Volume 54 Number 23 21 March 2018 Pages 2815–2932

ChemComm
Chemical Communications
rsc.li/chemcomm

ISSN 1359-7345

COMMUNICATION
Yi Xiao, Wei Yang, Xuhong Qian, Youjun Yang et al.
Super-resolution imaging of lysosomes with a nitroso-caged rhodamine
 ChemComm
View Article Online
COMMUNICATION View Journal | View Issue
Published on 23 January 2018. Downloaded by Southeast University - Jiulonghu Campus on 1/21/2024 8:29:56 AM.

Super-resolution imaging of lysosomes with

a nitroso-caged rhodamine†
Cite this: Chem. Commun., 2018,
54, 2842
Haihong He,‡a Zhiwei Ye,‡b Ying Zheng,b Xiu Xu,a Chunlei Guo,a Yi Xiao,*b
Received 18th November 2017, Wei Yang,*b Xuhong Qian*a and Youjun Yang *a
Accepted 18th January 2018

DOI: 10.1039/c7cc08886h

rsc.li/chemcomm

Caged-fluorophores are potentially suitable for photo-activated
localization microscopy (PALM) for super-resolution imaging.
N-Nitroso is a simple and robust photo-cage with biocompatible
nitric oxide as the only byproduct upon photolysis. We herein
reported a novel PALM probe (NOR535) for super-resolution imaging
of lysosomes with high localization precision.
The emergence of various super-resolution microscopic techniques,
e.g. stimulated emission depletion (STED),1 photoactivated
localization (PALM),2 and stochastic optical reconstruction
(STORM),3 have enabled imaging of biomacromolecules4–7 or
subcellular organelles8–13 with sub-diffraction resolutions. These
endeavors were often carried out with photoactivatable/switch-
able fluorescent proteins.12–15 However, the sub-optimal photo-
stability of most fluorescent proteins represents a major limita-
tion. As an alternative to fluorescent proteins, small-molecule
organic fluorophores with good localization specificity have
often been employed for live cell super-resolution imaging.8–13
PALM imaging of subcellular organelles in live cells with photo-
activatable probes is sought after because it does not require as
high an intensity laser as STED for bleaching, or a redox buffer to
facilitate photo-switching as in the case of STORM.
A photoactivatable probe for PALM is usually constructed by
caging a bright and photo-stable fluorophore with a photolabile
protective group. Although a wide variety of photo-cages have
been developed,16 only o-nitrobenzyl groups and few others have
found applications in developing PALM probes (Fig. 1B).17–22
In fact, o-nitrobenzyl groups are not ideal photo-cages for
developing probes, either. They release toxic and highly colored
o-nitrosobenzaldehyde upon photo-activation. The scarcity
of compatible photo-cages for super-resolution imaging
applications has spurred intense efforts towards searching for
novel photo-cages. Recently, spirocyclic diazoketone caged
rhodamines23–26 (Fig. 1C) and azido-substituted push–pull type
fluorophores27,28 (Fig. 1D) were reported to be feasible for
live-cell localization microscopy. Unfortunately, these two
photo-cages have two photo-decomposition pathways, one of
which leads to a non-fluorescent species.
Recently, we have discovered that non-fluorescent N-nitroso-
naphthalimides exhibited fluorescence turn-on upon irradia-
tion by UV light, and the endogenous and hence biocompatible
nitric oxide is the only photolysis byproduct (Fig. 1E).29 Follow-
ing these previous findings, we presume that N-nitrosated
fluorophores could be a potential class of photoactivatable
fluorophores for super-resolution imaging. Also, rhodamine

a
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
Chemical Biology, School of Pharmacy, East China University of Science and
Technology, Shanghai, 200237, China. E-mail: xhqian@ecust.edu.cn,
youjunyang@ecust.edu.cn
b
State Key Laboratory of Fine Chemicals, Dalian University of Technology,
Liaoning, 116024, China. E-mail: xiaoyi@dlut.edu.cn, yw917@dlut.edu.cn
† Electronic supplementary information (ESI) available. CCDC 1586365. For ESI Fig. 1 (A) The design of photoactivatable fluorophores; (B) structure
and crystallographic data in CIF or other electronic format see DOI: 10.1039/ of o-nitrobenzyl rhodamines; (C) structure of spirocyclic diazoketone
c7cc08886h rhodamines; (D) structure of azido-substituted dyes; and (E) structure of
‡ H. He and Z. Ye contributed equally to this work. NOD545.

2842 | Chem. Commun., 2018, 54, 2842--2845 This journal is © The Royal Society of Chemistry 2018

Communication
Published on 23 January 2018. Downloaded by Southeast University - Jiulonghu Campus on 1/21/2024 8:29:56 AM.
Fig. 2 Synthesis of NOR535 and its ORTEP drawing.
has been widely acknowledged for its feasibility for super-
resolution imaging, due to its brightness and photostability.
The two aforementioned considerations led to the design of
NOR535 (Fig. 2). Considering the irreplaceable importance of
lysosomes in trafficking, catabolite export and energy and
nutrient sensing,30 two morpholine moieties were introduced
to warrant the lysosomal specificity.31–34 Super-resolution
imaging of cell lysosomes has previously been achieved with
typically STORM and STED based techniques. However, only
one small-molecule based PALM probe has been reported for
lysosome, which is an o-nitrobenzyl protected rhodamine.35
There is a pressing need for PALM probes of improved
biocompatibility for super-resolution imaging of lysosome.
NOR535 is conveniently prepared via a three-step cascade
starting from fluorescein in high overall yield (Fig. 2). The
single-crystal X-ray diffraction of the NOR535 crystal shows
some interesting structural information (CCDC #: 1586365†).
First, NOR535 adopts the lactone conformation and hence is
expected not to absorb visible light. Second, it is interesting to note
that the two morpholine moieties point away from each other,
presumably to avoid steric interactions. Third, both nitrosamine
moieties are in good conjugation with the benzene ring.
NOR535 does not absorb appreciably in the visible region
and is non-fluorescent (blue line in Fig. 3A and C). Compound 1
has a maximal absorption at 520 nm with an extinction
coefficient of 88 000 cm1 M1. Upon excitation, an emission
band at 550 nm was observed with a fluorescence quantum
yield of 0.09. Presumably, the morpholine has quenched the
fluorescence of 1 via the photo-induced electron transfer (PET)
effect. Upon protonation, the absorbance band of 1 blue-shifted
by 10 nm to 510 nm, with the extinction coefficient remaining
unchanged at 92 900 cm1 M1. The emission maximum
moved by 15 nm to 535 nm. Notably, the fluorescence quantum
yield of 1 0 was high at 0.97. Therefore, a fluorescence turn-on
This journal is © The Royal Society of Chemistry 2018
View Article Online
ChemComm
Fig. 3 The change of the UV-Vis absorption and emission spectra of
NOR535 (10 mM) in (A) phosphate buffer (pH = 7.4) and (C) citrate buffer
(pH = 4.7) containing 1% DMSO upon irradiation by 365 nm. (B and D) The
fluorescence turn-on of NOR535 with respect to time.
of 25-fold is anticipated upon photoactivation of 1 into 1 0
(Fig. S3, ESI†).
The photo-decomposition of NOR535 was investigated using
UV-Vis absorption and fluorescence spectroscopies at two dif-
ferent pH values. In neutral phosphate buffer, the irradiation of
a NOR535 solution (10 mM) at 365 nm induced the appearance
of an intense absorption band with a maximum at 520 nm.
Excitation at 520 nm gave an emission band with a maximum
at 550 nm, in agreement with the spectral properties of
compound 1. The fluorescence enhancement was completed
in 600 s (Fig. 3B). However, in citrate buffer at 4.7, mimicking
the acidic environment of the lysosome region, the fluores-
cence turn-on was much more dramatic and also completed in
a shorter period of 300 s (Fig. 3D).
The generation of fluorophore 1 from the photo-triggered
decomposition of NOR535 was also verified by NMR. NOR535
was dissolved in methanol and the solution was irradiated by
365 nm light until the complete photolysis as monitored by
TLC. Then the solution was evaporated to afford the final red solid
for 1H NMR analysis. As shown in Fig. 4B, the red photodecom-
position product had only one set of peaks, which was in agree-
ment of the synthesized fluorophore 1. This indicated that
Fig. 4 Overlay of the 1H-NMR spectra of (A) NOR535, (B) 1 upon
UV-irradiation of NOR535, and (C) 1 from independent synthesis.
Chem. Commun., 2018, 54, 2842--2845 | 2843

ChemComm
NOR535 had a clean photo-triggered conversion to form
fluorophore 1.
The NO released from NOR535 upon photo-irradiation was
further confirmed by a nitric oxide probe (DAN), which could be
converted to another fluorophore (NAT) in the presence of NO
Published on 23 January 2018. Downloaded by Southeast University - Jiulonghu Campus on 1/21/2024 8:29:56 AM.
and oxygen. A solution containing both NOR535 (10 mM) and
DAN (10 mM) in phosphate buffer (50 mM, 10% DMSO) was
irradiated by 365 nm light, and the fluorescence spectra were
collected intermittently (Fig. S1, ESI†). The emission band of
NAT at 435 nm and that of fluorophore 1 at 550 nm were
simultaneously enhanced, while the emission band of DAN at
390 nm was decreased. This verifies that NO is indeed released
from NOR535 upon photo-irradiation at 365 nm.
The stability of NOR535 toward nucleophilic attack at the
N-nitroso is important for avoiding undesired fluorescence turn-
on via transnitrosation and improving localization specificity. Redu-
cing agents are another group of potentially interfering reagents,
which might induce reductive denitrosation. Therefore, the stability
of NOR535 (10 mM) in phosphate buffer (50 mM at pH = 7.4) with
1% DMSO was tested in the dark by incubating with glutathione,
cysteine, ascorbic acid, tryptophan and hydroquinone. The solution
absorption spectra remained unchanged after 30 min (Fig. S2, ESI†),
suggesting that NOR535 was chemostable and could potentially
avoid the transnitrosation and reductive denitrosation.
The cytotoxicity of NOR535 and 1 was checked with the MTT
assay. HeLa cells were incubated with NOD535 and 1 (up to 40 mM)
for 24 h. As shown in Fig. S5 (ESI†), the cell viability was higher than
90%, suggesting low cytotoxicity of both NOR535 and 1. The feasibility
of the photoactivation behaviour of NOR535 was further evaluated in
live cells. HeLa cells were incubated with NOR535 (10 mM) for 5 min.
HeLa cells were irradiated by a mercury lamp equipped with a
330–385 nm filter and emission in the 520–560 nm range was
collected (Fig. S6, ESI†). A dark image was obtained since 1 was not
present in the HeLa cells. In contrast, after photo-activation by UV
light from a mercury lamp, a bright fluorescence image of HeLa cells
was obtained and this indicated that NOR535 was photo-decomposed
to yield fluorophore 1. This experiment confirmed that NOR535 could
release the parent fluorophore 1 in vitro.
The intracellular distribution of NOR535 was studied via
co-localization. HeLa cells loaded with NOR535 (10 mM) and
Lysotracker Red (1 mM) were irradiated by a mercury lamp to
induce NO release from NOR535 and concomitant formation of
the corresponding fluorophore, 1. Lysotracker Red was chosen
for the low spectra overlap with fluorophore 1 so that the cells
could be imaged with the green and red channels, respectively
(Fig. 5A and B). Overlay of the signals from the two channels
with a Pearson’s coefficient of 0.81  0.01 (n = 4) suggests that
NOR535 localizes in cell lysosomes (Fig. 5C and E).
The feasibility of NOR535 for super resolution (SR) PALM
imaging of lysosomes in HeLa cells was investigated using a total
internal reflection fluorescence microscope (TIRFM). An activation
laser of 405 nm and a localization laser of 532 nm were employed in
this study. HeLa cells were incubated with 500 nM NOR535 for
5 min and then rinsed with PBS for three times. The addition of
an oxygen depletion medium or reductants was not necessary.18
The wide field (WF) confocal image is shown in Fig. 6A and the
2844 | Chem. Commun., 2018, 54, 2842--2845
View Article Online
Communication
Fig. 5 Colocalization analysis of NOR535 with Lysotracker Red. (A) The green-
channel image of fluorophore 1 from the photolysis of NOR535. (B) The red-
channel image of Lysotracker Red. (C) Merged image of the two channels.
(D) Bright-field image. (E) The line intensity profile of (C). Scale bar: 30 mm.
reconstructed super resolution PALM image of the same field of
view is displayed in Fig. 6B. The resolution of the PALM image
compared to the wide-field image was greatly improved and gave
the detailed lysosomal morphology. Fig. 6C and D are enlarged
boxed areas in Fig. 6A and B, respectively. The transverse profiles of
a single lysosome revealed that the full width from super-resolution
imaging was 86.9 nm (Fig. 6E). The number of mean photons
emitted from each fluorophore was B577 with a good localization
precision of 14.3 nm (Fig. 6F and G). Compared to the use of a UV
laser of 365 nm, the use of the 405 nm laser is advantageous due to
the higher biocompatibility.19,36
The photodecomposition of NOR535 is overall a photo-induced
hydrolysis, and therefore redox-neutral. A short-lived nitrogen based
radical species was generated. Radicals are routinely scavenged by
the reductive species in vitro, e.g. vitamin C, glutathione, vitamin E,
etc. However, it should not be taken for granted especially in
scenarios when the cellular redox homeostasis is disrupted. Also,
it should be noted that nitric oxide is involved in a range of
biological processes and may elicit facile biological responses. This
could potentially be harnessed for good. However, the impact of
nitric oxide on the structures and functions of the to-be-imaged
biological entities warrants further investigations.
In summary, we have developed a novel photo-activatable fluoro-
phore (NOR535) via N-nitrosation of a rhodamine dye. NOR535 is
cell-membrane permeable, passively localizes to lysosomes, and
feasible for imaging lysosomes in live cells with sub-diffraction
resolution. NOR535 is particularly attractive from the following
three aspects. First, PALM imaging with NOR535 can be performed
in a normal culture medium without an oxygen scavenging system
or other redox buffering agents. This allows cell imaging in their
physiological state. Second, the 405 nm laser line is more cell-
compatible than the use of UV. Third, 1 is a bright fluorophore and
gives a good localization precision of 14.3 nm. Therefore, NOR535
represents a superior molecular tool for lysosomal related biological
studies. We also note that N-nitroso is a practical and robust photo-
cage and could potentially be exploited to develop a broad range of
photo-activatable fluorophores for super-resolution microscopy.
The work is financially supported by the National Natural Science
Foundation of China (No. 21372080, 21572061, 21236002, 21376038,
This journal is © The Royal Society of Chemistry 2018

Communication
Published on 23 January 2018. Downloaded by Southeast University - Jiulonghu Campus on 1/21/2024 8:29:56 AM.
Fig. 6 The utility of NOR535 for super-resolution imaging of lysosomes in live HeLa cells. Wide-field image (A) and PALM image (B) of the lysosomes.
(C and D) Enlarged maps of the boxed areas of images A and B, respectively, showing a single lysosome. (E) Transverse profile of a single lysosome along
the white line in C and D. Fitting of the profile by two Gaussian functions (black line for C and orange line for D). (F) Histograms of the number of photons
per single-molecule event. (G) Histograms of the localization precision. Scale bars: 2 mm (A and B); 300 nm (C and D).
21421005, 21576040 and 21776037), National Basic Research Pro-
gram of China (No. 2013CB733702) and the Fundamental Research
Funds for the Central Universities (No. WY1514053, WY1516017 and
DUT17LK43).
Conflicts of interest
The authors declare no competing financial interests.
Notes and references
1 T. A. Klar, S. Jakobs, M. Dyba, A. Egner and S. W. Hell, Proc. Natl.
Acad. Sci. U. S. A., 2000, 97, 8206–8210.
2 E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych,
J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz and
H. F. Hess, Science, 2006, 313, 1642–1645.
3 M. J. Rust, M. Bates and X. Zhuang, Nat. Methods, 2006, 3, 793–795.
4 A. N. Boettiger, B. Bintu, J. R. Moffitt, S. Wang, B. J. Beliveau,
G. Fudenberg, M. Imakaev, L. A. Mirny, C. Wu and X. Zhuang,
Nature, 2016, 529, 418–422.
5 E. Lubeck and L. Cai, Nat. Methods, 2012, 9, 743–748.
6 L. Xue, I. A. Karpenko, J. Hiblot and K. Johnsson, Nat. Chem. Biol.,
2015, 11, 1–7.
7 I. Chamma, O. Rossier, G. Giannone, O. Thoumine and M. Sainlos,
Nat. Protoc., 2017, 12, 748–763.
8 X. Gu, E. Zhao, T. Zhao, M. Kang, C. Gui, J. W. Y. Lam, S. Du,
M. M. T. Loy and B. Z. Tang, Adv. Mater., 2016, 28, 5064–5071.
9 L. Carlini and S. Manley, ACS Chem. Biol., 2013, 8, 2643–2648.
10 S.-H. Shim, C. Xia, G. Zhong, H. P. Babcock, J. C. Vaughan,
B. Huang, X. Wang, C. Xu, G.-Q. Bi and X. Zhuang, Proc. Natl. Acad.
Sci. U. S. A., 2012, 109, 13978–13983.
11 G. Park, S. K. Chakkarapani, S. Ju, S. Ahn and S. H. Kang, Chin.
Chem. Lett., DOI: 10.1016/j.cclet.2017.08.018.
12 H. Takakura, Y. Zhang, R. S. Erdmann, A. D. Thompson, Y. Lin,
B. Mcnellis, F. Rivera-molina, S. Uno, M. Kamiya, Y. Urano,
J. E. Rothman, J. Bewersdorf, A. Schepartz and D. Toomre, Nat.
Biotechnol., 2017, 35, 773–780.
13 A. N. Butkevich and S. W. Hell, J. Am. Chem. Soc., 2017, 139, 12378–12381.
14 J. B. Grimm, A. K. Muthusamy, Y. Liang, T. A. Brown, W. C. Lemon,
R. Patel, R. Lu, J. J. Macklin, P. J. Keller, N. Ji and L. D. Lavis, Nat.
Methods, 2017, 14, 987.
This journal is © The Royal Society of Chemistry 2018
View Article Online
ChemComm
15 L. D. Lavis, Biochemistry, 2017, 56, 5165–5170.
16 P. Klán, T. Solomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina,
V. Popik, A. Kostikov and J. Wirz, Chem. Rev., 2013, 113, 119–191.
17 S. R. Adams and R. Y. Tsien, Annu. Rev. Physiol., 1993, 55, 755–784.
18 M. Fernández-Suárez and A. Y. Ting, Nat. Rev. Mol. Cell Biol., 2008, 9,
929–943.
19 L. M. Wysocki and L. D. Lavis, Curr. Opin. Chem. Biol., 2011, 15, 752–759.
20 S. Banala, D. Maurel, S. Manley and K. Johnsson, ACS Chem. Biol.,
2012, 7, 289–293.
21 J. B. Grimm, T. Klein, B. G. Kopek, G. Shtengel, H. F. Hess, M. Sauer
and L. D. Lavis, Angew. Chem., Int. Ed., 2016, 128, 1755–1759.
22 S. Hauke, A. von Appen, T. Quidwai, J. Ries and R. Wombacher,
Chem. Sci., 2017, 8, 559–566.
23 V. N. Belov, C. A. Wurm, V. P. Boyarskiy, S. Jakobs and S. W. Hell,
Angew. Chem., Int. Ed., 2010, 49, 3520–3523.
24 J. B. Grimm, B. P. English, H. Choi, A. K. Muthusamy, B. P. Mehl,
P. Dong, T. A. Brown, J. Lippincott-Schwartz, Z. Liu, T. Lionnet and
L. D. Lavis, Nat. Methods, 2016, 13, 985–988.
25 V. N. Belov, G. Y. Mitronova, M. L. Bossi, V. P. Boyarskiy, E. Hebisch,
C. Geisler, K. Kolmakov, C. A. Wurm, K. I. Willig and S. W. Hell,
Chem. – Eur. J., 2014, 20, 13162–13173.
26 E. A. Halabi, Z. Thiel, N. Trapp, D. Pinotsi and P. Rivera-Fuentes,
J. Am. Chem. Soc., 2017, 139, 13200–13207.
27 H. D. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams,
H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao
and W. E. Moerner, J. Am. Chem. Soc., 2010, 132, 15099–15101.
28 S. J. Lord, H. D. Lee, R. Samuel, R. Weber, N. Liu, N. R. Conley,
M. A. Thompson, R. J. Twieg and W. E. Moerner, J. Phys. Chem. B,
2010, 114, 14157–14167.
29 Z. Zhang, J. Wu, Z. Shang, C. Wang, J. Cheng, X. Qian, Y. Xiao, Z. Xu
and Y. Yang, Anal. Chem., 2016, 88, 7274–7280.
30 H. Xu and D. Ren, Annu. Rev. Physiol., 2015, 77, 57–80.
31 H. Yu, Y. Xiao and L. Jin, J. Am. Chem. Soc., 2012, 134,
17486–17489.
32 L. Wang, Y. Xiao, W. Tian and L. Deng, J. Am. Chem. Soc., 2013, 135,
2903–2906.
33 Q. Wan, S. Chen, W. Shi, L. Li and H. Ma, Angew. Chem., 2014, 126,
11096–11100.
34 H. Zhu, J. Fan, J. Du and X. Peng, Acc. Chem. Res., 2016, 49, 2115–2126.
35 D. Pan, Z. Hu, F. Qiu, Z.-L. Huang, Y. Ma, Y. Wang, L. Qin, Z. Zhang,
S. Zeng and Y.-H. Zhang, Nat. Commun., 2014, 5, 5573.
36 T. Klein, A. Löschberger, S. Proppert, S. Wolter, S. van de Linde and
M. Sauer, Nat. Methods, 2011, 8, 7–9.
Chem. Commun., 2018, 54, 2842--2845 | 2845