Switchable Solvatochromic Probes for Live-Cell Super-resolution Imaging of Plasma Membrane Organization

Article abstract of DOI:10.1002/anie.201907690

Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super-resolution microscopy probes that combines specific membrane targe

Full text of DOI:10.1002/anie.201907690

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Accepted Article
Title: Tailor-made switchable solvatochromic probes for live-cell super-
resolution imaging of plasma membrane organization
Authors: Dmytro I. Danylchuk, Seonah Moon, Ke Xu, and Andrey S
Klymchenko
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201907690
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10.1002/anie.201907690
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Tailor-made switchable solvatochromic probes for live-cell super-
resolution imaging of plasma membrane organization
Dmytro I. Danylchuk,[a]‡ Seonah Moon,[b], [c]‡ Ke Xu,[b], [c]* Andrey S. Klymchenko[a]*
be distinguished in model membranes. Previously, based on Nile
Red, we designed probe NR12S that reports lipid order selectively
at the outer leaflet of cell plasma membranes[12]. Due to its high
brightness and capacity to work at nanomolar concentrations, it
became a common tool to study transmembrane asymmetry,[13]
Abstract: Visualization of the nanoscale organization of cell
membranes remains challenging because of the lack of appropriate
fluorescent probes. Here, we introduce a new design concept for
super-resolution microscopy probes that combines specific
membrane targeting, ON/OFF switching, and environment sensing
functions. In this design, we propose a functionalization strategy for
solvatochromic dye Nile Red that improves its photostability. The dye
apoptosis,[14]
maturation
of
endosomes,[15]
membrane
potential,[16] lipid trafficking,[17] etc.[8] The current challenge is to
adapt this type of probes for super-resolution microscopy.[18]
is grafted to
a newly developed membrane-targeting moiety
Super-resolution
microscopy,
including
single-molecule
localization microscopy (SMLM), is making a revolution in the
composed of a sulfonate group and an alkyl chain of varied lengths,
which ensures high brightness and lipid-order sensitivity of the probes
and tunes their binding/unbinding-induced ON/OFF switching
required for single-molecule localization microscopy. While the long-
chain probe with strong membrane binding, NR12A, is suitable for
conventional microscopy, the short-chain probe NR4A, due to the
reversible binding, enables first nanoscale cartography of the lipid
order exclusively at the surface of live cells. The latter probe reveals
in plasma membranes the presence of nanoscopic protrusions and
invaginations of lower lipid order, suggesting a subtle connection
between membrane morphology and lipid organization.
structural and functional characterization at the subcellular level.[3,
19]
Numerous efforts have been made to develop fluorescent
probes for super-resolution techniques,[20] where ON/OFF
switching, required by SMLM, was implemented through photo-
switching,[7a, 21] isomerization,[22] transient binding of dyes[23] and
proteins[24] to the membrane, and DNA hybridization.[25] A largely
underexplored direction, which can open a new dimension in
SMLM, is to implement sensing function into this type of probes.
It became possible with recently introduced spectrally resolved
PAINT
(SR-PAINT)
for
super-resolution
imaging
of
biomembranes.[26] It exploits solvatochromic dye Nile Red, which
(i) lights-up upon reversible binding to biomembranes[23a] and (ii)
changes its emission color in response to the lipid order[6a, 8, 12, 26a,
26b]. However, this dye is not plasma membrane specific, as it
binds all lipid components of cells. Here, based on Nile Red
fluorophore, we designed the first probe combining together
plasma membrane specificity, controlled ON/OFF switching, and
sensing functions, which enabled imaging of both nanoscale
morphology and lipid order in biomembranes.
The complex nature of cell plasma membranes raised intensive
research in the last decades, stimulated by the hypothesis of
membrane microdomains (lipid rafts).[1] In model membranes,
these domains are identified in terms of lipid order,[2] where liquid-
ordered phases (Lo) rich in sphingomyelin and cholesterol “swim”
in a pool of disordered phase (Ld) formed by unsaturated lipids.
The challenge to visualize lipid domains in cells is linked to their
highly dynamic nature, nanoscopic size, and non-flat geometry of
plasma membranes with protrusions and invaginations.[3]
To develop probes for SR-PAINT, we fundamentally changed the
design strategy vs the original NR12S (Fig. 1a). First, we inverted
the position of Nile Red modification and thus removed phenolic
oxygen from the dye (NR12A and NR4A), which was
hypothesized to cause lower photostability. Second, we propose
here a new membrane-targeting moiety based on an anionic
sulfonate head group and a lipophilic alkyl chain of varied lengths.
We reasoned that dyes with long alkyl chains (NR12A and
NR12S) should label plasma membranes quasi-irreversibly (Fig.
1b), which is suitable for conventional fluorescence microscopy.
By contrast, a short alkyl chain (in NR4A) may enable reversible
labeling (Fig. 1c) and thus the desired ON/OFF switching required
for PAINT.[23a] Compounds NR12A and NR4A were thus
synthesized in six steps starting from m-anizidine (Fig. S1).
One approach to visualize membrane organization is fluorescent
labeling of lipids, such as sphingomyelin[4] and ceramide,[5] but
labeling may alter properties of lipids.[6] Another approach is to
label proteins that specifically target sphingolipids,[7] although the
effect of these large molecules on lipid order is still unclear. New
possibilities are offered by the environment-sensitive probes,[8]
sensitive to polarity (solvatochromic dyes), viscosity (molecular
rotors),[9] and membrane tension (flippers).[10] The emission color
of solvatochromic probes, like Laurdan,[2b] di-4-ANEPPDHQ,[11]
and Nile Red,[12] directly reflects the lipid order, because it is linked
with the local polarity and hydration, so that Lo and Ld phases can
In organic solvents of varying polarity, the absorption and
emission characteristics of NR4A and NR12A were close to Nile
Red (Fig. S2, Table S1), indicating that the functionalization did
not perturb the fluorophore properties. All studied probes showed
fluorescence intensity increase (fluorogenic response) on binding
to large unilamellar vesicles (LUVs) composed of DOPC, but the
titration curves varied strongly (Fig. 1d). NR12A showed
significantly higher affinity to LUVs than the parent Nile Red,
displaying rapid intensity growth followed by a plateau at higher
concentrations, similarly to NR12S. In sharp contrast, NR4A
displayed a slow increase in the fluorescence intensity with
liposomes concentration, even weaker than the parent Nile Red.
These results confirmed that we obtained a weak (NR4A) and a
strong (NR12A) binders of lipid membranes.
[a]
[b]
[c]
D. I. Danylchuk; Dr. A. S. Klymchenko
Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS
Université de Strasbourg, 74 route du Rhin, 67401, Illkirch, France
E-mail: andrey.klymchenko@unistra.fr
S. Moon; Dr. K. Xu
Department of Chemistry, University of California, Berkeley
University of California, Berkeley, California, 94720, United States
E-mail: xuk@berkeley.edu
S. Moon; Dr. K. Xu
Chan Zuckerberg Biohub, San Francisco, CA 94158, United States
‡These authors contributed equally.
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10.1002/anie.201907690
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Figure 1. Design of probes and their characterization in model membranes. (a) Chemical structures of NR12A and NR4A as well as the previously reported NR12S
and Nile Red. (b,c) Design concept of the fluorogenic probes that irreversibly (b) or reversibly (c) bind to biomembranes due to the presence of long or short
hydrophobic chains, respectively. (d) Titration of the four membrane probes (100 nM) with increasing concentrations of lipid vesicles: fluorescence intensity at 620
nm vs. lipid concentration. Errors are s.d.m. (n = 3). (e) Fluorescence spectra of NR12A (2 µM) in buffer (20 mM phosphate buffer, pH 7.4) and liposomes (LUVs)
of different lipid composition (1 mM lipids). Excitation wavelength was 520 nm. All the spectra were corrected via dividing by absorbance at the excitation wavelength.
(f) Fluorescence intensity of the different probes (400 nM) under continuous illumination vs. time in DOPC liposomes (200 µM). Excitation and emission wavelengths
were 540 and 620 nm, respectively. (g) Ratiometric confocal microscopy of giant vesicles composed of DOPC/SM/Chol, 1/1/0.7, stained with NR12A, NR4A, NR12S,
or Nile Red (200 nM). (h) Typical single-molecule raw images recorded at 9 ms integration time (110 frames per second) in DOPC supported bilayers, for the four
probes (10 nM). (i) Time-dependent counts of single molecules detected per µm2 per second for the four probes over the full camera view. (j) Distribution of single-
molecule brightness of probes in DOPC supported bilayers, expressed in terms of photon count after 50% splitting to the imaging channel. The average value for
each probe is given in the legend.
Fluorescence spectra of both NR4A and NR12A (Fig. 1e, S3)
exhibited blue shift by about 45-50 nm in Lo phase vesicles,
composed of sphingomyelin/cholesterol (SM/Chol), compared to
Ld phase vesicles, made of dioleoylphosphatidylcholine (DOPC).
exhibited large number of single-molecule turn-on events that
lasted well through >104 frames (Fig. 1hi, Movie S1), thanks to the
continuous probe exchange between the solution and the
membrane. By contrast, the strong binders, NR12A and NR12S,
showed few turn-on events (Fig. 1hi), probably because most
molecules were quasi-irreversibly bound to the membrane and
then photobleached before single-molecule images could be
recorded. Remarkably, single-molecule brightness histograms
further showed that NR4A surpassed all other probes, including
Nile Red, by the number of photons collected per molecule (Fig.
1j), in line with our photostability data in solution (Fig. 1f). Finally,
single-molecule spectroscopy confirmed sensitivity of NR4A to
lipid order in membranes (Fig. S5).
This behavior is similar to NR12S,[12] Nile Red (Fig. S3), and other
11]
solvatochromic
probes,[2b,
reflecting
lower
local
polarity/hydration in the Lo phase.[6a] Remarkably, the intensity
ratio of blue to red part of emission band for both new probes was
more sensitive than NR12S to lipid order (from Ld to Lo phase it
increased 5.9-, 4.9- and 2.9-fold for NR4A, NR12A and NR12S,
respectively), as their emission bands were narrower (Table S2).
In phosphate buffer, NR12A displayed blue-shifted absorption
compared to Nile Red (Table S1) and negligible fluorescence (Fig.
1e), similarly to NR12S, indicating that the NR12A forms self-
quenched aggregates in water. By contrast, NR4A showed red-
shifted absorption (Table S1) and non-negligible fluorescence
(Fig. 1e), similarly to Nile Red, suggesting that NR4A is well
dissolved and does not aggregate in water. Importantly, NR12A
and NR4A displayed high fluorescence quantum yields in LUVs
(39-53%, Table S1). Moreover, in DOPC LUVs they were
significantly more photostable than NR12S (Fig. 1f), confirming
our hypothesis on the negative effect of the phenolic oxygen on
the photostability of Nile Red fluorophore. In giant unilamellar
vesicles (GUVs), using two-color confocal microscopy, NR12A
and NR4A provided excellent contrast between Lo phase,
characterized by high intensity ratio I550-600/I600-650 (in yellow, Fig.
1g, S4), and Ld phase with low I550-600/I600-650 (in blue). The ratio
changes between the two phases were higher than that of NR12S
(pseudo-color change from yellow to green), in line with the
spectral data in LUVs.
Next, the probes were incubated with HeLa cells and imaged
using fluorescence microscopy (Fig. 2a). In contrast to Nile Red,
showing intracellular fluorescence, NR12A exclusively stained the
plasma membranes, being >2-fold brighter than NR12S. NR4A
also showed specific staining for plasma membranes, but the
signal was >10-fold lower, in line with its weak affinity to
biomembranes. Among studied dyes only NR4A showed no signs
of photobleaching (Fig. S6), which confirmed that it binds
reversibly to cell membranes, so that bleached probe species are
quickly replaced by intact ones, as required by PAINT.[23a] Overall,
NR12A appears as a significantly improved analog of NR12S for
conventional imaging, which enables, for instance, obtaining high-
quality three-dimensional (3D) confocal imaging of membrane
surface (Fig. 2b). Meanwhile, NR4A, for its reversible binding
capability, appears promising for PAINT. NR4A is also attractive
for stimulated emission depletion microscopy,[19a] because
exchangeable probes solve the problem of photobleaching.[27]
Single-molecule microscopy of the probes in supported DOPC
lipid bilayers showed that the weak binders, NR4A and Nile Red,
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Figure 2. Cellular studies show NR12A and NR4A as superior probes for conventional and super-resolution microscopy, respectively. (a) Spinning disk confocal
microscopy images of HeLa cells stained with NR12A, NR4A, NR12S, and Nile Red (20 nM) after 10 min incubation at r.t. (b) A 3D stacked image of KB cells
stained with NR12A. (c) Zoom-out views of 3D PAINT results of COS-7 cells with NR4A (10 nM) and NR12S (20 nM). (d) Time-dependent counts of single molecules
detected per µm2 per second for the two probes in representative regions: 5×104 frames were recorded for >450 s at 110 frames per second. (e) 3D PAINT image
of the top plasma membrane of a HeLa cell stained by NR4A. (f) Zoom-in of the white box in (e). (g-i) Virtual cross sections in the xz plane along lines 1-3 in (f). (j)
Zoom-in in-plane (xy) image, and a time series of the vertical (xz) images of the endocytosis site marked by the arrow in (f). All 3D PAINT images are rendered with
color to present the depth (z) information [color scale bar in (e)].
We then performed 3D PAINT super-resolution imaging of live
cells. With 10 nM dye in the imaging medium, NR4A reversibly
bound to the plasma membrane, thus achieving high counts of
single molecules over 5×104 frames (Fig. 2d), as in supported
bilayers (Fig. 1h), hence good PAINT images within 1 min
acquisition (Fig. 2ce and Fig. S7). In contrast, NR12S performed
poorly with low counts of single molecules (Fig. 2d), and so
resulted in very dim final PAINT images (Fig. 2c). Although Nile
Red also allowed decent image quality, it strongly labeled internal
membranes (Fig. S8), consistent with confocal data (Fig. 2a) and
previous PAINT results.[26b] We also examined CM-DiI and DiD,
two common plasma membrane probes for SMLM,[28] and found
both to nonspecifically label internal membranes in live cells (Fig.
S8).
of minutes (Fig. 2j and Fig. S7), indicative of endocytosis. Similar
membrane features were also observed in COS-7 cells (Fig S9).
We next exploited solvatochromism of NR4A to examine the
nanoscale distribution of local chemical polarity in plasma
membranes, reflecting lipid order. To this end, we performed SR-
PAINT, where the fluorescence spectrum of every single
molecule of NR4A was measured together with its super-localized
position.[26b, 29] The obtained spectral mean, calculated as the
intensity-weighted average of the emission wavelengths of each
probe molecule and presented in pseudo-color, allowed us to
describe local lipid order with nanometer-scale spatial resolution
and minute-scale temporal resolution[26b]. In live COS-7 cells,
NR4A showed a variation of pseudo-color from blue to cyan (Fig.
3ab), with the averaged single-molecule spectra peaked at ~610
nm (Fig. 3d), similar to that of Nile Red in the plasma
membrane.[26b] Interestingly, upon chemical fixation, NR4A
became cell permeable and so labeled internal organelle
membranes with a distinct ~20 nm redshift (Fig. 3cf), similar to the
results obtained earlier with Nile Red,[26b] indicating lower lipid
order for the organelle membranes.
Owing to its high specificity, NR4A helps unveil rich nanoscale
structural features of the plasma membrane (Fig. 2e-j). For HeLa
cells, 3D PAINT showed numerous outward curves of the plasma
membrane, including mildly bulging sites that raised up ~100 nm
in height over ~1 µm lateral distances (Fig. 2fg), tube-like
protrusions with higher curvatures (~100 nm radius; Fig. 2fh), as
well as tall protrusions above the limit of our working z range
(>400 nm above the focal plane), so that they appear dark at the
center (Fig. 2fi). The observed nanoscale protrusions of the
plasma membrane were highly dynamic, showing significant
structural changes on the time scale of 1 minute (Fig. S8).
Meanwhile, we also noted 150-200 nm-sized, pit-like invagination
structures that dynamically moved into the cell on the time scale
The high specificity of NR4A to the plasma membrane in live cells
enabled nanoscale cartography of lipid order of cell surface, which
has been a challenge so far as Nile Red exhibits strong
background from internal membranes. Remarkably, for the tube-
like nanoscale protrusions, in cyan pseudo-color, the locally
averaged single-molecule spectra showed a ~4 nm redshift when
compared to smooth parts of the plasma membrane,
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Figure 3. Nanoscopic mapping of lipid order in cell membranes with NR4A. (a,b)
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[20]
Thus, we introduce the concept of a fluorescent probe for super-
resolution microscopy that combines target specificity, ON/OFF
switching, and polarity sensing. It was realized based on
solvatochromic dye Nile Red and a newly introduced low-affinity
membrane binder that ensures reversible bind/unbinding events
and targets specifically cell plasma membranes. The developed
probe (NR4A) revealed lipid order heterogeneity at the cell
surface and its connection to nanoscale membrane topology.
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Acknowledgements
This work was supported by the European Research Council ERC
Consolidator grant BrightSens 648528 and the National Science
Foundation (CHE-1554717). K.X. is a Chan Zuckerberg Biohub
investigator. S.M. acknowledges support from a Samsung
Scholarship. Bohdan Wasylyk is acknowledged for providing the
KB cells.
[25]
[26]
Keywords: Fluorescent probes • solvatochromism • membranes
• super-resolution microscopy • lipid order
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10.1002/anie.201907690
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Targeting, switching and sensing
were combined together in one
fluorescent probe to achieve
Dmytro I. Danylchuk, Seonah Moon, Ke
Xu,* Andrey S. Klymchenko*
Page No. – Page No.
nanoscale cartography of cell plasma
membranes. The proposed design is
based on a new functionalization
strategy of solvatochromic dye Nile
Red, sensitive to lipid order, and a
special membrane-targeting moiety
that controls binding/unbinding-
induced ON/OFF switching for super-
resolution microscopy.
Tailor-made switchable
solvatochromic probes for live-cell
super-resolution imaging of plasma
membrane organization
This article is protected by copyright. All rights reserved.