Organelle-selective energy transfer: A fluorescent indicator of intracellular environment

Article abstract of DOI:10.1016/j.bmcl.2011.01.040

A dye cassette fluoresces green (ca 520 nm) in the cytoplasm, endoplasmic reticulum (ER), and lysosomes, but red in mitochondria, that is, it illustrates 'organelle specific energy transfer'. This phenomenon may open new horizons in intracellular imaging.

Full text of DOI:10.1016/j.bmcl.2011.01.040

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1849–1851
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Organelle-selective energy transfer: A fluorescent indicator
of intracellular environment
Aurore Loudet a, Yuichiro Ueno a, Liangxing Wu a, Jiney Jose a, Rola Barhoumi b,
Robert Burghardt b, Kevin Burgess a,
⇑
a Department of Chemistry, Texas A&M University, PO Box 30012, College Station, TX 77842-3012, United States
b Department of Veterinary Integrative Bioscience, Texas A&M University, College Station, TX 77843-4458, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A dye cassette fluoresces green (ca 520 nm) in the cytoplasm, endoplasmic reticulum (ER), and lyso-
somes, but red in mitochondria, that is, it illustrates ‘organelle specific energy transfer’. This phenomenon
may open new horizons in intracellular imaging.
Received 22 November 2010
Revised 8 January 2011
Accepted 11 January 2011
Available online 15 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probes
Cell imaging
Energy transfer
Organelle-selective
Confocal microscopy
Visualization of intracellular organelles is based on two
approaches. Some organelles have distinctive shapes/cellular loca-
tions and are easily recognizable. Others are made conspicuous
using fluorescent dyes known to target specific organelles, for
example, LysoTracker, ER-Tracker, MitoTracker, and TubulinTrac-
ker.1–3 Generally, these dyes absorb in one wavelength range and
emit in another; there are limited cases where the fluorescence
emission wavelength of a dye changes significantly with its intra-
cellular environment. Probes with fluorescence emission wave-
lengths that do vary with cellular location, however, could be
valuable when monitoring dynamic organelle specific functions.
Our group has an ongoing interest in ‘energy transfer cassettes’.
Briefly, these are molecules with donor components that absorb
strongly at a convenient wavelength (e.g., 488 nm), and acceptor
fragments that emit efficiently at significantly longer wavelengths
(Fig. 1).4–7 Until now the objectives for designing these cellular
imaging probes were based solely on predictable photophysical
phenomena, for example, minimization of the impact of back-
scattering from the excitation source, possibilities for multiplexing.
However, in the work described here an unexpected issue was
encountered: the proportions of donor/acceptor emissions can be
dependent on the cellular environment causing the cassettes to change
color. This is described in the context of cassette 1 that does not
display this phenomenon, and 2 that does.
Cassettes 1 and 2 were prepared via Sonogashira coupling
BODIPY or cyanine-derived fragments as described in the Supple-
mentary data. Salient features of their photophysical properties
are indicated in Figure 1. The cassettes each have two distinct
absorbance maxima corresponding to their donor and acceptor
fragments. We have defined the ratio of the fluorescence quantum
yields of cassettes when excited at the donor to that when excited
at the acceptor as the energy transfer efficiency (ETE).7 The overall
quantum yield for 2 is lower than for cassette 1, but both are
bright fluorescent dyes. When excited at 488 nm, they glow red
corresponding to fluorescence emission from the acceptor part.
After treatment of Clone 9 rat-liver cells with cassette 1 for
30 min the probe permeated inside and accumulated in the lyso-
somes and ER ( Fig. 2a; colocalization with LysoTrackerÒ Green
DND-26, and with ER-Tracker™ Blue-White DPX, respectively,
see Supplementary data). As in EtOH, no fluorescence was ob-
served from the donor part of this cassette upon excitation at
488 nm, that is, there was no fluorescent signal in the green chan-
nel. However, strong fluorescence was observed in the red channel
indicating efficient energy transfer in both the lysosomes and in
the ER; thus cassette 1 was largely unaffected by its cellular
environment.
Cassette 2 permeated Clone 9 cells and accumulated in the
mitochondria, lysosomes and cytoplasm. The wavelength of the
⇑
Corresponding author.
E-mail address: burgess@tamu.edu (K. Burgess).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.040

1850
A. Loudet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1849–1851
energy transfer cassettes
hv
emits relatively
emits relatively
long
wavelengths
if energy is
transferred
short
donor
acceptor
wavelengths
if energy is
not transferred
acceptor
MeO
λmax ab 498, 543 nm
λmax emiss 600 nm
ΦD 0.46+/-0.02
CO2H
ΦA 0.48+/-0.01
donor
O
ETE 96 %
O
N
N
N
N
(EtOH)
BF2
BF2
N
MeO
NaO3S
donor
1
λmax ab 504, 569 nm
λmax emiss 590 nm
ΦD 0.20+/-0.01
ΦA 0.22+/-0.01
ETE 90 %
acceptor
(EtOH)
N
OMe
F2B
N
HO2C
N
I–
4
Figure 2. Cellular uptake of cassettes 1 and 2 in Clone 9 cells. Part a shows that
cassette 1 accumulates in the lysosomes and the ER, and perfect energy transfer is
observed. Part b shows that cassette 2 labels the lysosomes, cytoplasm, ER and
mitochondria, and the extent of energy transfer is organelle specific. Throughout
the cassettes were excited at 488 nm, that is, donor part. Fluorescence images for
the donor and acceptor emissions were collected with Band Pass (BP) 500–530 and
BP 565–615 emission filters, respectively.
N
2
Figure 1. Concept of energy transfer cassettes, and probes 1 and 2.
indicative of good energy transfer) was observed in the mitochon-
dria. Conversely, only green emission was observed in the cyto-
plasm and lysosomes (Fig. 2b).
fluorescence observed was organelle specific (excitation at 488 nm,
throughout). Thus, red emission (fluorescence from the acceptor,
Figure 3. Fluorescence of cassette 2 when excited at the donor part (488 nm, top row) and the acceptor part (543 nm, bottom). It emits from the donor in the cytoplasm, ER
and lysosomes, and from the acceptor in the mitochondria when excited at 488 nm. Direct excitation at the acceptor part (543 nm, bottom row) proves the emission from the
acceptor is quenched in the cytoplasm, ER, and lysosomes.

A. Loudet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1849–1851
1851
Two possibilities were envisaged to account for the fact that the
Acknowledgments
fluorescence of the acceptor is not observed for cassette 2 in the
cytoplasm, lysosomes and in the ER. The first is that the energy
transfer from the donor to the acceptor became inefficient in the
environment of those intracellular regions. The second hypothesis
is that fluorescence from the acceptor becomes quenched in the
cytoplasm, lysosomes and in the ER, but not in the mitochondria.
These alternatives could be distinguished by exciting the localized
cassettes at the acceptor absorption maximum; under these condi-
tions the acceptor would be visible if the first hypothesis applied,
but not in the second case. In the event, when 2 was excited di-
rectly at 543 nm, that is, at the acceptor part, only red emission
from the mitochondria could be observed (Fig. 3, bottom). No red
signal was seen in the ER, cytosol or the lysosomes, suggesting
the fluorescence from the cyanine acceptor is quenched in these
environments. However, the energy transfer cannot be complete
otherwise residual fluorescence from the donor parts would not
be seen in the intracellular regions that fluoresce green.
We thank The National Institute of Health (GM087981) and The
Robert A. Welch Foundation (A-1121) for the financial support for
this work.
Supplementary data
Supplementary data (experimental procedures for the prepara-
tion of cassettes 1 and 2, photophysical studies, and additional cell
imaging experiments (particularly colocalization)) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.01.040.
References and notes
1. http://probes.invitrogen.com. In Molecular Probes, pH Indicators, Chapter 20,
Invitrogen Corporation, 2006.
In summary, cassette 1 targeted the ER and lysosomes, the en-
ergy transfer observed for this cassette was near-perfect in both
these environments, that is, it always fluoresced red. Conversely,
cassette 2 targeted a different set of organelles and fluoresced with
different colors: mitochondria (red), cytoplasm including the ER
(green), and lysosomes (green).
2. Oakley, B. R.; Xiang, X. Mycol. Ser. 2008, 26, 513.
3. Zorov, D. B.; Kobrinsky, E.; Juhaszova, M.; Sollott, S. J. Circ. Res. 2004, 95,
239.
4. Jiao, G.-S.; Thoresen Lars, H.; Burgess, K. J. Am. Chem. Soc. 2003, 125, 14668.
5. Burgess, K. U.S. Patent 2005/0032120 A1, The Texas A&M University System,
USA, US, 2005, 7.
6. Burgess, K. U.S. Patent 2005/0032120 The Texas A&M University System US,
2009; 24.
Cassette 1 has a neutral BODIPY-based acceptor fragment, while
2 has a cationic cyanine; this structural difference is most likely the
origin of acceptor fluorescence quenching in some organelles, but
just for the cyanine-based cassette 2. Further, the fluorescence of
cyanines tends to be influenced by membrane potential effects.8–13
Overall, these data lead to a general conclusion that may be obvi-
ous, but is not often considered: some organelle specific dyes
may be widely distributed in cells but only seen in regions where
their fluorescence is not quenched. In general, organelle-selective
energy transfer such as observed here may open new horizons
for intracellular imaging.
7. Wu, L.; Loudet, A.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. J. Am. Chem. Soc.
2009, 131, 9156.
8. Waggoner, A. S. Methods Enzymol. 1979, 55, 689.
9. Benson, R. C.; Kues, H. A. J. Chem. Eng. Data 1977, 22, 379.
10. Johnson, L. V.; Walsh, M. L.; Bockus, B. J.; Chen, L. B. J. Cell Biol. 1981, 88,
526.
11. Wilson, H. A.; Seligmann, B. E.; Chused, T. M. J. Cell. Physiol. 1985, 125, 61.
12. Liu, Z.; Bushnell, W. R.; Brambl, R. Plant Physiol. 1987, 84, 1385.
13. Patsenker, L. D.; Tatarets, A. L.; Terpetschnig, E. A. In Advanced Fluorescence
Reporters in Chemistry and Biology I: Fundamentals and Molecular Design;
Demchenko, A. P. Ed.; 2010; pp 65–104.