 24 h following which, they
were incubated with MTT for 4 h. DMSO was then added to solubilize the formazan, and the
absorbance was recorded at 570 nm using BioTek SYNERGYH1 plate reader.
Fluorescence Live-cell imaging
For live-cell studies, 0.1 million Cos-7/McA-RH7777 cells were seeded on 35 mm glass-
bottom plates. The cells were treated with 200 nM of the respective probes (J-1 and C-1) for
10 and 30 minutes, respectively. The plates were then washed thrice with Fluorobrite imaging
medium and images were obtained on an inverted epifluorescence Nikon Ti2 Eclipse
microscope, equipped with 60X 1.49 NA objective and an EMCCD camera (Andor iXon
897Ultra, Andore Technologies, USA). The images were acquired at a speed of 10 fps and
stored in the form of movies. For co-distribution studies with Oil Red O, 0.1 million Cos-7
cells were seeded on 35 mm glass-bottom plates. Cells were first incubated with Oil Red O and
images were captured in the red channel. Following this, the cells were incubated with 200 nM
of the respective probes (C-1 and J-1) and images were taken in green channel.
Data analysis
The image analysis and quantification were performed using ImageJ software (nih.gov). Cells
were randomly selected to perform the quantification. The value of maximum fluorescence
intensity was obtained from ImageJ. Background fluorescence was subtracted from this
maximum value to get the fold change in intensity.

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DOI: 10.1039/D0OB01277G
Results and Discussion
Synthesis of fluorophores 1 and 2
Two fluorescent probes bearing coumarin and pyridine scaffolds (C-1 and J-1) were
synthesized by a condensation reaction of pyridine acrylonitrile with the corresponding
aldehyde 4 & 7 (Fig-1). The aldehydes (4 and 7) used were synthesized by following published
literature 35, 40. The aldehydes 2 and 5 were cyclized with malonic ester in the presence of
piperidine to achieve coumarin scaffold 3 and 6 with diethylamine and julolidine donating
groups respectively. Vilsmeier-Haack formylation of 3 and 6 yields the desired aldehydes 4
1
13
and 7. All the structures have been characterized using H-NMR, C-NMR & HRMS. The
synthesized derivatives differ with each other: a diethylamine donor in C-1 and a julolidine
group in J-1. A cyanovinyl -spacer separates both the coumarin and pyridyl group.
Fig 1: Synthesis of coumarin styrylpyridine probes C-1 and J-1. (i) CH2(COOEt)2, Piperidine,
ethanol, reflex; (ii) POCl3, DMF, 60C, 8h; (iii) 4-pyridine acrylonitrile, piperidine, ethanol,
reflux, 8h.
Photophysical characterization
The synthesized compounds were investigated for their optical properties in various organic
solvents & water. C-1 contains a diethylamine donor while J-1 has julolidine electron donating
group. The presence of julolidine may help in improve the electron-donating ability and
improving the ICT absorption and emission. The compounds absorb strongly in the visible
region with C-1 absorbing in the range of 477 nm-488 nm and J-1 absorbing at 498 nm. The
higher absorption maxima are due to increased electron-donating ability of the julolidine
moiety [Fig-2]. Moderate bathochromic absorption shifts (~10 nm) were noted with an increase
in solvent polarity and attributed to solute-solvent interactions and energy level stabilization.
Broad emission is recorded in water due to its weaker solubility and may also be due to the

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formation of aggregates41, 42. The emission maxima show bathochromic shifts with an increase
DOI: 10.1039/D0OB01277G
in solvent polarity. C-1 emits at 545nm in dioxane with a change of + ~35 nm upon increasing
the solvent polarity (Fig-3 & Table-1). The stronger electron-donating amino group in J-1
yields slightly larger shifts (39 nm) in the emission wavelength. The compounds are also
characterized by as substantial Stokes shift 95 nm for C-1 and 93 nm in J-1.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1,4-Dioxan
THF
ACN
a
b
1,4-Dioxane
THF
CH3CN
DMF
DMF
DMSO
MeOH
H2O
DMSO
MeOH
H2O
300
350
400
450
500
550
600
650
700
300
350
400
450
500
550
600
650
700
Wavelength (nm)
Wavelength(nm)
Fig 2: Absorption of a) C-1 and b) J-1 in different organic solvents
b
a
Dioxane
THF
CH3CN
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Dioxane
THF
CH3CN
DMF
DMF
DMSO
MeOH
H2O
DMSO
MeOH
H2O
500
550
600
650
700
750
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
Fig 3: Emission of C-1 and J-1 in different organic solvents.

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DOI: 10.1039/D0OB01277G
Table-1 Absorption & emission maxima of C-1 & J-1.
C-1
J-1
Stokes
Shift
(nm)
Stokes
Solvents
ab
(nm)
em
(nm)
f
ab
(nm)
em
(nm)
f
Shift
(cm-1)
65
72
86
86
93
97
Dioxane
THF
CH3CN
DMF
DMSO
H2O
477
545
68
64
85
86
95
102
0.059
0.051
0,043
0.038
0.043
0.012
499
564
0.015
0.015
0.014
0.017
0.021
0.010
484
481
488
485
478
548
566
574
580
580
508
509
515
512
502
580
595
601
605
599
f: was calculated using 0.1 M fluorescein and excited at 470 nm
Effect of Viscosity
The donor-acceptor molecular systems exhibiting strong emission due to intramolecular charge
transfer have been used to study the impact of viscosity. Of particular relevance to the paper is
to correlate the viscosity effects towards the localization in lipid droplets, for LDs provide
hydrophobic and viscous environment compared to that of other cellular organelles. We,
therefore, correlated the emission intensity of the probe with the change in viscosity of the
solution. The viscosity of LDs (∼50 cP) is significantly higher than that of cytosol (∼1 cP)43
and an increase in viscosity is expected to decrease the non-radiative transition yielding
significant emission enhancement. We thus studied the emission changes of the fluorophores
C-1 and J-1 in a methanol-glycerol binary mixture [Fig-4]. With increased glycerol fractions,
the molecular motions are hindered yielding gradual emission intensity increments. C-1 shows
a ~11 folds intensity increment while J-1 shows a ~7 folds rise in intensity along with a ~20
nm bathochromic emission shift. Distinct color changes also accompany the enhancement in
strength. The titration of fluorophores with different surfactant (CTAB) concentrations show
strong intensity enhancement with changes in micelle concentration indicating the preference
to interact with viscous environment [Fig S1].

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DOI: 10.1039/D0OB01277G
0% Glycerol
b
a
1.6x105
1.4x105
1.2x105
1.0x105
8.0x104
6.0x104
4.0x104
2.0x104
0.0
2.5x106
2.0x106
1.5x106
1.0x106
5.0x105
0.0
20% Glycerol
40% Glycerol
60% Glycerol
80% Glycerol
100% Glycerol
0% glycerol
20% glycerol
40% glycerol
60% glycerol
70% glycerol
80% glycerol
90% glycerol
100% glycerol
500
550
600
650
700
750
800
500
550
600
650
700
750
800
Wavelength(nm)
Wavelength(nm)
Fig-4: Increase in viscosity yields vigorous intensity and color changes: Emission of a) C-1
and b) J-1 in methanol-glycerol binary mixtures.
Effect of pH:
Considering the presence of favorable lone pair of electrons on the pyridine nitrogen and their
role in controlling the photophysical properties, we studied the pH effect on C-1 and J-1. The
protonation of nitrogen is expected to significantly alter the charge transfer properties.
Subsequently as shown in Fig 5, increasing acidity leads to protonation of pyridine nitrogen
yielding pyridinium ion that acts as strong acceptor shifting the absorption maximum to ~550
nm for C-1 and 585 nm in J-1 associated with strong color changes. The emission spectra
shown in Fig S2 indicate shifting of the emission band to ~635 nm (C-1) in strongly acidic
conditions associated with emission quenching for both molecules (Fig S2). This loss of
emission in acidic media is attributed to formation of twisted intramolecular charge transfer
excited state disturbing the planar delocalization44.

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DOI: 10.1039/D0OB01277G
C
d
Fig 5. Absorption of C-1 and J-1 at different pH conditions. Associated color changes are
shown in Fig 5c (C-1) and 5d (J-1). Fig S2 gives emission spectra

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DOI: 10.1039/D0OB01277G
Fig 6: Probes efficiently label lipid droplets: Cos-7 cells incubated with 200 nM of A) C-1 for
30 minutes and B) J-1 for 10 minutes respectively. The highly refractile spherical structures
under DIC illumination (Left panels) and fluorescently labelled bright lipid droplets (Middle
panels) showed significant overlapping (Right panels) when imaged in Cos-7 cells. Scale bar
represents 10 µm.
Coumarin-based probes for live-cell lipid droplet imaging.
Considering the solvatochromic emission and enhanced intensity of the fluorophores in the
viscous solvents, we examined the fluorescent-tagging of lipid droplets (LDs). Cos-7 cells
incubated with these fluorescent dyes (C-1 and J-1) rapidly crossed the cell membrane and
showed distinct bright uniform round punctate localization distributed throughout the
cytoplasm (Figure-6). Interestingly, when these cells were imaged under differential
interference contrast (DIC) illumination, the above bright fluorescent punctate structures
showed significant overlapping with spherical, highly refractile vesicle structures in the
cytoplasm resembling lipid droplets (Fig-6). Lipid droplets, due to their considerable light
diffraction properties appear as highly refractile particles under differential interference
contrast (DIC) microscopy45, 46. To further confirm the identity of these refractile particles, we
transiently transfected cells with a plasmid coding for C-terminal mCherry-tagged HPos

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(HPos-mCherry), a marker for nascent lipid droplets47. As expected, the brigDhOtI: r10e.f10ra39c/tDi0lOeB01277G
particles showed a significant overlap with lipid droplets that are fluorescently labelled with
HPos-mCherry (Fig. 7A). In addition, we also performed co-distribution experiments of these
probes with the well-known lipid droplet stain Oil Red (Fig. 7B & C) 48. Together, our results
from the co-localization studies indicate that both the probes selectively localize to and stain
the lipid droplets (PCC for C-1: 0.97 ± 0.01; PCC for J-1: 0.96 ± 0.02) with a sizeable signal-
to-noise ratio (Fig 7B & C). Moreover, such localization signals were observed even in cells
treated with as low as 200 nM concentrations of the probes for the durations as short as 10
minutes.
Furthermore, to potentiate the use of these fluorescent probes across the cell types, we imaged
lipid droplets in liver cancer cells. The liver is a crucial organ that functions as a part of different
physiological processes, including digestion, metabolism and hormone production. Also, the
liver is the main storage and metabolic center for the lipids and hence the lipid droplet levels
are strictly regulated in the hepatocytes 49, 50. Therefore it would be exciting and clinically
relevant to know whether these fluorescent probes can be used to image the lipid droplets in
the hepatocytes. To test this, we incubated McA-RH7777, rat hepatoma cells with the above
fluorescent probes and imaged under epifluorescence and DIC illumination. Similar to the Cos-
7 cells, both the fluorophores showed distinct fluorescently labelled bright punctate localization
distributed
in
the
cytoplasm
of
McA-RH7777 cells that were highly refractile and spherical under DIC illumination
(Figure- 8A & B), consistent with previous work45. Together, the data suggest that the probes
J-1 and C-1 have great specificity for the lipid droplets due to the strong hydrophobic core of
the lipid droplets and stain the lipid droplets with the large signal-to-noise ratio. These results
show good promise in extrapolating the use of these probes to clinical research.

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Fig 7: Probes efficiently colocalize with known lipid droplet markers: A) Cos-7 cells
transfected with HPos-mCherry. Lipid droplets stained with Oil Red O (B& C: Left panels)
and 200 nM of B) C-1 or C) J-1 respectively (Middle panels) showed significant overlapping
(Right panels) in Cos-7 cells. (PCC for C-1: 0.97 ± 0.01; PCC for J-1: 0.96 ± 0.02) Scale bar
represents 10 µm.

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DOI: 10.1039/D0OB01277G
Fig 8: Probes efficiently label lipid droplets: McA-RH7777 cells incubated with 200 nM of A)
C-1 and B) J-1, respectively. The highly refractile spherical structures under DIC illumination
(Left panels) and fluorescently labelled bright lipid droplets (Middle panels) showed significant
overlapping (Right panels) when imaged in McA-RH7777 cells. Scale bar represents 10 µm.
Novel increase in the fluorescent intensity of probes under progressive excitation
Lipid droplets are highly dynamic structures that undergo constant restructuring51. This raises
the fundamental need for any fluorophore to be able to capture the dynamics of these molecules
at the cellular timescale. Having known that our probes could stain the lipid droplets, we
performed live-cell imaging to find out whether they were capable enough to track the
dynamics of these structures for longer time-scale. Our results from real-time tracking studies
of these probes showed the rapid movement and dynamics of the lipid droplets at sub-second
timescales [Video 1 (C1) and Video-2 (J1): Supporting Information]. Surprisingly, unlike the
conventional fluorophores that photobleach progressively upon continuous excitation, our
coumarin-based probes do not photobleach over time. Instead, the fluorescent probes showed
an increase in their intensity with time under constant excitation. To quantitatively analyse the
progressive increase in the intensity, we recorded the fluorescent intensities of individual lipid

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droplets frame-by-frame and measured the percentage change in their fluorescenDcOeI:i1n0.t1e0n39s/iDt0yO.B01277G
Surprisingly, we observed that there was a significant increase (P< 0.0001; t-test) in the
fluorescence signal with time (Figure- 9A & B).
Fig 9: Remarkable increase in the fluorescent intensity of probes with progressive excitation:
A) C-1 Average intensity of individual lipid droplets at 0 mins and 10 min. B) J-1: Average
intensity of individual lipid droplets at 0 mins and 5 min. n= 65; *** P<0.0001 (Paired t-test).
Both J-1 and C-1 showed potent application in staining lipid droplets inside the cell. However,
for any probe to be effectively used for bio-applications, their effect on the cell viability is an
important parameter. To check the effect of these probes on cell viability, we performed an
MTT assay across a wide range of concentrations, ranging from 100 nM to 1000 nM. Our
results indicated that while C-1 was toxic at higher concentrations (Figure- 10A), J-1 was not
cytotoxic even after 24 h of treatment (Fig. 10B).

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DOI: 10.1039/D0OB01277G
Fig. 10: Cell viability assays. A) C-1 and B) J-1. HeLa cells incubated with varying
concentrations of the probes for 24 h were used for the MTT assay. The values are represented
as mean ± SD (n=3; #: not significant; **P < 0.01 (Student t-test); *** P<0.0001 (ANOVA).
Conclusions
In this work, styryl derivatives containing coumarin and pyridine scaffolds were designed and
synthesised. The fluorophores show significant Stokes shift with emission > 550 nm. Live-cell
imaging experiments demonstrate selective staining of the lipid droplets at lower
concentrations of the fluorophores with a high signal-to-noise ratio at low concentrations. The
fluorophores also show strong biocompatibility and excellent photostability. This work
provides a simpler way to visualize the lipid droplets and provide powerful tools to unravel the
dynamics of lipid droplets.
Supporting Information
1
13
Comparative T=Table of recently published literature (Table S1), H and C NMR data and
lipid droplet localization videos.
Acknowledgments
A financial grant from the SERB (CRG/2018/004020) is appreciated. VS, AS acknowledge
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. PJ acknowledges a fellowship
from IIT Gandhinagar. We also thank reviewers for the insightful suggestions.

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DOI: 10.1039/D0OB01277G
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