Please do not adjust margins
ChemComm
Page 2 of 4
DOI: 10.1039/C5CC06277B
COMMUNICATION
Journal Name
the 0→1 or 0→2 vibronic transitions.13 These absorption properties
makes them excellent candidates as NMDAR-targeted probes for
MSOT, with the maximum wavelength of absorption falling in the
near-infrared (NIR) window where the majority of endogenous
chromophores do not strongly absorb. In this region, light scattering
is relatively low resulting in greater light penetration, allowing
images to be obtained from deeper within the body. The emission
spectra of L1 and L2 were measured (λexc 776 nm) and were
structurally the same, with one main emission band at 801 nm,
giving a Stokes’ shift of 25 nm (Scheme 1). The quantum yields (5-
10%) fell in the range of similar dye structures.14
The in cellulo studies were performed on differentiated NSC-
34 cells expressing receptor subunits NMDAR-2B and NMDAR-1 at
high densities, as shown earlier.11,15 None of these probes exhibited
significant cytotoxicity after 24 h (MTT) , at concentrations of up to
100 µM. To determine the live cell-surface co-localisation profile of
L1 and L2, two optical microscopic techniques were explored to
visualise NMDARs on live neuronal cells (NSC-34). The probes were
first visualised upon cell surface NMDARs-tagging using
epifluorescence microscopy following direct excitation (λexc
>750nm). Differentiated NSC-34 cells expressing NMDARs were
incubated with the probes [5 (as a control), L1 or L2 (10 μM, 30 min)]
and then washed with Hank’s balanced salt solution (HBSS) to
remove unbound probe. L1 and L2 showed complete NMDAR-probe
visualisation in fluorescence mode (Fig. S3). Larger signal intensity
was detected from cells treated with L1, suggesting a higher
receptor affinity. No signal was obtained from cells upon incubation
with the control dye 5, consistent with specific binding of L1 and L2
on the NMDARs (Fig. S3).
Fig. 1. L1 dye absorption (green) and [Eu.L]3- emission (red)
spectra, (H2O, pH 7.2, 295 K). Table: quenching studies for [Eu.L]3-
with L1 or Dy-647-NH2 as acceptor. (data (± 5%), 295 K, 50 mM
HEPES, 50 mM NaCl, pH 7.4; λex 332 nm). Weak Eu transitions at
750 and 800 nm (ꢀJ = 5 and ꢀJ = 6 manifolds) are not shown.
The cellular NMDAR-probe binding specificity was further
proven by studies on NMDAR-negative cells (NIH 3T3 mouse skin
fibroblast cells). These cells were incubated with a solution of r L1 or
L2 (10 µM 30 min.), washed to remove unbound probe and treated
with [Eu.L]3- (20 µM). No localisation profile was observed,
strengthening the case for receptor-mediated localisation of L1 and
L2 with differentiated NSC-34 cells.
One further characteristic for these probes is the ability to
bind reversibly at the NMDAR, with an affinity competitive with
endogenous Glu. Since L1 was visualised on the cell surface
receptors, the binding of this probe after a simulated ‘Glu burst’
was assessed. The differentiated NSC-34 cells were consecutively
incubated with L1 (10 µM, 30 min, 3X HBSS wash), Cell Mask Orange
(5 μg mL−1, 5 min, 2X HBSS wash) and then treated with fresh buffer
containing 20 µM of [Eu.L]3-. The cells were imaged and the average
intensity was recorded in triplicate. These cells were washed with a
A second microscopic technique was used to confirm that the
probes were at the cell surface. Using a FRET-based assay, L1 and L2
were acceptors, excited by a non-internalised Eu(III) complex16
[Eu.L]3- (Fig. 1), as the donor. Excitation of [Eu.L]3- at 365 nm led to
population of the dye excited state. Spectral overlap of the ΔJ = 4
emission band of the donor with the absorption spectrum of the
acceptor led to excitation of L1 and L2, indicating donor and
acceptor in close proximity (Fig. 1A). Emission from the acceptor
was also observed above 780 nm.
Glu-rich (2X,
1 mM) medium and then treated with buffer
containing 20 µM of [Eu.L]3. This sequence resulted in a ten-fold
drop in fluorescence intensity (Fig. 2F and 2G), compared to the
original cell staining experiment (Fig. 2B), whilst the non-specific
membrane dye maintained the same intensity (Fig. 2E).
A study of the energy transfer process was first performed in
vitro to confirm this possibility. As a reference NIR dye, the
commercially available Dy-647-NH2 was chosen as the acceptor.
Changes in the [Eu.L]3- emission lifetime were monitored as a
function of the concentration of NIR dyes [L1 and Dy-647-NH2].
Stern-Volmer analysis showed 65% energy transfer efficiency
between [Eu.L]3- and L1, less than that calculated for Dy-647-NH2
(93%), due to reduced spectral overlap (Fig. 1B). The differentiated
NSC-34 cells were incubated with a 10 µM solution of either L1 or L2
for 30 minutes and washed to remove unbound probe. The cells
were treated with fresh buffer containing 20 µM of [Eu.L]3-, and
To demonstrate the suitability of the probes for OA detection,
the probes were assessed by MSOT after interaction with cell
surface NMDARs on differentiated NSC-34 cells. These experiments
were conducted using a small animal MSOT scanner. In brief, about
106 differentiated NSC-34 cells were treated with 20 µM of either 5,
L1 or L2 for 30 minutes, washed thoroughly to remove any unbound
probe and were then re-suspended in a 1:1 mixture of fresh buffer
and a 3% agar solution. The agar solution generated a scattering
pattern mimicking tissue scattering. Images were obtained by
scanning wavelengths between 700-900 nm in 5 nm steps, with 20
averages each (Fig. 3 and S4).
imaged using
a
modified Zeiss Axiovert 200M inverted
microscope.17 Excitation at 365 nm and observation of the emission
above 780 nm demonstrated that both L1 and L2 gave rise to a
localisation profile at the cell surface (Fig. 2B and 2D), with L1
appearing to label the cells more readily than L2. Live-cell imaging
studies were showed the extracellular probe-receptor tagging (Fig.
2D). Confirmation of cell-surface localisation was achieved by
repeating the loading experiment with a co-incubation of the
commercially available, plasma membrane stain, CellMask Orange
(5 μg mL−1 for 5 min) (Fig 2A and 2C).
L1 clearly labels NSC-34 cells, generating a strong optoacoustic
signal (Fig. 3C). As a control, untreated cells (Fig. 3A) and cells
treated with the non-targeted heptamethine cyanine dye, 5 (Fig.
3B) did not show any OA signal, suggesting that the receptor-
binding moiety of L1 binds to the cell surface NMDARs and is
responsible for the observed OA signal. However, cell labelling with
L2 (20 µM, 30 min) gave rise to only a faint OA signal (Fig. 3D)
replicating results obtained from fluorescence microscopy when
2 | Chem. Commun., 2015, 00, 1-4
This journal is © The Royal Society of Chemistry 2015
Please do not adjust margins