Selective Detection of Cu+ Ions in Live Cells via Fluorescence Lifetime Imaging Microscopy

Article abstract of DOI:10.1002/anie.202109349

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is impor

Full text of DOI:10.1002/anie.202109349

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Selective detection of Cu+ ions in live cells via fluorescence
lifetime imaging microscopy
Authors: Ramon Vilar, Martin Priessner, Peter A. Summers, Ben Lewis,
Magdalena Sastre, Liming Ying, and Marina K. Kuimova
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202109349
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10.1002/anie.202109349
Angewandte Chemie International Edition
Selective detection of Cu+ ions in live cells via fluorescence lifetime
imaging microscopy
Martin Priessner,[a] Peter A. Summers,[a] Benjamin W. Lewis,[a] Magdalena Sastre,[b] Liming Ying,[c] Marina K.
Kuimova,*[a] Ramon Vilar*[a]
[a] Department of Chemistry
Imperial College London
White City Campus
London W12 0BZ
E-mail: r.vilar@imperial.ac.uk, m.kuimova@imperial.ac.uk
[b] Department of Brain Sciences
Imperial College London
Hammersmith Campus
London W12 0NN
[c] National Heart and Lung Institute
Imperial College London
Molecular Sciences Research Hub
London W12 0BZ.
Abstract: Copper is an essential trace element in living organisms with its levels and
localisation being carefully managed by the cellular machinery. However, if misregulated,
deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to
have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a
new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity
upon binding to copper(I), but not in the presence of high concentration of other physiologically
relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the
fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in
lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).
1
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10.1002/anie.202109349
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Copper is an essential trace element for living organisms. It can cycle between copper(II) and
copper(I), with the latter being the predominant oxidation state present under physiological
conditions. In a cellular environment copper ions can coordinate to a range of nitrogen- and
sulphur-based ligands.[1] It participates in single-electron transfer reactions and, therefore, is
involved in a wide range of biological processes including respiration, free radical scavenging,
iron metabolism, connective tissue biogenesis and synthesis of neuropeptides.[2–4] It is tightly
regulated by a system of intracellular chaperones and transporters (e.g. CTR-1, ATOX-1,
GSH)[5] that prevent the accumulation of free copper ions. Cellular copper ions are generally
found in two forms: either in a static pool, where the metal is tightly bound to proteins and
other macromolecules, or in a labile pool, where it is weakly bound to cellular ligands and,
hence, mobile.[6] Mismanagement of cellular copper pools can result in oxidative stress with
the formation of reactive oxygen species (ROS) through Fenton-type reactions.[7]
Due to the important biological roles that copper plays, its misregulation can have significant
implications for health and disease. Excess copper causes toxicity and compromises the redox
homeostatic state of cells. The toxicity of a copper overload becomes particularly clear in
Wilson disease, a pathological condition caused by a mutation in the ATP7B gene (which
encodes a copper-transporting ATPase), causing a build-up of copper in the liver and several
other tissues.[8] On the other hand, Menkes disease is a genetic disorder associated with
impaired copper efflux from enterocytes into the blood and inadequate transport of copper to
the brain.[9] Furthermore, copper is believed to play a crucial role in the progression of several
neurogenerative diseases such as Parkinson’s,[10] Alzheimer’s[11] and Huntington’s[12] disease.
Copper’s biological relevance has motivated the development of techniques to measure its
cellular levels. These include inductively coupled plasma mass spectrometry (ICP-MS),[13] X-
ray fluorescence microscopy (XFM)[14] and nano-secondary ion mass spectrometry (Nano-
SIMS),[15] amongst others. While these techniques have provided important insights into the
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biology of copper, they are not capable of measuring and tracking it in living cells. Therefore,
there is significant interest in the development of fluorescent probes that can selectively
respond to copper ions, enabling its direct visualisation in live cells. Over the last two decades
several such fluorescent probes for copper(I) have been reported in the literature.[16–21] Most of
these probes turn-on their fluorescence intensity upon binding to copper(I) via a photoinduced
electron transfer (PET) process or a related charge transfer (CT) pathway.[22] However,
intensity-based probes have the disadvantage that they are concentration dependent, i.e. their
signal is strongly dependent on probe uptake to a particular intracellular location.
An attractive alternative to intensity-based probes are fluorophores that change their
fluorescence lifetime upon binding to a target analyte. Since this parameter is concentration
independent, it can provide a more realistic map of a cellular distribution of a target of interest.
Thus, by using fluorescence lifetime imaging microscopy (FLIM), in conjunction with a
fluorophore with lifetime-sensitivity to copper(I), it should be possible to detect and image true
levels of copper(I), even in the presence of competing species.
FLIM has been previously used to investigate the behaviour of intracellular copper(II).[23–27] In
contrast, to the best of our knowledge there are no previous studies where this approach has
been employed to visualize copper(I) in live cells. Herein we report a new fluorescent probe
(FLCS1, Scheme 1), which switches-on its intensity in the presence of copper(I). Additionally,
it displays a distinct fluorescence lifetime change upon copper(I) binding, which can be used
to visualize this metal ion in live cells via FLIM.
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Scheme 1. Synthetic scheme for the preparation of the new BODIPY-based optical probe FLCS1 (for
full reaction scheme, see ESI)
The design of our new probe (FLCS1 – see Scheme 1) is based on linking the known NS4 copper(I)
receptor pioneered by Chang et. al.[28] with a highly conjugated BODIPY-based fluorophore, which
displays good photostability, high quantum yield and a red shifted emission, in the spectral range suitable
for cell and tissue imaging.[29] We expected PET from the electron-rich NS4 chelating fragment to the
BODIPY core to quench its emission in the absence of copper(I).[30] Fluorescence should switch-on upon
copper(I) binding, due to a reduction of the electron-donating ability of the NS4 receptor towards the
BODIPY fluorophore.
FLCS1 was obtained via the synthetic pathway shown in Scheme 1. The tetrathia-aldehyde building block
4 and the BODIPY precursor 2 were synthesised following previously reported procedures (see ESI for
full reaction scheme and experimental details).[29] To synthesise the new compound 5, it was first
necessary to deprotect 1 with trifluoroacetic acid (TFA) to yield 2, which was immediately reacted with
compound 4. This was followed by solvent exchange (to THF) and addition of DDQ yielding 5. In the
final step, a crude sample of 5 was reacted with DIEA and BF3·Et2O to yield FLCS1 which was purified
4
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by column chromatography and preparative TLC. The new probe was characterised by 1H and 13C NMR
spectroscopy and MALDI-TOF (see ESI for spectroscopic data).
With the new BODIPY-based probe in hand, a series of studies were performed to establish its response
to copper(I). FLCS1 displayed characteristic optical features of a BODIPY chromophore with extended
conjugation, showing the main absorption band at 646 nm (ε = 2.48 × 105 M-1 cm-1) and a broad shoulder
at 594 nm (ε = 8.07 × 104 M-1 cm-1), Figure 1a. Addition of copper(I) to a methanolic solution of FLCS1
leads to a slight bathochromic shift (4 nm) of the λmax in the UV/vis spectrum. Importantly, addition of
copper(I) to FLCS1 switches on its fluorescence (Figure 1b). While a methanolic solution of the probe
in the absence of copper(I) is practically non-fluorescent (Φ = 0.032), increasing amounts of copper(I)
lead to a clear enhancement of the emission band centred at 660 nm.
Figure 1. Spectroscopic studies of the interaction of copper(I) with FLCS1 (1 µM) in methanol upon addition of
increasing amounts of copper(I), up to 1.6 equivalents. a) UV/vis spectra; b) Fluorescence spectra (λex = 610 nm);
inset shows the binding plot recorded at the fluorescence peak maximum, λemis = 660 nm. c) Time-resolved
fluorescence decays of FLCS1 (λex = 630 nm, λemis = 660±10 nm); instrument response function (IRF) is shown in
dark red; inset shows calculated intensity-weighted average lifetime vs. Cu+/FLCS1 ratio. d) Response of FLCS1
(1 μM in methanol) to the addition of different metal ions with bars representing the integrated fluorescence
response of the probe. Black bars represent the response to the addition of excess metal ions (2 mM Ca2+, Mg2+,
Zn2+ and Na+; 20 μM Co2+, Fe2+, Mn2+, Ni2+, Zn2+, K+ and Cu2+); red bars represent the response to the subsequent
addition of Cu+ (3 μM) to the corresponding metal ion solutions.
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Saturation was reached after the addition of approximately one equivalent of copper(I), at which point the
fluorescence intensity increased by ca. 20-fold (Φ = 0.66). The one-to-one binding stoichiometry between
FLCS1 and copper(I) was confirmed by the method of continuous variation (i.e. a Job plot), which
showed a clear maximum at 0.5 mol fraction (see Figure S4a). The Kd between FLCS1 and copper(I) was
determined in a buffered thiourea solution (in methanol) as previously reported (see Figure S4b,c),[31,32]
giving a value of 2.6*10-9 M (this value cannot be directly compared to the Kd values previously reported
for similar copper(I) probes since different solvents were used). To assess the reversibility of the process,
the FLCS1-Cu+ complex was treated with 2 equivalents of PSP-2, which is known to have a very strong
binding constant to copper(I).[33] This led to a decrease of the fluorescence intensity down to the basal
level, observed for the free probe. Addition of excess copper(I) to this solution led to a complete
restoration of the fluorescence intensity, initially observed for the FLCS1-Cu+ complex, confirming that
the binding event is reversible (see Figure S5).
Interestingly, the fluorescence lifetime of FLCS1 increased significantly upon binding to copper(I) (see
Figure 1c). The time-resolved fluorescence traces were best fitted using a biexponential decay function,
with two lifetime components (τ1 = 0.39 ns and τ2 = 3.08 ns) extracted from a global fit. The full set of
fitted data is presented in the ESI (Figure S6). We assign the short lifetime component (τ1 = 0.39 ns) to
free FLCS1 and the long lifetime component (τ1 = 3.08 ns) to copper-bound FLCS1, which is supported
by the change in fitted amplitudes of the two components upon copper(I) titration (ESI Table S1 and
Figure S7).
To determine the selectivity of FLCS1 for copper(I), we studied the fluorescence response of the probe
upon addition of physiologically relevant concentrations of various metal ions usually present in the cell.
Pleasingly, no switch-on effect of the fluorescence of FLCS1 was observed upon addition of 2 mM CaCl2,
NaCl, MgCl2 and ZnCl2, nor upon the addition of 20 μM concentrations of KCl, CoCl2, FeSO4, MnBr2,
Ni(OAc)2, and CuSO4. A very slight increase was observed upon addition of 20 μM copper(II), which is
consistent with previously published literature on probes with the same NS4 receptor (see Figure 1d).[34,35]
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Having established the excellent in vitro copper(I)-sensing capabilities of FLCS1, we investigated its
cellular properties using neuroblastoma SH-SY5Y cells. Cells were incubated with a 60 nM solution of
FLCS1 in the presence of 0.2% lipofectamine in DMEM for 24 hours at 37 °C. Lipofectamine was needed
for cellular uptake of FLCS1 since this probe has poor water solubility and hence is not readily cell
permeable. As can be seen in Figure 2, under these conditions the probe is taken up by cells and localises
throughout the cytoplasm in specific cellular compartments with punctate distribution. After incubation
with FLCS1 cells showed to be fully viable, growing and replicating for over 48 hours (see Figure S8).
Co-staining experiments with Lysotracker Green (a well-established dye for staining lysosomes) showed
close colocalization of both probes (Figure 2). To confirm that the probe is not sensitive to acidic pH
typically found in lysosomes, cells were treated with bafilomycin A1, which stops lysosomal acidification,
thus increasing the pH. Pleasingly, confocal microscopy showed that this treatment did not alter the
fluorescence intensity coming from FLCS1 localised in lysosomes suggesting that these changes in pH
do not affect the probe (see Figure S11).
Figure 2. Colocalization FLCS1 with LysoTracker Green in SH-SY5Y cells. Cells were incubated with FLCS1 60
nM with 0.2% Lipofectamine for 24 h in DMEM and LysoTracker Green was used at 50 nM concentration in DMEM
for 15 minutes before imaging. Images are shown at 63x magnification with zoomed-in sections highlighted by
white boxes; the zoomed in sections are shown in the bottom row (a) brightfield image; (b) imaging FLCS1 (lex =
635 nm; lemis = 645–770 nm); c) imaging LysoTracker Green (lex = 500 nm; lemis = 510–530 nm); d) overlayed images
b) and c), showing colocalization of LysoTracker Green with FLCS1 (scale bare 5 μm).
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In recent years, a growing body of evidence has indicated that lysosomes, which are involved in the
degradation and recycling of cellular waste and energy metabolism,[36,37] also play a vital role in the
regulation of transition metal homeostasis including copper ions.[38,39] Our microscopy studies are
consistent with this theory and indicate the presence of copper(I) ions in lysosomes, since the fluorescence
of FLCS1 is clearly switched-on in these organelles. However, as discussed above, intensity-based probes
are not concentration-independent and, thus, it is not possible to assess based on the intensity alone,
whether the high fluorescence observed is indeed due to the presence of large pools of copper(I) in
lysosomes, or simply a consequence of high accumulation of the probe in this organelle. To address this,
we recorded fluorescence lifetime imaging microscopy (FLIM) images of SH-SY5Y live cells incubated
with FLCS1. Since the cell-free assays discussed above showed that FLCS1 displays significantly longer
fluorescence lifetimes when bound to copper(I) as compared to the metal-free probe (see Figure 1c),
FLIM should provide an unambiguous confirmation of the presence of copper(I) in lysosomes.
The FLIM images (see Figure 3a for a representative example) show biexponential time-resolved decays
(Figure 3g) of FLCS1 in cells, which is consistent with the presence of a mixture of bound and unbound
probe, as in the cell-free data (see Figure 1c and ESI Figure S6). Fitting the images resulted in cellular
distribution of τ1 centred around 1.1 ns and τ2 around 2.6 ns (see Figure S9g and i). These values deviate
slightly from the observed in vitro values for FLCS1 (copper free τ1 = 0.39 ns, copper bound τ2 = 3.08
ns). This difference could be due to the complex intracellular environment, as compared to the cell-free
assays (e.g. crowding, viscosity, polarity, refractive index). Indeed, we established that in vitro the probe
is affected by various parameters; for example, adding increasing amounts of water to methanolic
solutions of FLCS1 decreases its fluorescence lifetime; increasing viscosity or decreasing the pH shifts
the fluorescence lifetime of the probe to slightly lower values (see Figure S10). It is important to
emphasise that none of these changes are large, but together with other factors such as the differences in
the refractive index, they are sufficient to explain the small discrepancy in the lifetime values between
the in vitro and in cellulo data.
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Figure 3. FLIM imaging of SH-SY5Y cells treated with FLCS1. FLIM data recorded before (a, c, d) and after (b, e,
f) incubation with 5 μM Cu-GTSM for 20 min; a, b, c, e show the lifetime maps (τavg); (d) and (f) show fluorescence
intensity images; c, d, e, f show the zoomed in sections highlighted by white squares in (a) and (b); (g) and (h)
show typical time-resolved fluorescence decay curves from the regions indicated by white crosses in c and e; fitting
parameters (inserts) and residuals (bottom panels) are also shown; (i) amplitude of the longest lifetime component
histograms before and after Cu-GTSM incubation; (j) average lifetime and (k) brightfield image of cells with
fluorescence image overlayed; (l) statistics of average lifetime change before and after treatment recorded for 12
cells, over 3 independent biological repeats.
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The resulting weighted average lifetime (τavg = 2.06 ns, τ1 = 1.1 ns, τ2 = 2.6 ns, α1=0.41, α2= 0.59, χ2 =
1.18; see Figure S9c and compare to cell-free data in Table S1) in lysosomes indicates that the probe is
partially bound to copper(I). To confirm further the ability of FLCS1 to image copper(I) in cellulo, we
artificially increased the cellular concentration of copper(I) using Cu-GTSM. This is a well-established
reagent that upon cellular uptake undergoes a bio-reduction process releasing copper(I) inside a cell.[40]
Comparing the FLIM images of cells before and after treatment, the most suitable fitting model remains
a biexponential decay. The fitting (Figure S9a,b) shows significant changes in the amplitudes of the
fluorescence lifetimes following the addition of Cu-GTSM, without significant changes to the distribution
of the short and long lifetime components (Figure S9). To reduce the uncertainty in the determination of
the fitting parameters, the two lifetime components were fixed at τ1 = 0.7 ns and τ2 = 2.7 ns for copper(I)
treatment analysis. This did not influence the goodness of fit parameter (χ2) distribution (see Figure S13)
but allowed us to clearly visualise the changes of the amplitudes of two lifetimes before and after the
treatment (see Figure S12).
Consistent with the release of additional copper(I), an upwards shift in the average fluorescence lifetime
(or, in other words, an upshift in the longer component amplitude, Figure S12a,b) of the FLCS1 probe
was observed upon treating SH-SY5Y cells with Cu-GTSM (5 μM for 20 minutes) (see Figures 3b and
3e). This increase in the amplitude of the longer lifetime component α2 (see in Figure S12a,b), rather than
its lifetime, is consistent with an increase in the fraction of the bound copper(I) seen by FLIM in cells
following the treatment. It should be noted that the fluorescence intensity did not undergo significant
changes following Cu-GTSM treatment (see Figures 3d and 3f), confirming that FLIM is a superior
technique compared to intensity-based imaging, enabling us to monitor small changes in copper(I)
abundance in cells. As controls, treatment of cells with DMSO as well as the repeated exposure of the
cells cultured in the presence of FLCS1 to the laser irradiation did not lead to significant changes in
fluorescence lifetime compared to cells treated with Cu-GTSM (see Figure S14 and S15).
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In conclusion, the new BODIPY-based FLCS1 probe shows excellent in vitro selectivity for copper(I)
ions over all other physiologically abundant metal ions. Coordination of copper(I) to FLCS1 not only
leads to a large switch-on of the probe’s fluorescence intensity, but also to a significant increase in its
fluorescence lifetime. Incubation of FLCS1 with live SH-SY5Y cells showed that at concentrations used
for imaging the probe is non-toxic and largely localised in lysosomes. Our studies are consistent with the
direct detection of copper(I) in lysosomes of live cells confirming previous reports indicating that
lysosomes have high concentration of this metal ion. Our data is also consistent with direct detection of
an increase of copper(I) concentration in live cells via FLIM upon treatment with copper(I) releasing
agent Cu-GTSM.
Acknowledgements
M.P. is thankful to the UK Engineering and Physical Sciences Research Council (EPSRC) for a
studentship as part of the Centre of Doctoral Training Neurotechnology (EP/L016737/1). M. K. K. is
thankful to the EPSRC for the Career Acceleration Fellowship (EP/I003983/1). L.Y. to the Leverhulme
Trust (RPG-2015-345). We acknowledge the financial support from the Excellence Fund for Frontier
Research, Imperial College London. Prof Christoph Fahrni is thanked for donating a sample of PSP-2 and
for very insightful discussions. Prof Peter Faller and Dr. Paulina Gonzalez are thanked for providing a
sample of GTSM.
Keywords: FLIM • Copper(I) • fluorescence • TCSPC • lifetime
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10.1002/anie.202109349
Angewandte Chemie International Edition
Entry for the Table of Contents
Copper is an essential trace element in living organisms and therefore it is important to have reliable
methods to visualize it in cells. Herein we report a new fluorescent probe that upon binding to copper(I)
(but not any other physiologically relevant metal ions), displays an intensity switch-on as wells as a
significant change in its fluorescence lifetime. The later has allowed us to visualize copper(I) in live
cells via fluorescence lifetime imaging microscopy.
15
This article is protected by copyright. All rights reserved.