Neuo: A fluorescent chemical probe for live neuron labeling

Article abstract of DOI:10.1002/anie.201408614

To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neur

Full text of DOI:10.1002/anie.201408614

Angewandte
Chemie
DOI: 10.1002/anie.201408614
Neuron Imaging
NeuO: a Fluorescent Chemical Probe for Live Neuron Labeling**
Jun Cheng Er, Cheryl Leong, Chai Lean Teoh, Qiang Yuan, Paolomi Merchant, Matthew Dunn,
David Sulzer, Dalibor Sames, Akshay Bhinge, Dongyoon Kim, Seong-Min Kim, Myung-
Han Yoon, Lawrence W. Stanton, Shawn H. Je, Seong-Wook Yun,* and Young-Tae Chang*
Abstract: To address existing limitations in live neuron
imaging, we have developed NeuO, a novel cell-permeable
fluorescent probe with an unprecedented ability to label and
image live neurons selectively over other cells in the brain.
NeuO enables robust live neuron imaging and isolation in vivo
and in vitro across species; its versatility and ease of use sets the
basis for its development in a myriad of neuronal targeting
applications.
differential adhesion[7] protocols. The same selective probe
can also be used with automated sorting methods for easy
isolation of live neurons. Herein, we present NeuO, a live cell
imaging probe that selectively stain neurons fluorescently in
the presence of other brain cells. NeuO can be applied to
multiple imaging and cellular platforms for the real-time
imaging of neurons and is observed to be useful both in vitro
and in vivo.
In view of the lack of mechanistic cues to rationally design
probes for neurons, we envisioned that high-throughput
screening will be necessary to assist us in identifying promis-
ing leads that fluorescently label live neurons.[8] A subset of
5040 fluorescent compounds was selected from our in-house
diversity-oriented fluorescence libraries (DOFL; Supporting
Information (SI), Figure S1). The dyes were then applied to
a primary brain cell screening platform,[9] which employed
mouse primary neurons, astrocytes, and microglia cultures
isolated by differential adhesion methods from P1–P3 neo-
natal mouse brains (Figure S2).[7] These primary cells were
prepared in 384-well microplates and cultured for 5 days
in vitro before incubation with the DOFL compounds at
500 nm concentrations for 1 h. Through multiple rounds of
automatic intensity-based analysis and image-based evalua-
tion (Figure S3), we identified a lead fluorescent probe 1 that
selectively stained neurons fluorescently (Table 1, Entry 1).
To improve the neuron selective response, a structure–
activity relationships (SAR) study was initiated to evaluate
the chemical groups important for conferring the dyeꢀs
neuronal response. In total, 21 derivatives were prepared
N
eurons are responsible for information processing and
transmission in the brain by electrical and chemical signals.
They are organized into complex neural networks that
underlie the basic brain functions (e.g., cognitive, emotional,
and motor).[1] Over the past century, various tools have been
developed to label neurons. However, these methods either
lack intrinsic selectivity or compromise cell viability. Nissl and
Golgi stains work only for frozen or fixed brain tissue
preparations;[2] membrane staining dyes such as DiI and DiO
are not selective for neurons and require manual injection for
retrograde labeling.[3] On the other hand, neurotransmitter
mimics work only for subtypes of neurons and under selective
environments.[4] Expression of fluorescent proteins driven by
neuron-specific promoters requires genetic manipulation
which may induce undesirable effects to native protein
functions and can be less straightforward.[5] Thus, a chemical
probe that is widely applicable for generic staining of live
neurons in vitro and in vivo would thus represent a valuable
tool for researchers. Current methods to selectively isolate
live neurons are based mainly on density centrifugation[6] or
[*] J. C. Er,[+] Prof. Y.-T. Chang
Dr. A. Bhinge, Dr. L. W. Stanton
Department of Chemistry, National University of Singapore
3 Science Drive 2, 117543 Singapore (Singapore)
E-mail: chmcyt@nus.edu.sg
Genome Institute of Singapore
Agency for Science, Technology and Research
60 Biopolis Street, 138672 Singapore (Singapore)
Dr. C. Leong,[+] Dr. C. L. Teoh, Dr. S.-W. Yun, Prof. Y.-T. Chang
D. Kim, S.-M. Kim, Prof. M.-H. Yoon
Singapore BioImaging Consortium
Materials Science and Engineering
Agency for Science, Technology and Research
11 Biopolis Way, 138667 Singapore (Singapore)
E-mail: emailtoyun@gmail.com
Gwangju Institute of Science and Technology
261 Cheomdan-gwagiro, Gwangju 500712 (Korea)
[+] These authors contributed equally to this work.
Q. Yuan, Prof. S. H. Je
[**] This work was supported by an intramural funding from the A*STAR
Biomedical Research Council and Singapore Ministry of Education
Academic Research Fund Tier 2 (MOE2010-T2-1-025). J.C.E. was
supported by a NGS Scholarship. C.L. was supported by an A*STAR
Graduate Scholarship. Q.Y. was supported by a Duke-NUS SRP
Phase 2 Research Block Grant given to H.S.J. D.S. thanks the
G. Harold & Leila Y. Mathers Charitable Foundation. All animal
work was carried out under the approved IACUCs no.100564 and
no.100575 in compliance with NACLAR Guidelines.
Duke-NUS Graduate Medical School
National University of Singapore
8 College Road, 169857 Singapore (Singapore)
P. Merchant, M. Dunn, Prof. D. Sames
Department of Chemistry, Columbia University
3000 Broadway MC3101, New York, NY 10027 (USA)
Prof. D. Sulzer
Departments of Neurology and Psychiatry, Columbia University
650 W 168th Street BB308, New York, NY 10032 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408614.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
Table 1: Structure Activity Relationships.[a]
On the other hand, compounds 18–21 and 23, which carry
various “head” groups, have significantly lower or no
selectivity, highlighting the importance of the hydroxyacetyl
for optimal neuron recognition. From the SAR, we may
deduce that a compound having the general structure shown
in Table 1 would make a feasible live neuron-selective probe.
This information will be relevant to future probe designs for
imaging live neurons. Compound 3, which showed the highest
selectivity toward live neurons, was thus chosen as the final
fluorescent probe and named Neuron Orange (NeuO, lex/
lem = 470 nm/555 nm; Figure 1a).
Dye R1
R2
R3
R4
logP[b] SLI
Within 1 h after application of 500 nm of NeuO, live
neurons were seen to be selectively stained over other non-
neuronal cells in the background (Figure 1c, left), with
a distinct cytoplasmic perinuclear staining pattern. In addi-
tion, high magnification imaging of NeuO-stained primary
neuron cultures processes showed that NeuO staining extends
to the fine processes of the neurites, with continuous dye
staining throughout some parts of the neurite or otherwise
being localized to discrete granules along the branches
(Figure 1c, right). Confocal imaging of NeuO demonstrates
that the dye does not co-localize significantly with any of the
known organellar trackers (Mitotracker, Lysotracker, ER
tracker, Golgi tracker; Figure S5a). To confirm that the dye
was selectively staining neurons over other neural cells,
NeuO-stained cells (Figure 1d) were fixed and immuno-
stained with antibodies specific for neurons (b-III-tubulin),
astrocytes (glial fibrillary acidic protein, GFAP), and oligo-
dendrocytes (O4; Figure 1e). This was further verified by
analysis of characteristic neuronal gene expressions in the
FACS-sorted populations of mixed primary neural cells from
neonatal mouse brains separated using NeuO (Figure 1b,
middle); the isolated NeuO-positive cells expressed a high
level of the neuronal marker, b-III-tubulin (Figure 1b, right).
Immunostaining with antibodies specific to other neuronal
markers, including MAP2, NCAM, and NeuN, similarly
supported neuronal identity (Figure S6b). NeuO also labeled
most neuronal cell types including glutamatergic, GABAer-
gic, cholinergic, dopaminergic, and serotinergic neurons as
demonstrated by post-NeuO immunostaining with specific
antibodies (Figure S5c). We tested whether NeuO labeling
triggered any changes in cell viability or neuronal property.
The application of 10 mm of NeuO to cultured neurons did not
affect the cell survival rate when measured by the MTS assay
(Figure S6a), nor neuronal morphology based on Scholl and
Fractal analysis (Figure S6b).[12] Furthermore, NeuO-stained
neurons showed indistinguishable changes in spiking pattern
(Figure S7a and S7b), resting membrane potential (Fig-
ure S7c), capacitance (Figure S7d), and membrane resistance
(Figure S7e), indicating that NeuO is not detrimental to
neuron function.
1[c]
2
(CO)CH2OH
2-Me-C6H4-CH2-
2-Me-C6H4-CH2-
Bn
Bn
Bn
Bn
Bn
2-MeO-C6H4-CH2-
3-MeO-C6H4-CH2-
4-MeO-C6H4-CH2-
4-Me-C6H4-CH2-
4-tBu-C6H4-CH2-
4-AcNH-C6H4-CH2-
nBu
H
Cy-CH2-
Bn
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
sPe
nPr
nPr
nHp 4.7
tBu
Ph
4.5
3.2
2.8
17
23
26
23
22
20
13
19
21
19
17
11
24
17
17
10
8
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
(CO)CH2OH
Ac
3[d]
4
5
6
7
8
3.1
3.3
1.8
3.0
2.8
2.7
3.2
4.2
1.4
2.9
1.2
3.8
3.1
3.4
3.8
3.6
3.3
H
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
9
10
11
12
13
14
15
16
17
18
19
20
21
Me nPr
Bn
Bn
H
H
H
H
nPr
nPr
nPr
nPr
0
0
6
0
(CO)CH2Cl
(CO)CH2OMe Bn
CH2CH2OH Bn
[a] Abbreviations: sPe, sec-pentyl; nHp, n-heptyl. [b] Relative hydropho-
bicity, logP as determined by the HPLC method (Table S2).[11] [c] Primary
hit from DOFL screening. [d] NeuO.
which differ systematically in the chemical groups, namely,
the acetyl “head”, and the amino and triazolyl “arms”
(Table 1). These fluorescent dyes were then assessed for
their selectivity index (SLI), which is a normalized functional
measure of the dyeꢀs selective staining of neurons over its
nonselective staining (Table 1). Like the stain index,[10] SLI is
defined as D/W, in which D is the difference between positive
and negative populations and W is 2 standard deviations
(S.D.) of the negative population (Figure 1b, left); higher SLI
compounds brightly stain live neurons more selectively and
segregate neurons more distinctly on flow cytometry.
Our measurements revealed that compounds 2 to 7—
derivatives substituted with various alkyl and aryl moieties on
the triazolyl “arm”—were able to sort neurons with excellent
selectivities. Compounds 8–11 and 13–15, which vary in the
substituents on the “amino” arm, similarly showed high
selectivities. However, compounds with bulky amino “arms”,
such as 12 and 16, displayed largely reduced neuronal
staining. Together, these observations suggest that both the
triazolyl and amino “arms” can accommodate a range of
chemical modifications with the former having greater scope
than the latter.
NeuO exhibits excellent cell permeability due to its ability
to diffuse rapidly into and stain neuronal cells passively upon
loading into the cell media. In Video S1, mixed primary
neural cell cultures isolated from P1–P3 neonatal brains were
allowed to grow to confluence before staining with NeuO and
CDr10 (a microglia-selective dye)[9a] for dual color imaging. A
large neuron was clearly visible amidst a dense bed of
neuroglia and NeuO staining remained stable for up to 36 h.
2
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
Figure 1. Properties of NeuO. a) Chemical structure and optical properties of NeuO (10 mm, DMSO). b) Left: neuronal population segregation by
flow cytometry as represented by a histogram; calculation of SLI: D/W=(meanpositive–meanbackground)/(2ꢀS.D.). Middle: neuronal population
segregation by flow cytometry as represented by a pseudocolor bivariate plot; x and y-axes are flow cytometry measures of the NeuO fluorescence
signal (FL1, 530/30 nm) and granularity, respectively. Right: b-III-tubulin expression analyzed in the unsorted, bright, and dim fraction of cells
NeuO-sorted neonatal mouse brain cells. c) Confocal images of live NeuO-stained neurons on a bed of astrocytes (left) and a high magnification
image of a neuron dendrite (right); scale bar: 10 mm and 5 mm, respectively. d,e) Selective staining of neurons in live neural cell cultures using
NeuO; d) phase contrast and fluorescence image of live mixed primary neural cell culture stained with 500 nm of NeuO; e) live NeuO stained
cells were subsequently fixed and permeabilized for immunocytochemistry; NeuO-positive live cells were positive for the neuron marker b-III-
tubulin, but not for other neural cell type markers (GFAP for astrocytes, O4 for oligodendrocytes); scale bar: 20 mm.
DIV3 primary rat hippocampal neurons cultured in the
presence of NeuO also showed normal neurite formation
in vitro (Video S2). In conditions requiring multiple washes,
the dye is rapidly washed out from live neurons in the absence
of excess dye (Figure S8a). However, neuronal staining
remained intact after the stained cells were fixed by
paraformaldehyde, although the dye staining was susceptible
to cell permeabilization and organic solvent washing (Fig-
ure S8b). Hence NeuO staining is best applied in live neuron
samples. This is in contrast with conventional Nissl and Golgi
staining, which require membrane permeabilization by deter-
gents before the stains can be visualized in neurons. However,
NeuO is not selective for neurons in dead cells as seen from
the dye staining all cells nonselectively post fixation (Fig-
ure S8b).
brain tissue, two-photon microscopy imaging was carried out
in the hippocampal regions of mouse brain slices, because
these regions are densely populated with glutamatergic
neuronal cell bodies which can be easily identified by their
characteristic s-shaped organization. NeuO densely labeled
the principle cell layer of the DG, the stratum granulosum,
and sparsely labeled the principle cell layer of CA1, the
stratum pyramidale (Figure 2a). The cell labeling pattern was
similar to P1 neonatal whole-brain staining through which
clear neuronal structures were visible when viewed under
confocal microscopy (Figure S9a). This staining pattern
differed from the one of Sulforhodamine 101 (SFR 101),
which stains live astrocytes (Figure S9a).[14] Neuron-selective
staining was also effectively demonstrated in neurons derived
from human embryonic stem cells, whereby NeuO positive
cells were identified to be b-III tubulin-positive compared to
NeuO negative cells which consisted mostly of S100b positive
astrocytes (Figure S9b). In a 3 dpf zebrafish larvae, selective
fluorescence staining of neurons by NeuO was observed in the
neuromasts.[15] Neuromasts make up part of the lateral lines
system in zebrafish, which are comprised of a set of rosette-
Two-photon ex vivo or in vivo imaging of the brain has
become increasingly popular over the years due to its deep
penetration with low photobleaching and phototoxicity. Two-
photon imaging also provides high-resolution and efficient
fluorescence detection,[13] thereby making it possible to image
cells in the live brain tissue. To examine NeuO labeling in
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
Figure 2. Applications of NeuO. a) Left: NeuO staining in the hippocampus shows dense labeling; scale bar: 200 mm. Right: NeuO staining of
cell bodies in the dentate gyrus shows a perinuclear labeling pattern; scale bar: 20 mm. b) Images of the hippocampus, cerebellum, and spinal
cord regions of the mouse brain showing neuronal staining after 1 h intravenous injection of NeuO; scale bar: 50 mm. c) The same NeuO-stained
regions were immunostained with NeuN (green), GFAP (red), and Hoechst (blue). NeuO-stained regions correspond to NeuN immunostaining
(green); scale bar: 50 mm.
like organs near the head and the body (Figure S9c).[16] These
data demonstrate that NeuOꢀs neuronal staining is conserved
across species.
NeuO provides a convenient tool for the selective labeling of
live neurons which will be valuable for prospective studies in
various areas of neuron development, networking, and
degeneration.
To determine the selectivity of NeuO for neurons in vivo,
the dye was administered by intravenous injection into the tail
vein of mice. Brains and spinal cords were harvested at time
points of 10 min, 1 h, 3 h, and 6 h and snap-frozen for
microscopic analysis of frozen sections. Control images were
also taken from non-injected mouse tissues at the same
exposure (Figure S10a). The optimal time point was deter-
mined to be 1 h (Figure S10b) as observed from the improved
neuron–glia signal ratio (Figure S10c). Brains isolated 1 h
after NeuO injection showed clear fluorescence signals in
fresh frozen tissue sections from the neurons of the hippo-
campus, cerebellum, and spinal cord (Figure 2b) with mini-
mal background. Cell bodies of large neurons and their
processes could also be observed in cross sections of the spinal
cord. Immunostaining of the neuronal nuclear marker, NeuN,
confirmed that the dye selectively stained neurons in the brain
and spinal cord (Figure 2c). NeuO staining was also similar to
the staining pattern of the Nissl dyes (Figure S10d). The
successful detection of NeuO in the neurons of the brain and
spinal cord demonstrates that the dye effectively bypasses the
blood brain barrier and that neuronal staining is conserved
when the dye is administered in vivo.
Received: August 27, 2014
Revised: November 11, 2014
Published online: && &&, &&&&
Keywords: BODIPY · fluorescent probes · imaging agents ·
.
live neurons
[1] C. Wiedemann, Nat. Rev. Neurosci. 2010, 11, 74.
[2] A. Kꢁdꢁra, G. Wittmanna, Z. Lipositsa, C. Fekete, J. Neurosci.
Methods 2009, 184, 115 – 118.
[3] M. G. Honiga, R. I. Hume, Trends Neurosci. 1989, 12, 333 – 341.
[4] P. C. Rodrigueza, D. B. Pereirab, A. Borgkvistb, M. Y. Wongb, C.
Barnardb, M. S. Sonders, H. Zhang, D. Sames, D. Sulzer, Proc.
Natl. Acad. Sci. USA 2013, 110, 870 – 875.
[5] I. K. Sariyer, Methods Mol. Biol. 2013, 1078, 133 – 139.
[6] G. J. Brewer, J. Neurosci. Methods 1997, 71, 143 – 155.
[7] M. Jana, A. Jana, U. Pal, K. Pahan, Neurochem. Res. 2007, 32,
2015 – 2022.
[8] S.-W. Yun, N.-Y. Kang, S.-J. Park, H.-H. Ha, Y. K. Kim, J.-S. Lee,
Y.-T. Chang, Acc. Chem. Res. 2014, 47, 1277 – 1286.
In conclusion, we described the systematic discovery of
NeuO for the selective staining of live neurons of different
species. NeuO represents the first selective fluorescent dye for
specific live neuron labeling. The utility of NeuO for neuronal
labeling has been demonstrated in a variety of imaging
techniques and neuronal cell isolation procedures. Using
NeuO, neurons can be selectively visualized and separated
from mixed neural cell cultures. Overall, the discovery of
[9] a) C. Leong, S. C. Lee, J. Ock, X. Li, P. See, S. J. Park, F.
Ginhoux, S.-W. Yun, Y.-T. Chang, Chem. Commun. 2014, 50,
1089 – 1091; b) S.-W. Yun, C. Leong, D. Zhai, Y. L. Tan, L. Lim,
X. Bi, J.-J. Lee, H. J. Kim, N.-Y. Kang, S. H. Ng, L. W. Stanton,
Y.-T. Chang, Proc. Natl. Acad. Sci. USA 2012, 109, 10214 – 10217.
[10] H. Maecker, J. Trotter, Nat. Methods 2008, 5, 0.
[11] a) J. Sangster, J. Phys. Chem. Ref. Data 1989, 18, 1111 – 1229;
b) A. Romieu, C. Massif, S. Rihn, G. Ulrich, R. Ziessel, P.-Y.
Renard, New J. Chem. 2013, 37, 1016 – 1027.
4
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
[12] L. Billeci, C. Magliaro, G. Pioggia, A. Ahluwalia, Front. Neuro-
inform. 2013, 7, 0.
[13] Z. F. Mainen, M. Maletic-Savatic, S. H. Shi, Y. Hayashi, R.
Malinow, K. Svoboda, Methods 1999, 18, 231 – 239.
[14] A. Nimmerjahn, F. Kirchhoff, J. N. D. Kerr, F. Helmchen, Nat.
Methods 2004, 1, 31 – 37.
[15] A. F. Sarrazina, E. J. Villablancaa, V. A. NuÇeza, P. C. Sando-
vala, A. Ghysenb, M. L. Allende, Dev. Biol. 2006, 295, 534 – 545.
[16] Y. He, H. Mei, H. Yu, S. Sun, W. Ni, H. Li, Exp. Mol. Med. 2014,
46, e94.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Neuron Imaging
Selective labeling of live neurons over
other brain cells was achieved with
a novel fluorescent probe, NeuO. It
enables stable, live neuron imaging
in vivo and in vitro across species, thus
setting the stage for various neuronal
targeting applications including the study
of neuron development and degenera-
tion.
J. C. Er, C. Leong, C. L. Teoh, Q. Yuan,
P. Merchant, M. Dunn, D. Sulzer,
D. Sames, A. Bhinge, D. Kim, S.-M. Kim,
M.-H. Yoon, L. W. Stanton, S. H. Je,
S.-W. Yun,* Y.-T. Chang*
&&& — &&&
NeuO: a Fluorescent Chemical Probe for
Live Neuron Labeling
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!